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
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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
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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
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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
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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
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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
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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
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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
10. 제목서식
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
