carotene in cultured mouse embryos exposed to nicotine

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 575287 11 pageshttpdxdoiorg1011552013575287

Research ArticleAntiteratogenic Effects of 120573-Carotene in Cultured MouseEmbryos Exposed to Nicotine

Chunmei Lin Jung-Min Yon A Young Jung Jong Geol Lee Ki Youn JungBeom Jun Lee Young Won Yun and Sang-Yoon Nam

College of Veterinary Medicine and Research Institute of Veterinary Medicine Chungbuk National UniversityCheongju 361-763 Republic of Korea

Correspondence should be addressed to Sang-Yoon Nam synamcbuackr

Received 28 January 2013 Accepted 9 April 2013

Academic Editor Alfredo Vannacci

Copyright copy 2013 Chunmei Lin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

After maternal intake nicotine crosses the placental barrier and causes severe embryonic disorders and fetal death In this studywe investigated whether 120573-carotene has a beneficial effect against nicotine-induced teratogenesis in mouse embryos (embryonicday 85) cultured for 48 h in a whole embryo culture system Embryos exposed to nicotine (1mM) exhibited severe morphologicalanomalies and apoptotic cell death as well as increased levels of TNF-120572 IL-1120573 and caspase 3mRNAs and lipid peroxidation Thelevels of cytoplasmic superoxide dismutase (SOD) mitochondrial manganese-dependent SOD cytosolic glutathione peroxidase(GPx) phospholipid hydroperoxide GPx hypoxia inducible factor 1120572 and Bcl-x

119871mRNAs decreased and SOD activity was reduced

compared to the control group However when 120573-carotene (1 times 10minus7 or 5 times 10minus7120583M) was present in cultures of embryos exposedto nicotine these parameters improved significantly These findings indicate that 120573-carotene effectively protects against nicotine-induced teratogenesis in mouse embryos through its antioxidative antiapoptotic and anti-inflammatory activities

1 Introduction

Cigarette smoking can increase the risk of adverse out-comes during pregnancy including fetal growth restrictionincreased rates of spontaneous abortion premature placentalabruption perinatal lethality decreased birth weight andsudden infant death syndrome [1] Although themechanismslinking fetal exposure to cigarette smokewith cellular damageare not clearly understood smoking induces a toxic statethat increased oxidative stress and modulates inflammatoryresponses [2] Human and animal studies have demonstratedthat cigarette smoking causes oxidative damage and growthretardation in the embryo via production of excess reactiveoxygen species (ROS) [3] Nicotine a major toxic componentof cigarette smoke crosses the placental barrier and actsdirectly on the fetus with fetal concentrations generally 15higher than maternal levels [1]

Maternal cigarette smoking during pregnancy may alterthe micronutrient status in both the fetal and maternalenvironment [4] Recently we found that resveratrol apolyphenol from red wine can prevent nicotine-induced

teratogenesis in culturedmouse fetuses [5] Dietarymicronu-trients such as vitamin C vitamin E and 120573-carotene con-tribute to the antioxidant defense system [6] In particular theprovitamin A carotenoid 120573-carotene is not only an essentialsource of vitaminA [7] but also shows antioxidative and anti-inflammatory activities in various tissues [8]

Since early embryonic antioxidant systems are immatureand antioxidants are present at considerably lower levelsthan in adults the developing embryo may be more sus-ceptible to ROS-induced damage [9] Recently exogenousantioxidants introduced through diet have become popularThere has been a growing interest in identification of possibledietary antioxidants to treat or prevent diseases caused byROS Therefore we hypothesized that the nicotine-inducedexcessive ROS that leads to embryonic and fetal oxidativestress could be effectively counteracted by 120573-carotene andoxidative damage could be attenuated In the present studywe investigated the potential protective effects of 120573-caroteneagainst nicotine-induced teratogenesis in cultured mouseembryos using a whole embryo culture system

2 Evidence-Based Complementary and Alternative Medicine

(a) (b1) (b2) (b3)

(c1) (c2) (d1) (d2)

Figure 1 Representative images of mouse embryos exposed to nicotine and 120573-carotene Normal control group (a) Embryos treated with1mM nicotine alone show typical abnormalities such as exposed brain reduced forebrain abnormal heart deformed posterior trunk andregressed forelimbs (b1minus3) Embryos treated with nicotine plus 120573-carotene [1 times 10minus7 120583M (c1 and c2) and 5 times 10minus7 120583M (d1 and d2)] appearmorphologically similar to the control group

2 Materials and Methods

21 Experimental Animals Male and female ICR mice (8ndash10 weeks old) were purchased from a commercial breeder(BioGenomics Co Seoul Republic of Korea) The animalswere housed in a climate controlled facility with an ambienttemperature of 21 plusmn 2∘C relative humidity of 55 plusmn 10air ventilation rate of 10 cycles per hour and a 12 12 hlight dark cycle The animals were fed standard mouse chow(Samyang Ltd Incheon Republic of Korea) and tap waterad libitum throughout the experimental period One maleand three female mice were housed in a cage for matingPregnancies were confirmed the following morning (0800)by the presence of vaginal plugs or spermatozoa detected ina vaginal smear after mating the previous evening (2000)this was considered embryonic day (E) 05 Pregnant micewere sacrificed and embryos were obtained at E85 Allexperiments were approved by the Chungbuk National Uni-versity Animal Care Committee and carried out accordingto the Guide for Care and Use of Animals (ChungbukNational University Animal Care Committee according toNIH number 86ndash23)

22 Rat Serum Preparation Serum of Sprague-Dawley malerats (10ndash12 weeks old) was prepared for embryo cultures asfollows After collection blood samples were immediatelycentrifuged for 10min at 3000 rpm and 4∘C to clear the

plasma fraction of cellsThe supernatant was then transferredto new tubes and centrifuged for 10min at 3000 rpm and 4∘Cto remove remaining blood cellsThe clear serum supernatantwas decanted and pooled and the pooled serum was heat-inactivated for 30min at 56∘C in a water bath It was theneither used immediately or stored at minus70∘C Serum wasincubated at 37∘C and filtered through a 02 120583m filter priorto use in the whole embryo culture

