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  • 1. Lab. of Vet. Anat. Preventive effects of natural supplements against embryotoxicity induced by alcohol and nicotine Sang-Yoon Nam, DVM, PhD (synam@cbu.ac.kr) Laboratory of Veterinary Anatomy, College of Veterinary Medicine, CHUNGBUK NATIONAL UNIVERSITY
  • 2. 제목서식 텍스트 서식 Lab. of Vet. Anat. Fetal Alcohol Syndrome (FAS) is characterized by pre- and postnatal growth retardation, neuronal damage, a specific pattern of craniofacial abnormalities, and limb and cardiac defects (Jones and Smith, 1973). Fetal Alcohol Syndrome (FAS) (http://www.faslink.org/fasmain.htm) The insult on the brain by ethanol affects intelligence, cognitive function, and behavior (Abel, 1990). In the United States and Europe, the FAS prevalence rate is estimated to be between 0.2-2 in every 1000 live births (Vaux and Keith, 2012).
  • 3. 제목서식 텍스트 서식 Lab. of Vet. Anat. Ethanol-induced toxic mechanism Maternal alcohol consumption induces an excess production of ROS in fetal tissues which is responsible for some toxic responses to alcohol (Cohen-Kerem and Koren , 2003; Lee et al 2005). Therefore, antioxidants may significantly improve adverse developmental outcomes and diminish the incidence and severity of major malformations that result from ethanol exposure in utero.
  • 4. 제목서식 텍스트 서식 Lab. of Vet. Anat. Effects on pregnancy and fetus by smoking 자연유산, 조산, 전치태반, 태반 조기박리, 신생아사망, 구순열, 구개열, 저체중아, 정신장애 수정능력 저하, 유산, 조기폐경, 골다공증, 암발생 및 심혈관계 질환 증가 등 A single cigarette smoking provides about 1 mg of nicotine for human and produces a peak increment of plasma nicotine concentration of 5–30 ng/ml within minutes (Benowitz, 1988). Nicotine readily gains access to fetal compartment via the placenta, with fetal concentrations generally 15% higher than maternal levels (Lambers and Clark, 1996).
  • 5. 제목서식 텍스트 서식 Lab. of Vet. Anat. Nicotine toxic mechanisms To reduce embryotoxicity caused by cigarette smoking during pregnancy, exogenous antioxidants that counteract the damaging effects of cigarette smoke have received increasing attention (Eiserich et al., 1995). Nicotine induces excessive generation of reactive oxygen species (ROS) that perturbs the oxidant/antioxidant balance, and leads to the development of embryonic and fetal oxidative stress during pregnancy (Zhao and Reece, 2005; Bruin et al., 2008).
  • 6. 제목서식 텍스트 서식 Lab. of Vet. Anat. Objectives To search the preventive natural supplements against embryotoxicities induced by alcohol and nicotine and demonstrate their regulatory mechanisms using a whole embryo culture system
  • 7. 제목서식 텍스트 서식 Lab. of Vet. Anat. Whole Embryo Culture System Ed 8.5 (Ikemoto Rika Kogyo, Japan) 1) 5% O2, 5% CO2, 90% N2: 17 hrs 2) 20% O2, 5% CO2, 75% N2: 7 hrs 3) 40% O2, 5% CO2, 55% N2: 24 hrs Ed 10.5 : 3.75 mg/embryo Rat serum Organogenesis is the stage of development during which the embryo is most sensitive to environmental insults (Lambers and Clark, 1996).
