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Lab. of Vet. Anat.
Preventive effects of natural supplements against embryotoxicity induced by
alcohol and nicotine
Sang-Yoon Nam, DVM, PhD ([email protected])
Laboratory of Veterinary Anatomy, College of Veterinary Medicine,
CHUNGBUK NATIONAL UNIVERSITY
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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).
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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.
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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).
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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).
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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
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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).
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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
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Results
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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/ml Ethanol 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
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Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos (Reprod Toxicol. 2008;26(3-4):292-7)
SOD activity GPx1
GPx4
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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).
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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
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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).
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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
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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).
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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
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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
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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 D2 C2
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
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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
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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).
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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
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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).
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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)
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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.
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충북대학교 수의과대학 해부학교실
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Conclusion (I