23 Whole Embryo Culture and Nicotine and 120573-CaroteneTreatments The whole embryo culture system was based ona previously described procedure [10] Animals were sacri-ficed between 0900 and 1000 h via cervical dislocation whenembryos reached E85 Only embryos with 4ndash8 somites wereutilized After removing the decidua and Reichertrsquos mem-branes embryos with intact visceral yolk sacs and ectopla-cental cones were placed randomly into sealed culture bottles(three embryosbottle) containing 3mL of culture mediumand different concentrations (1 times 10minus7 or 5 times 10minus7 120583M)of 120573-carotene (Sigma St Louis MO USA) dissolved indimethyl sulfoxide (DMSO Sigma) andor 1mM nicotine(1638 120583gmL serum Sigma) The nicotine concentrationused here was determined by previous studies [5 11]The finalconcentration of DMSO in the medium was less than 01Embryos were randomized into four treatment groups (1)control (2) nicotine (3) nicotine plus 1times 10minus7 120583M120573-caroteneand (4) nicotine plus 5 times 10minus7 120583M 120573-carotene The embryos

Evidence-Based Complementary and Alternative Medicine 3M

DA

pro

tein

(nm

olm

g)

50

40

30

20

10

0Con N

lowast

1 times 10minus7

5 times 10minus7

N + 120573-car N + 120573-car

Figure 2 Protective effects of 120573-carotene against oxidative damageinduced by nicotine in E85 mouse embryos treated in vitro for 2days Lipid peroxidation was evaluated by measuring the malon-dialdehyde (MDA) concentration in embryos treated with 1mMnicotine in the absence or presence of 1 times 10minus7 or 5 times 10minus7 120583M 120573-carotene (120573-car) Results are presented as mean plusmn SEM (119899 = 12)Significant differences (lowastcontrol versus nicotine alone nicotineversus 120573-car + nicotine) were evaluated by one-way ANOVA at119875 lt 005

SOD

activ

ityp

rote

in (U

mg)

1

08

06

04

02

0Con N

lowast

1 times 10minus7

5 times 10minus7

N + 120573-car N + 120573-car

Figure 3 Superoxide dismutase (SOD) activity levels in E85 mouseembryos exposed to nicotine and120573-carotene for 2 days in vitro SODactivity in embryos treated with 1mM nicotine in the absence orpresence of 1times 10minus7 or 5times 10minus7 120583M120573-carotene (120573-car) wasmeasuredResults are presented as mean plusmn SEM (119899 = 6) Significant differences(lowastcontrol versus nicotine alone nicotine versus 120573-car + nicotine)were evaluated by one-way ANOVA at 119875 lt 005

were incubated at 37 plusmn 05∘C in sealed culture bottles (threeembryosbottle) and rotated at 25 rpm The culture bottleswere initially gassed with a mixture of 5 O

2 5 CO

2 and

90 N2over a 17 h period at a flow rate of 150mLmin

Subsequent gassing was performed at the same rate over 7 h(20 O

2 5 CO

2 and 75 N

2) and 24 h (40 O

2 5 CO

2

and 55 N2) All embryos were cultured for 48 h using a

whole embryo culture system (Ikemoto Rika Kogyo Japan)

24 Morphological Scoring At the end of the 48 h cultureperiod the morphology of the embryos was evaluatedaccording to a previously described scoring system [12] Onlyviable embryos with yolk sac circulation and a heartbeatwere used for morphological scoring Measurements of eachviable embryo were obtained for 17 standard scoring itemsas well as the yolk sac diameter crown-rump length andhead length The morphological features that were assessedincluded embryonic flexion heart caudal neural tube brain(forebrain midbrain and hindbrain) otic and optic systemsolfactory organs branchial arch maxilla mandible limbbuds (forelimb and hindlimb buds) yolk sac circulationallantois and somites

25 Lipid Peroxidation Measurements Lipid peroxidationwas determined using thiobarbituric acid (TBA) as describedby Ohkawa et al [13] with minor modifications The levelof malondialdehyde (MDA) a secondary by-product of lipidperoxidation was measured spectrophotometrically afterreaction with TBA The results are expressed as nmolmgprotein Briefly embryos (10ndash16) in each group were homog-enized in chilled 10mM phosphate buffer and were thenmixed thoroughly with 81 sodium dodecyl sulfate 20acetic acid and 075 2-thiobarbituric-acid solution Thesolution was heated for 30min in a 95∘C oven After cool-ing insoluble material was removed by centrifugation at3500 rpm for 15min The absorbance of the supernatant wasmeasured at 532 nmwith a spectrophotometer and comparedto the prepared 1133-tetramethoxypropane standard curveThe total protein content of the embryos was determinedaccording to the method of Lowry et al [14] using bovineserum albumin as the standard

26 SODActivityAssay Total SODactivitywas assayed usinga SOD Assay kit-WST (Dojindo Laboratories KumamotoJapan) Briefly 5ndash8 mouse embryos were homogenizedand the protein concentrations of the supernatants wereanalyzed by the Bradford method [15] The supernatantswere incubated with an assay reagent containing xanthinexanthine oxidase and a water-soluble tetrazolium salt WST-1 The superoxide free radicals generated from the xanthineby xanthine oxidase reduced WST-1 to WST-1 diformazanwhich absorbs maximally at 450 nm SOD in the embryosinhibits the WST-1 reduction since the enzyme catalyzesthe dismutation of superoxide ions to molecular oxygen andhydrogen peroxide The reduction of WST-1 was measuredspectrophotometrically at 450 nm SOD activity was calcu-lated as an inhibition rate in which 1 U was defined as a 50decrease from the control value over a period of 30min at37∘C

27 Nile Blue Staining Embryonic cell death was detectedby a classic technique using Nile blue staining to observeapoptotic nuclei and dead cells in blue color E105 embryoswere dissected into PBS Embryos were placed in 15 Nileblue (Sigma) in PBS incubated at 37∘C for 45 minutesand then monitored every 15min using a light microscopeuntil staining reached the desired level Pale blue staining


12

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SOD

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(x-fo

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N + 120573-car N + 120573-car

(a)

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SOD

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(b)

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(x-fo

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(c)

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GPx

4120573

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expr

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N + 120573-car N + 120573-car

(d)

Figure 4 Gene expression levels of antioxidant enzymes in E85 mouse embryos exposed to nicotine and 120573-carotene for 2 days in vitroLevels of mRNA for cytoplasmic superoxide dismutase (SOD1 (a)) manganese SOD (SOD2 (b)) cytoplasmic glutathione peroxidase (GPx1(c)) and phospholipid hydroperoxide GPx (GPx4 (d)) in embryos exposed to 1mM nicotine in the absence or presence of 1 times 10minus7 or 5times 10minus7 120583M 120573-carotene (120573-car) were measured by quantitative RT-PCR Results are mean plusmn SEM (119899 = 8) 120573-actin was used as an internalstandard to normalize target transcript expression Significant differences (lowastcontrol versus nicotine alone nicotine versus 120573-car + nicotine)were evaluated by one-way ANOVA at 119875 lt 005