  • 8. 제목서식 텍스트 서식 Lab. of Vet. Anat. Morphological Scoring: Van Maele-Fabry et al (1990) 17 standard scoring items (differentiation) along with the yolk sac diameter, crown- rump length, and head length (growth). Yolk sac circulation Crown-rump length (mm) Flexion Midbrain
  • 9. 제목서식 텍스트 서식 Lab. of Vet. Anat. Results
  • 10. 제목서식 텍스트 서식 Lab. of Vet. Anat. Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos (Reprod Toxicol. 2008;26(3-4):292-7.) Capsaicin 1×10−8 or 1×10−7 μg/ml/embryo = 0.16 or 1.6 μg/ 60 kg BW Cap 1×10−8 μg/mlEthanol 1ul/ml Cap 1×10−8 μg/ml E+Cap 1×10−8 or 1×10−7 μg/ml Normal The major pungent ingredient found in hot peppers of the plant genus Capsicum. The capsaicin content in peppers ranges from 0.1 to 2.5 mg/g (Parrish, 1996). Alcohol 1 μl/ml = 0.1% 면허취소/6개월이하 징역/ 300만원이하 벌금
  • 11. 제목서식 텍스트 서식 Lab. of Vet. Anat. Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos (Reprod Toxicol. 2008;26(3-4):292-7) SOD activity GPx1 GPx4
  • 12. 제목서식 텍스트 서식 Lab. of Vet. Anat. Black ginseng(BG) 1, 10 and 100 μg/ml/embryo = 16, 160, 1600 g/ 60kg BW Ethanol 1 μl/ml E+ BG (1, 10 and 100 μg/ml) Normal Black ginseng inhibits ethanol-induced teratogenesis in cultured mouse embryos through its effects on antioxidant activity (Toxicol In Vitro. 2009 ;23(1):47-52.) Black ginseng is produced from red ginseng by 9 cycles of steam treatment, at which point it becomes black in color (Lee et al., 2006). During the steaming process, the ratio of major bioactive components, including ginsenosides, saponin, phenolics, and proteins, is altered as newly produced components (Kim et al., 2007).
  • 13. 제목서식 텍스트 서식 Lab. of Vet. Anat. Black ginseng inhibits ethanol-induced teratogenesis in cultured mouse embryos through its effects on antioxidant activity (Toxicol In Vitro. 2009 ;23(1):47-52) GPx1 Selenoprotein p GPx4
  • 14. 제목서식 텍스트 서식 Lab. of Vet. Anat. Ethanol 5 μl/3 ml GIN 10−8 & 10−7 μg/ml Protective Effect of [6]-Gingerol on the Ethanol-induced Teratogenesis of Cultured Mouse Embryos (Arch Pharm Res. 2012;35(1):171-8) [6]-Gingerol (GIN) 1 ×10−8 or 1 × 10−7 μg/ml = 0.16 or 1.6 μg/ 60 kg BW Normal Ginger rhizome (Zingiber officinale), commonly known as ginger, is consumed worldwide as a spice and a flavoring agent which contains pungent phenolic substances and aromatic ketones known as [6]- gingerols (Kim et al., 2005).
  • 15. 제목서식 텍스트 서식 Lab. of Vet. Anat. Protective Effect of [6]-Gingerol on the Ethanol-induced Teratogenesis of Cultured Mouse Embryos (Arch Pharm Res. 2012;35(1):171-8) GPx1 SOD2 SOD1 SOD activity
  • 16. 제목서식 텍스트 서식 Lab. of Vet. Anat. Normal Ethanol(E) E+Emo 10−5 , 10−4 μg/ml Emodin Prevents Ethanol-Induced Developmental Anomalies in Cultured Mouse Fetus through Multiple Activities (Birth Defects Res B Dev Reprod Toxicol. 2013;98(3):268-75) Emodin 1 × 10−5 or 1 × 10−4 μg/ml = 0.16 or 1.6 mg/ 60 kg BW A naturally occurring anthraquinone that is found in many herbs and vegetables, such as aloe, cabbage, lettuce, beans, and peas (Singh and Trigun, 2011).