(background level) shows the normal live cells and dark bluestaining reveals regions of cell death

28 Quantitative Real-Time Polymerase Chain Reaction (PCR)Analysis Total RNA was extracted from six to nine cul-tured mouse embryos using the Trizol Reagent (InvitrogenCarlsbad CA USA) The RNA was further purified using anRNA clean-up kit (Macherey-Nagel Bethlehem USA) TotalRNA (2 120583g) was used for cDNA synthesis (Invitrogen) Real-time PCR was carried out in a 20 120583L reaction volume usingthe SYBR Green Master Mix (Applied Biosystems FosterCity CA USA) and mouse embryonic cDNA (16 120583g) as thetemplate Reactions were performed using a 7500 Real-TimePCR System (Applied Biosystems) according to the manu-facturerrsquos instructions Gene-specific primers were designedby TIB Mol-Bio Synthesis (Berlin Germany) Primers to

mouse cytoplasmic superoxide dismutase (SOD1) man-ganese SOD (SOD2) cytoplasmic glutathione peroxidase(GPx1) phospholipid hydroperoxide glutathione peroxidase(GPx4) hypoxia inducible factor 1120572 (HIF-1120572) Bcl-119909

119871 caspase

3 and cytokines (TNF-120572 and IL-1120573) were used (Table 1) 120573-actin primers were used as an internal standard to normalizetarget transcript expression Data from nine independentruns were analyzed using the comparative Ct method [16]

29 Statistical Evaluation Group differences in gene expres-sion lipid peroxidation and SOD activity were assessed viaone-way ANOVA followed by Tukeyrsquos multiple comparisontest Morphological data were compared using the Kruskal-Wallis nonparametric ANOVA and Dunnrsquos multiple compar-ison post hoc test A 119875 lt 005 was considered significantAll data are expressed as mean plusmn SEM All analyses were

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Primer sequences used in the study

Gene Primer sequence (51015840-31015840) Accession number

120573-actin Forward TTT CCA GCC TTC CTT CTT GGG TAT G NM 007393Reverse CAC TGT GTT GGC ATA GAG GTC TTA C

SOD1 Forward TGC GTG CTG AAG GGC GAC NM 011434Reverse GTC CTG ACA ACA CAA CCT GGT TC

SOD2 Forward GGA GCA AGG TCG CTT ACA GA NM 013671Reverse GTG CTC CCA CAC GTC AAT C

GPx1 Forward TGT TTG AGA AGT GCG AAG TG NM 008160Reverse GTG TTG GCA AGG CAT TCC

GPx4 Forward TAA GAA CGG CTG CGT GGT NM 008162Reverse GTA GGG GCA CAC ACT TGT AGG

HIF-1120572 Forward CAC CAG ACA GAG CAG GAA NM 010431Reverse TCA GGA ACA GTA TTT CTT TGA TTC A

TNF-120572 Forward TACCTTGTTGCCTCCTCTT NM 013693Reverse GTCACCAAATCAGCGTTATTAAG

IL-1120573 Forward TCACAAGCAGAGCACAAG NM 008361Reverse GAAACAGTCCAGCCCATAC

Bcl-119909119871

Forward TGACCACCTAGAGCCTTGGA NM 009743Reverse TGTTCCCGTAGAGATCCACAA

Caspase 3 Forward AAA GCC GAA ACT CTT CA TCA T NM 009810Reverse GTC CCA CTG TCT GTC TCA

conducted using the SPSS forWindows software version 100(SPSS Inc Chicago IL USA)

3 Results

31 Effect of 120573-Carotene on Nicotine-Induced DevelopmentalArrest in Mouse Embryos Growth parameters includingyolk sac diameter and circulation size of the allantois crown-rump length head length and number of somites and devel-opmental parameters including morphology of the hearthind- mid- and forebrain otic optic and olfactory systemsbranchial bars maxillary and mandibular processes fore-limb and hindlimb ofmouse embryos exposed to nicotine inthe presence or absence of 120573-carotene were scored accordingto an established scale [12] (Table 2 and Figure 1) All thegrowth anddevelopmental parameters of the nicotine-treatedgroup were significantly lower than the normal controls (119875 lt005) Furthermore the total morphological score (484 plusmn081) of embryos exposed to nicotine alone was significantlylower than that of control embryos (750 plusmn 046 119875 lt 005)However when 120573-carotene (1 times 10minus7 or 5 times 10minus7 120583M) wasadded to the culture medium in the presence of nicotine(1mM) the embryos showed significant improvement in allembryonic growth and developmental parameters (119875 lt 005compared to nicotine alone) with the exception of the caudalneural tube score Furthermore the totalmorphological score(616 plusmn 054 or 624 plusmn 072) for each concentration of 120573-carotene was significantly higher than the score for embryostreated with nicotine alone (119875 lt 005)

32 Effect of 120573-Carotene on Nicotine-Induced Oxidative Dam-age in Mouse Embryos Oxidative stress was analyzed in

whole embryos by measuring the MDA levels (Figure 2)Mouse embryos exposed to 1mM nicotine alone exhib-ited significantly increased lipid peroxidation (3663 plusmn057 nmolmg) compared to the control group (2981 plusmn048 nmolmg) (119875 lt 005) However embryos treated withnicotine plus 120573-carotene (1 times 10minus7 or 5 times 10minus7 120583M) exhibitedsignificantly reduced lipid peroxidation levels (3236plusmn134 or2801 plusmn 096 nmolmg) compared to the nicotine only group(119875 lt 005)

33 120573-Carotene Enhances SOD Activity in Mouse EmbryosTreated with Nicotine Mouse embryos exposed to 1mMnicotine exhibited significantly reduced SOD activity (058 plusmn003Umgprotein) compared to the control group (068plusmn004Umg protein) (119875 lt 005) However when the embryos weretreatedwith 1times 10minus7 or 5times 10minus7 120583M120573-carotene in the presenceof nicotine SOD activity (067 plusmn 002 Umg or 071 plusmn 005Umg) was significantly greater than in the nicotine onlytreatment group (119875 lt 005) (Figure 3)

34 120573-Carotene Upregulates the Expression of Antioxida-tive Enzyme Genes in Mouse Embryos Exposed to NicotineThe cytoplasmic SOD1 mRNA level (Figure 4(a)) in mouseembryos exposed to 1mM nicotine was 066-fold that of thecontrol group (1-fold) However when embryos were treatedwith 1 times 10minus7 or 5 times 10minus7 120583M 120573-carotene and 1mM nicotinethe embryo SOD1 mRNA levels (089-fold or 090-fold thatof controls resp) were significantly greater than with thenicotine only treatment (119875 lt 005)

The mitochondrial SOD2 mRNA level (Figure 4(b)) inmouse embryos exposed to 1mM nicotine was 065-fold thatof the control group (1-fold) However when embryos were


Table 2 Summary of morphological changes in cultured mouse embryos exposed to 1mM nicotine in the presence or absence of 1 times 10minus7 or5 times 10minus7 120583M 120573-carotene (120573-car)

Chemical (dose) Con N N + 120573-car (1 times 10minus7) N + 120573-car (5 times 10minus7)Number of embryos 33 33 34 31Yolk sac diameter (mm) 35 plusmn 030 24 plusmn 029a 30 plusmn 032b 29 plusmn 021b

Yolk sac circulation 43 plusmn 037 36 plusmn 064a 40 plusmn 026 39 plusmn 023b

Allantois 23 plusmn 044 16 plusmn 026a 19 plusmn 019b 20 plusmn 015b

Flexion 49 plusmn 012 32 plusmn 100a 47 plusmn 076b 49 plusmn 019b

Crown-rump length (mm) 30 plusmn 029 21 plusmn 029a 26 plusmn 022b 24 plusmn 025b

Head length (mm) 15 plusmn 021 09 plusmn 018a 13 plusmn 017b 12 plusmn 014b

Heart 48 plusmn 033 34 plusmn 055a 45 plusmn 036b 43 plusmn 035b

Hindbrain 47 plusmn 029 29 plusmn 060a 42 plusmn 029b 42 plusmn 020b

Midbrain 49 plusmn 019 29 plusmn 057a 42 plusmn 023b 42 plusmn 024b

Forebrain 58 plusmn 033 30 plusmn 056a 44 plusmn 033b 43 plusmn 036b

Otic system 49 plusmn 017 30 plusmn 058a 43 plusmn 025b 44 plusmn 033b

Optic system 50 plusmn 009 29 plusmn 057a 43 plusmn 024b 44 plusmn 030b

Branchial bars 37 plusmn 031 22 plusmn 038a 31 plusmn 036b 32 plusmn 038b

Maxillary process 29 plusmn 025 13 plusmn 041a 21 plusmn 025b 22 plusmn 045b

Mandibular process 28 plusmn 034 13 plusmn 039a 20 plusmn 030b 21 plusmn 039b

Olfactory system 28 plusmn 030 04 plusmn 050a 18 plusmn 040b 17 plusmn 056b

Caudal neural tube 50 plusmn 000 46 plusmn 072 50 plusmn 000 50 plusmn 000b

Fore limb 28 plusmn 021 17 plusmn 045a 26 plusmn 039b 27 plusmn 039b

Hind limb 12 plusmn 063 00 plusmn 000a 06 plusmn 045b 07 plusmn 052b

Somites 40 plusmn 000 35 plusmn 051a 40 plusmn 000b 40 plusmn 000b

Total score 750 plusmn 046 484 plusmn 081a 616 plusmn 054b 624 plusmn 072b

Each value represents the mean plusmn SEMaVersus normal control (Con) group at 119875 lt 005bVersus nicotine alone (N) group at 119875 lt 005

treated with 1 times 10minus7 or 5 times 10minus7 120583M 120573-carotene and 1mMnicotine the SOD2 mRNA levels (088-fold or 107-fold thatof the control group resp) were significantly greater thanwith the nicotine treatment alone (119875 lt 005)

The cytoplasmic GPx1 mRNA level (Figure 4(c)) inmouse embryos exposed to 1mM nicotine was 065-foldthat of the control group (1-fold) (119875 lt 005) Howeverwhen embryos were treated with 5 times 10minus7 120583M 120573-carotene and1mM nicotine the GPx1 mRNA level (099-fold that of thecontrol group)was significantly greater thanwith the nicotinetreatment alone (119875 lt 005)

The phospholipid hydroperoxide GPx4 mRNA level(Figure 4(d)) in mouse embryos exposed to 1mM nicotinedecreased significantly to 072-fold that of the control group(1-fold) (119875 lt 005) However when embryos were treatedwith 1 times 10minus7 or 5 times 10minus7 120583M 120573-carotene and 1mM nicotinethe GPx4 mRNA levels (093-fold or 091-fold that of thecontrol group resp) were significantly greater than with thenicotine treatment alone (119875 lt 005)

35 120573-Carotene Upregulates HIF-1120572 Gene Expression inNicotine-Treated Embryos TheHIF-1120572mRNA level inmouseembryos exposed to 1mM nicotine decreased significantlyto 066-fold that of the control group (1-fold) (119875 lt 005)However when embryos were treated with 1 times 10minus7 or 5 times10minus7 120583M 120573-carotene and 1mM nicotine HIF-1120572mRNA levels

(082-fold or 114-fold that of the control group resp) weresignificantly greater than with the nicotine treatment alone(119875 lt 005) (Figure 5)

36 120573-Carotene Downregulates Proinflammatory CytokinesGene Expression in Embryos Exposed to Nicotine The TNF-120572 mRNA level (Figure 6(a)) in mouse embryos exposed to1mM nicotine was 147-fold that of the control group (1-fold)(119875 lt 005) However when embryos were treated with 1 times10minus7 or 5 times 10minus7 120583M 120573-carotene and 1mM nicotine TNF-120572mRNA levels (068-fold or 059-fold that of the control groupresp) were significantly lower than with the nicotine onlytreatment (119875 lt 005)

The IL-1120573 mRNA level (Figure 6(b)) in mouse embryosexposed to 1mM nicotine was 131-fold that of the controlgroup (1-fold) (119875 lt 005) However when embryos weretreated with 1mM nicotine and 1 times 10minus7 or 5 times 10minus7 120583M 120573-carotene the IL-1120573mRNA level (032-fold or 027-fold that ofthe control group resp) was significantly lower than with thenicotine only treatment (119875 lt 005)

37 120573-Carotene Decreases Nicotine-Induced Apoptosis

371 Bcl-119909119871Gene Expression Pattern TheBcl-119909

119871mRNA level

in mouse embryos exposed to 1mM nicotine was 072-foldthat of the control group value (1-fold) (119875 lt 005) However