  • 17. 제목서식 텍스트 서식 Lab. of Vet. Anat. Emodin Prevents Ethanol-Induced Developmental Anomalies in Cultured Mouse Fetus through Multiple Activities (Birth Defects Res B Dev Reprod Toxicol. 2013;98(3):268-75) HIF-1a TNFa Caspase 3 SOD activity SOD1 GPx1 SOD2
  • 18. 제목서식 텍스트 서식 Lab. of Vet. Anat. Natural products Alcohol-induced teratogenesis Antioxidative effects Conclusion (I) Natural products (capsaicin, [6]-gingerol , black ginseng, and emodin) play an important role in the protection of ethanol-induced teratogenesis in mouse embryo through their antioxidative activities. Capsaicin Black ginseng [6]-Gingerol Emodin
  • 19. 제목서식 텍스트 서식 Lab. of Vet. Anat. Resveratrol prevents nicotine-induced teratogenesis in cultured mouse embryos (Reprod Toxicol. 2012; 34(3):340-6) Resveratrol 1×10−8 (C) & 1×10−7 (D) µM/ embryo = 36.8 & 368 ng/ 60 kg BW Nicotine + Resveratrol 1mM Nicotine C1 D1 D2C2 Normal A member of the phytoalexin family found in a wide diversity of plants including grapes, raspberries, mulberries, pistachios, and peanuts (Soleas et al 2001). Fresh grape skin contains about 50–100 mg of resveratrol per gram (Jeandet et al, 1991). 1mM Nicotine (162.23 µg/ml ) = 0.16 cigarette
  • 20. 제목서식 텍스트 서식 Lab. of Vet. Anat. Resveratrol prevents nicotine-induced teratogenesis in cultured mouse embryos (Reprod Toxicol. 2012; 34(3):340-6.) HIF-1a Hypoxia inducible factor-1a mRNA expression levels SOD1 SOD2 GPx1 GPx4 Gene expression levels of antioxidant enzymes Lipid peroxidation levels MDA Antioxidative enzyme (SOD) activity levels SOD activity Gene expression levels of apoptosis related factors Bcl-𝑥𝐿 Caspase 3
  • 21. 제목서식 텍스트 서식 Lab. of Vet. Anat. 1mM Nicotine Morphological scoring (growth and differentiation) in cultured mouse embryos Antiteratogenic effects of 𝛽-Carotene in cultured mouse embryos exposed to nicotine (Evid Based Complement Alternat Med. 2013;2013:575287) 𝛽-Carotene 1ⅹ10-7 (C) & 5ⅹ10-7 (D) µM = 0.88 & 4.4 mg/ 60 kg BW Normal Nicotine + 𝛽-Carotene The provitamin A carotenoid 𝛽-carotene is not only an essential source of vitaminA, but also shows antioxidative and antiinflammatory activities in various tissues (Bai et al., 2005).
  • 22. 제목서식 텍스트 서식 Lab. of Vet. Anat. Lipid peroxidation levels MDA Antioxidative enzyme (SOD) activity levels SOD activity Gene expression levels of proinflammatory cytokines TNF-𝛼 IL-1𝛽 normal control 1mM nicotine N+𝛽-car 1×10−7 Apoptotic embryos images by Nile blue staining N+𝛽-car 5×10−7 Antiteratogenic effects of 𝛽-Carotene in cultured mouse embryos exposed to nicotine (Evid Based Complement Alternat Med. 2013;2013:575287) Gene expression levels of antioxidant enzymes SOD1 SOD2 GPx1 GPx4 Gene expression levels of apoptosis related factors Bcl-𝑥𝐿 Caspase 3
  • 23. 제목서식 텍스트 서식 Lab. of Vet. Anat. 4-O-methylhonokiol inhibits serious embryo anomalies caused by nicotine via modulations of oxidative stress, apoptosis, and inflammation (Birth Defects Res B. 2014 (in press) Magnolia officinalis(후박나무) 4-O-methylhonokiol 1х 10-4 (C) & 1х 10-3 (D) µM/ embryo = 4.4 & 44 mg/ 60 kg BW 1mM Nicotine Nicotine + 4-O-methylhonokiol Normal Active components isolated from Magnolia officinalis. 4-O-methylhonokiol exhibits various biological activities including anticancer effects, anti-inflammatory, and antioxidant actions (Kong et al., 2008).
  • 24. 제목서식 텍스트 서식 Lab. of Vet. Anat. Lipid peroxidation levels MDA SOD activity SOD1 GPx1 GPx4 Gene expression levels of antioxidant enzymes HIF-1a Hypoxia inducible factor-1a mRNA expression levels Gene expression levels of proinflammatory cytokines TNF-𝛼 IL-1𝛽 Gene expression levels of apoptosis related factors Bcl-𝑥𝐿 Caspase 3 4-O-methylhonokiol inhibits serious embryo anomalies caused by nicotine via modulations of oxidative stress, apoptosis, and inflammation (Birth Defects Res B. 2014 (in press)
  • 25. 제목서식 텍스트 서식 Lab. of Vet. Anat. Natural products 4-O-methylhonokiol b-carotene Resveratrol Nicotine-induced teratogenesis anti-apoptotic, anti-inflammatory, anti-oxidative effects Conclusion (II) Natural products (resveratrol, β-carotene, and 4-O- methylhonokiol) play an important role in the protection of nicotine-induced teratogenesis in mouse embryo through anti- apoptotic, anti-inflammatory, and anti-oxidative effects.
  • 26. 제목서식 텍스트 서식 Lab. of Vet. Anat. 충북대학교 수의과대학 해부학교실
  • 27. 제목서식 텍스트 서식 Lab. of Vet. Anat. Conclusion (I