16

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HIF

-1120572

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lowast

Con N1 times 10

minus7

5 times 10minus7

N + 120573-car N + 120573-car

Figure 5 Hypoxia inducible factor-1 120572 expression levels in E85mouse embryos exposed to nicotine and 120573-carotene for 2 days invitro HIF-1120572 mRNA in embryos exposed to 1mM nicotine in theabsence or presence of 1 times 10minus7 or 5 times 10minus7 120583M 120573-carotene (120573-car)was measured by quantitative RT-PCR Results are mean plusmn SEM(119899 = 8) 120573-actin was used as an internal standard to normalizetarget transcript expression Significant differences (lowastcontrol versusnicotine alone nicotine versus 120573-car + nicotine) were evaluated byone-way ANOVA at 119875 lt 005

when embryos were treated with 1mM nicotine and 1 times 10minus7or 5 times 10minus7 120583M 120573-carotene the Bcl-119909

119871mRNA level (109-fold

or 094-fold that of the control group resp) was significantlygreater than with the nicotine only treatment (119875 lt 005Figure 7(a))

372 Caspase 3 Gene Expression Pattern The caspase 3mRNA level in mouse embryos exposed to 1mM nicotinewas 120-fold that of the control group (1-fold) (119875 lt 005)However when embryos were treated with nicotine in thepresence of 1 times 10minus7 or 5 times 10minus7 120583M 120573-carotene caspase 3mRNA levels (096-fold or 094-fold that of the control groupresp) were significantly lower than with the nicotine onlytreatment (119875 lt 005 Figure 7(b))

373 120573-Carotene Reduces Nicotine-Induced Apoptosis inMouse Embryos To determine whether 120573-carotene antag-onizes nicotine-induced apoptosis the Nile blue stainingtechnique was used Normal cells were stained pale blue incontrol embryos (Figure 8(a)) By contrast apoptotic cellsappeared dark blue in color especially in the heart opticand olfactory pits brain otic stalk cranial nerve nucleiand tail bud in the nicotine-treated embryos (Figure 8(b))Cotreatment with 120573-carotene resulted in a marked reductionin the levels of apoptosis induced by nicotine (Figures 8(c)and 8(d))

4 Discussion

The popularity of smoking during pregnancy is between 13and 25 in high-income countries and is increasing rapidlyin low- andmiddle-income countries [17] Although effective

smoking cessation strategies during pregnancy are importantfor maternal and fetal health previous studies have suggestedthat an alternative therapy may be to use natural antioxidanttreatments that can protect against nicotine-induced embryotoxicity [5 18] In the present study we expanded uponthis concept not only to demonstrate the beneficial effectsof 120573-carotene against nicotine-induced damage but also todistinguish themechanisms of nicotine damage further usingan embryo culture system

In previous studies maternal smoking affected the devel-opment of many fetal organs and tissues including thenervous cardiovascular and skeletal systems [19ndash24] Nico-tine increases the fetal heart rate reduces fetal breathingmovements and is associated with deficiencies in braincell number [25] In the current study embryonic growthas measured by yolk sac diameter and circulation size ofthe allantois crown-rump length head length and numberof somites as well as development of the heart centralnervous system sensory organs branchial bars maxillaryand mandibular processes and limbs were inhibited andmorphological features of the embryos were significantlyaltered by nicotine treatment However when nicotine-treated embryos were concurrently exposed to 120573-carotenemost of the morphological anomalies including abnormalheart development deformed posterior trunk regressedlimbs and brain malformations were significantly improvedcompared to embryos treatedwith nicotine aloneThese find-ings indicate that 120573-carotene can effectively protect embryosfrom nicotine-induced defects in organogenesis

Cell membranes contain substantial levels of polyun-saturated fatty acids that are highly vulnerable to perox-idative breakdown [26] Oxidative stress characterized byincreased ROS and impaired antioxidant defenses acts as animportant mediator of defective embryo development andgrowth retardation [9] However both enzymatic (SODGPxand catalase) and nonenzymatic (GSHGSSG peroxiredoxinthioredoxin vitamin C and vitamin E) antioxidant systemsexist to combat excessive ROS generation [27] Nicotineinduces oxidative stress both in vivo and in vitro [9] Recentlywe found that resveratrol a natural polyphenol compoundprevents nicotine-induced teratogenesis in cultured mouseembryos through its potent antioxidative activity [5] Inthe current study nicotine increased the MDA level anddecreased the SOD activity in embryos However when theembryos were concurrently treated with nicotine and 120573-carotene these embryonic oxidative stress responses andimpaired antioxidant enzyme levels recovered to the controllevels The antioxidant 120573-carotene provides essential pro-tection against oxygen radical damage since it terminatesperoxidative chain reactions of unsaturated lipids in thebrain and other tissues [28] and effectively scavenges ROS incells exposed to oxidative stress [29] Therefore exogenous120573-carotene may improve the SOD status of embryos andneutralize the excess ROS generated by nicotine

SODs inactivate superoxide radicals and GPxs reducehydrogen peroxide to H

2O at the expense of glutathione

oxidation [30 31] During mouse embryogenesis antioxi-dant enzymes such as GPx1 GPx4 SOD1 and SOD2 arehighly expressed in metabolically active tissues [32ndash35] In


2

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Con N1 times 10

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TNF-120572

120573-a

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A ex

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(x-fo

ld o

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N + 120573-car N + 120573-car

(a)

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Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)

Figure 6 Gene expression levels of proinflammatory cytokines in E85mouse embryos exposed to nicotine and 120573-carotene for 2 days in vitroLevels of TNF-120572 (a) and IL-1120573 (b)mRNA in embryos exposed to 1mMnicotine in the absence or presence of 1times 10minus7 or 5times 10minus7 120583M120573-carotene(120573-car) were measured by quantitative RT-PCR Results are mean plusmn SEM (119899 = 8) 120573-actin was used as an internal standard to normalize targettranscript expression Significant differences (lowastcontrol versus nicotine alone nicotine versus 120573-car + nicotine) were evaluated by one-wayANOVA at 119875 lt 005

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)

Figure 7 Gene expression levels of apoptosis related factors in E85 mouse embryos exposed to nicotine and 120573-carotene for 2 days in vitroLevels of Bcl-119909

119871(a) and caspase 3 (b) mRNA in embryos exposed to 1mM nicotine in the absence or presence of 1 times 10minus7 or 5 times 10minus7 120583M

120573-carotene (120573-car) were measured by quantitative RT-PCR Results are mean plusmn SEM (119899 = 8) 120573-actin was used as an internal standardto normalize target transcript expression Significant differences (lowastcontrol versus nicotine alone nicotine versus 120573-car + nicotine) wereevaluated by one-way ANOVA at 119875 lt 005

the current study nicotine significantly decreased SOD1SOD2 GPx1 and GPx4 gene expression in cultured embryosbut the expression levels were restored by cotreatment with120573-carotene As early organogenesis occurs in a relativelyhypoxic environment embryos are sensitive to oxidativestress [9] Null mutations in HIF-1120572 cause cardiac vascularand neuralmalformations and result in fetal lethality on E105

[36] Hypoxia induces oxidative stress and abnormal organo-genesis in mouse embryos by downregulating HIF-1120572 andintracellular SOD gene expression [37] In the current studythe levels of HIF-1120572 mRNA in cultured embryos decreasedsignificantly following nicotine treatment but were restoredby co-treatment with 120573-carotene These results indicate that120573-carotene can protect embryos against nicotine-induced


(a) (b)

(c) (d)

Figure 8 Representative images of apoptotic embryos exposed to nicotine and 120573-carotene by Nile blue staining Nile blue staining wasperformed to observe apoptotic nuclei and dead cells which stained dark blue Normal control embryos (a) Embryos treated with 1mMnicotine exhibit increased levels of apoptosis (b) Embryos treated with 1mM nicotine plus 120573-carotene [1 times 10minus7 120583M (c) and 5 times 10minus7 120583M (d)]appear similar to the control group

oxidative damage through its antioxidative and antihypoxicactivities

Cigarette smoke alters a wide range of immunologicalfunctions and adversely influences humoral and cellularimmune responses in both humans and animals [38] ROSmediate these immune reactions through various proinflam-matory cytokines and can influence the function of oocytesperm and embryo [39] In the current study nicotine sig-nificantly increased gene expression of the proinflammatorycytokines TNF-120572 and IL-1120573 in cultured embryos but theselevels were significantly reduced to levels lower than controllevels by co-treatmentwith120573-caroteneThese results indicatethat 120573-carotene may protect the embryos by reducing theimmune response stimulated by nicotine treatment

Previous studies have confirmed that apoptosis plays animportant role in normal embryonic development Develop-mental apoptosis is a well-balanced process that is crucialfor formation of embryonic structures However interfer-ence with this balance induces morphological abnormalities

[40 41] In the current study Bcl-119909119871 one of several antiapop-

totic proteins that aremembers of the Bcl-2 family of proteinsdecreased significantly and caspase 3 a marker for cellsundergoing apoptosis [42] increased significantly followingnicotine treatment of cultured embryos Increased apoptosiswas also detected in embryos exposed to nicotine by Nileblue staining However these apoptotic changes inducedby nicotine were blocked by co-treatment with 120573-caroteneThese results indicate that 120573-carotene protects embryos fromnicotine-induced abnormal development via its antiapoptoticactivity

5 Conclusions

Nicotine induces excessive ROS and leads to fetal anomaliesand lethality The findings of the current study indicate thatthe antioxidative anti-hypoxic antiapoptotic and antiproin-flammatory functions of 120573-carotene may prevent nicotine-induced impairments of embryos and facilitate normal


embryonic development Although these data support thehypothesis that 120573-carotene obtained in the diet effectivelycounteracts the deleterious effects of nicotine during fetalorganogenesis an in vivo study usingmousewould be neededto compare the functions of 120573-carotene on nicotine-inducedembryotoxicities in future

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgment

This work was supported by Priority Research Centers Pro-gram through the National Research Foundation of Korea(NRF) funded by the Ministry of Education Science andTechnology (2011-0031403)

References

[1] D S Lambers and K E Clark ldquoThe maternal and fetalphysiologic effects of nicotinerdquo Seminars in Perinatology vol 20no 2 pp 115ndash126 1996

[2] S A A Comhair and S C Erzurum ldquoAntioxidant responses tooxidant-mediated lung diseasesrdquo American Journal of Physiol-ogy vol 283 no 2 pp L246ndashL255 2002

[3] A Ornoy ldquoEmbryonic oxidative stress as a mechanism ofteratogenesis with special emphasis on diabetic embryopathyrdquoReproductive Toxicology vol 24 no 1 pp 31ndash41 2007

[4] M E Cogswell PWeisberg and C Spong ldquoCigarette smokingalcohol use and adverse pregnancy outcomes implications formicronutrient supplementationrdquo Journal of Nutrition vol 133pp 1722Sndash1731S 2003

[5] C Lin J M Yon A Y Jung et al ldquoResveratrol preventsnicotine-induced teratogenesis in cultured mouse embryosrdquoReproductive Toxicology vol 34 no 3 pp 340ndash360 2012

[6] J Limon-Pacheco and M E Gonsebatt ldquoThe role ofantioxidants and antioxidant-related enzymes in protectiveresponses to environmentally induced oxidative stressrdquoMutation Research vol 674 no 1-2 pp 137ndash147 2009

[7] E Spiegler Y K Kim L Wassef V Shete and L QuadroldquoMaternal-fetal transfer and metabolism of vitamin A and itsprecursor beta-carotene in the developing tissuesrdquo BiochimicaEt Biophysica Acta no 1 pp 88ndash198 1821

[8] S K Bai S J Lee H J Na et al ldquo120573-carotene inhibitsinflammatory gene expression in lipopolysaccharide-stimulated macrophages by suppressing redox-based NF-120581Bactivationrdquo Experimental and Molecular Medicine vol 37 no4 pp 323ndash334 2005

[9] P A Dennery ldquoEffects of oxidative stress on embryonic devel-opmentrdquo Birth Defects Research C vol 81 no 3 pp 155ndash1622007

[10] D A New ldquoWhole-embryo culture and the study of mam-malian embryos during organogenesisrdquo Biological reviews of theCambridge Philosophical Society vol 53 no 1 pp 81ndash122 1978

[11] M A Joschko I E Dreosti and R S Tulsi ldquoThe teratogeniceffects of nicotine in vitro in rats a light and electron micro-scope studyrdquo Neurotoxicology and Teratology vol 13 no 3 pp307ndash316 1991

[12] G VanMaele-Fabry F Delhaise and J J Picard ldquoMorphogene-sis and quantification of the development of post-implantationmouse embryosrdquo Toxicology in Vitro vol 4 no 2 pp 149ndash1561990

[13] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[14] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[15] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[16] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[17] S Cnattingius ldquoThe epidemiology of smoking during preg-nancy smoking prevalence maternal characteristics and preg-nancy outcomesrdquo Nicotine and Tobacco Research vol 6 no 2pp S125ndashS140 2004

[18] T Coleman S Cooper J G Thornton et al ldquoA randomizedtrial of nicotine-replacement therapy patches in pregnancyrdquoTheNew England Journal of Medicine vol 366 no 9 pp 808ndash8182012

[19] P Czekaj A Pałasz T Lebda-Wyborny et al ldquoMorphologicalchanges in lungs placenta liver and kidneys of pregnant ratsexposed to cigarette smokerdquo International Archives of Occupa-tional and Environmental Health vol 75 no 1 pp S27ndashS352002

[20] K Kallen ldquoMultiple malformations and maternal smokingrdquoPaediatric and Perinatal Epidemiology vol 14 no 3 pp 227ndash233 2000

[21] M J Seller and K S Bnait ldquoEffects of tobacco smoke inhalationon the developing mouse embryo and fetusrdquo ReproductiveToxicology vol 9 no 5 pp 449ndash459 1995

[22] R R Resende and A Adhikari ldquoCholinergic receptor pathwaysinvolved in apoptosis cell proliferation and neuronal differen-tiationrdquo Cell Communication Signaling vol 7 no 20 pp 1ndash202009

[23] T A Slotkin ldquoFetal nicotine or cocaine exposure which one isworserdquo Journal of Pharmacology and Experimental Therapeu-tics vol 285 no 3 pp 931ndash945 1998

[24] M M Werler ldquoTeratogen update smoking and reproductiveoutcomesrdquo Teratology vol 55 no 6 pp 382ndash388 1997

[25] T A Slotkin ldquoCryptic brain cell injury caused by fetal nicotineexposure is associated with persistent elevations of c-fos pro-tooncogene expressionrdquo Brain Research vol 750 no 1-2 pp180ndash188 1997

[26] S A Amini R H Dunstan P R Dunkley and R N MurdochldquoOxidative stress and the fetotoxicity of alcohol consumptionduring pregnancyrdquo Free Radical Biology and Medicine vol 21no 3 pp 357ndash365 1996

[27] D Shao S Oka C D Brady J Haendeler P Eaton and JSadoshima ldquoRedox modification of cell signaling in the cardio-vascular systemrdquo Journal of Molecular and Cellular Cardiologyvol 52 no 3 pp 550ndash558 2011

[28] J J Mitchell M Paiva and M B Heaton ldquoThe antioxidantsvitamin e and 120573-carotene protect against ethanol- inducedneurotoxicity in embryonic rat hippocampal culturesrdquo Alcoholvol 17 no 2 pp 163ndash168 1999


[29] P Palozza ldquoCan 120573-carotene regulate cell growth by a redoxmechanism An answer from cultured cellsrdquo Biochimica etBiophysica Acta vol 1740 no 2 pp 215ndash221 2005

[30] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selenoenzymerdquoFEBSLetters vol 32 no 1 pp 132ndash134 1973

[31] J M McCord and I Fridovich ldquoSuperoxide dismutase Anenzymic function for erythrocuprein (hemocuprein)rdquoThe Jour-nal of Biological Chemistry vol 244 no 22 pp 6049ndash6055 1969

[32] J M Yon I J Baek S R Lee et al ldquoThe spatio-temporalexpression pattern of cytoplasmic CuZn superoxide dismutase(SOD1)mRNAduringmouse embryogenesisrdquo Journal ofMolec-ular Histology vol 39 no 1 pp 95ndash103 2008

[33] J M Yon I J Baek B J Lee Y W Yun and S Y NamldquoDynamic expression of manganese superoxide dismutase dur-ing mouse embryonic organogenesisrdquo International Journal ofDevelopmental Biology vol 55 no 3 pp 327ndash334 2011

[34] I J Baek J M Yon J L Beom et al ldquoExpression pattern ofcytosolic glutathione peroxidase (cGPx) mRNA during mouseembryogenesisrdquo Anatomy and Embryology vol 209 no 4 pp315ndash321 2005

[35] I J Baek D S Seo J M Yon et al ldquoTissue expression andcellular localization of phospholipid hydroperoxide glutathioneperoxidase (PHGPx)mRNA inmalemicerdquo Journal ofMolecularHistology vol 38 no 3 pp 237ndash244 2007

[36] D Yoon Y D Pastore V Divoky et al ldquoHypoxia-induciblefactor-1 deficiency results in dysregulated erythropoiesis signal-ing and iron homeostasis in mouse developmentrdquo The Journalof Biological Chemistry vol 281 no 35 pp 25703ndash25711 2006

[37] J M Yon I J Baek B J Lee YW Yun and S Y Nam ldquoEmodinand [6]-gingerol lessen hypoxia-induced embryotoxicities incultured mouse whole embryos via upregulation of hypoxia-inducible factor 1120572 and intracellular superoxide dismutasesrdquoReproductive Toxicology vol 31 no 4 pp 513ndash518 2011

[38] J D Johnson D P Houchens W M Kluwe D K Craig andG L Fisher ldquoEffects of mainstream and environmental tobaccosmoke on the immune system in animals and humans a reviewrdquoCritical Reviews in Toxicology vol 20 no 5 pp 369ndash395 1990

[39] E Jauniaux J Hempstock N Greenwold and G J BurtonldquoTrophoblastic oxidative stress in relation to temporal andregional differences in maternal placental blood flow in normaland abnormal early pregnanciesrdquo American Journal of Pathol-ogy vol 162 no 1 pp 115ndash125 2003

[40] A J Copp ldquoNeurulation in the cranial regionmdashnormal andabnormalrdquo Journal of Anatomy vol 207 no 5 pp 623ndash6352005

[41] F CecconiM Piacentini andGM Fimia ldquoThe involvement ofcell death and survival in neural tube defects a distinct role forapoptosis and autophagyrdquo Cell Death and Differentiation vol15 no 7 pp 1170ndash1177 2008

[42] A Semlali J Chakir J P Goulet W Chmielewski and MRouabhia ldquoWhole cigarette smoke promotes human gingivalepithelial cell apoptosis and inhibits cell repair processesrdquoJournal of Periodontal Research vol 46 no 5 pp 533ndash541 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(a) (b1) (b2) (b3)

(c1) (c2) (d1) (d2)

Figure 1 Representative images of mouse embryos exposed to nicotine and 120573-carotene Normal control group (a) Embryos treated with1mM nicotine alone show typical abnormalities such as exposed brain reduced forebrain abnormal heart deformed posterior trunk andregressed forelimbs (b1minus3) Embryos treated with nicotine plus 120573-carotene [1 times 10minus7 120583M (c1 and c2) and 5 times 10minus7 120583M (d1 and d2)] appearmorphologically similar to the control group

2 Materials and Methods

21 Experimental Animals Male and female ICR mice (8ndash10 weeks old) were purchased from a commercial breeder(BioGenomics Co Seoul Republic of Korea) The animalswere housed in a climate controlled facility with an ambienttemperature of 21 plusmn 2∘C relative humidity of 55 plusmn 10air ventilation rate of 10 cycles per hour and a 12 12 hlight dark cycle The animals were fed standard mouse chow(Samyang Ltd Incheon Republic of Korea) and tap waterad libitum throughout the experimental period One maleand three female mice were housed in a cage for matingPregnancies were confirmed the following morning (0800)by the presence of vaginal plugs or spermatozoa detected ina vaginal smear after mating the previous evening (2000)this was considered embryonic day (E) 05 Pregnant micewere sacrificed and embryos were obtained at E85 Allexperiments were approved by the Chungbuk National Uni-versity Animal Care Committee and carried out accordingto the Guide for Care and Use of Animals (ChungbukNational University Animal Care Committee according toNIH number 86ndash23)

22 Rat Serum Preparation Serum of Sprague-Dawley malerats (10ndash12 weeks old) was prepared for embryo cultures asfollows After collection blood samples were immediatelycentrifuged for 10min at 3000 rpm and 4∘C to clear the

plasma fraction of cellsThe supernatant was then transferredto new tubes and centrifuged for 10min at 3000 rpm and 4∘Cto remove remaining blood cellsThe clear serum supernatantwas decanted and pooled and the pooled serum was heat-inactivated for 30min at 56∘C in a water bath It was theneither used immediately or stored at minus70∘C Serum wasincubated at 37∘C and filtered through a 02 120583m filter priorto use in the whole embryo culture

23 Whole Embryo Culture and Nicotine and 120573-CaroteneTreatments The whole embryo culture system was based ona previously described procedure [10] Animals were sacri-ficed between 0900 and 1000 h via cervical dislocation whenembryos reached E85 Only embryos with 4ndash8 somites wereutilized After removing the decidua and Reichertrsquos mem-branes embryos with intact visceral yolk sacs and ectopla-cental cones were placed randomly into sealed culture bottles(three embryosbottle) containing 3mL of culture mediumand different concentrations (1 times 10minus7 or 5 times 10minus7 120583M)of 120573-carotene (Sigma St Louis MO USA) dissolved indimethyl sulfoxide (DMSO Sigma) andor 1mM nicotine(1638 120583gmL serum Sigma) The nicotine concentrationused here was determined by previous studies [5 11]The finalconcentration of DMSO in the medium was less than 01Embryos were randomized into four treatment groups (1)control (2) nicotine (3) nicotine plus 1times 10minus7 120583M120573-caroteneand (4) nicotine plus 5 times 10minus7 120583M 120573-carotene The embryos


DA

pro

tein

(nm

olm

g)

50

40

30

20

10

0Con N

lowast

1 times 10minus7

5 times 10minus7

N + 120573-car N + 120573-car


SOD

activ

ityp

rote

in (U

mg)

1

08

06

04

02

0Con N

lowast

1 times 10minus7

5 times 10minus7

N + 120573-car N + 120573-car



2 5 CO

2 and



2 5 CO

2 and 75 N

2) and 24 h (40 O

2 5 CO

2








12

1

08

06

04

02

0Con N

lowast

1 times 10minus7

5 times 10minus7

SOD

1120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

SOD

2120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(b)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

GPx

1120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(c)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

GPx

4120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(d)





119871 caspase














Bcl-119909119871




3 Results








































119871mRNA level



16

14

12

1

08

06

04

02

0

HIF

-1120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

lowast

Con N1 times 10

minus7

5 times 10minus7

N + 120573-car N + 120573-car







4 Discussion









2

15

1

05

0

lowast

Con N1 times 10

minus7

5 times 10minus7

TNF-120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)


16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)







(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DA

pro

tein

(nm

olm

g)

50

40

30

20

10

0Con N

lowast

1 times 10minus7

5 times 10minus7

N + 120573-car N + 120573-car


SOD

activ

ityp

rote

in (U

mg)

1

08

06

04

02

0Con N

lowast

1 times 10minus7

5 times 10minus7

N + 120573-car N + 120573-car



2 5 CO

2 and



2 5 CO

2 and 75 N

2) and 24 h (40 O

2 5 CO

2








12

1

08

06

04

02

0Con N

lowast

1 times 10minus7

5 times 10minus7

SOD

1120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

SOD

2120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(b)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

GPx

1120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(c)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

GPx

4120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(d)





119871 caspase














Bcl-119909119871




3 Results








































119871mRNA level



16

14

12

1

08

06

04

02

0

HIF

-1120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

lowast

Con N1 times 10

minus7

5 times 10minus7

N + 120573-car N + 120573-car







4 Discussion









2

15

1

05

0

lowast

Con N1 times 10

minus7

5 times 10minus7

TNF-120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)


16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)







(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















12

1

08

06

04

02

0Con N

lowast

1 times 10minus7

5 times 10minus7

SOD

1120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

SOD

2120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(b)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

GPx

1120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(c)

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

GPx

4120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(d)





119871 caspase














Bcl-119909119871




3 Results








































119871mRNA level



16

14

12

1

08

06

04

02

0

HIF

-1120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

lowast

Con N1 times 10

minus7

5 times 10minus7

N + 120573-car N + 120573-car







4 Discussion









2

15

1

05

0

lowast

Con N1 times 10

minus7

5 times 10minus7

TNF-120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)


16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)







(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Bcl-119909119871




3 Results








































119871mRNA level



16

14

12

1

08

06

04

02

0

HIF

-1120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

lowast

Con N1 times 10

minus7

5 times 10minus7

N + 120573-car N + 120573-car







4 Discussion









2

15

1

05

0

lowast

Con N1 times 10

minus7

5 times 10minus7

TNF-120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)


16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)







(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































119871mRNA level



16

14

12

1

08

06

04

02

0

HIF

-1120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

lowast

Con N1 times 10

minus7

5 times 10minus7

N + 120573-car N + 120573-car







4 Discussion









2

15

1

05

0

lowast

Con N1 times 10

minus7

5 times 10minus7

TNF-120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)


16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)







(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















16

14

12

1

08

06

04

02

0

HIF

-1120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

lowast

Con N1 times 10

minus7

5 times 10minus7

N + 120573-car N + 120573-car







4 Discussion









2

15

1

05

0

lowast

Con N1 times 10

minus7

5 times 10minus7

TNF-120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)


16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)







(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2

15

1

05

0

lowast

Con N1 times 10

minus7

5 times 10minus7

TNF-120572

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

IL-1120573

(b)


16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Bcl-x

L120573

-act

in m

RNA

expr

essio

n(x

-fold

of v

ehic

le)

N + 120573-car N + 120573-car

(a)

16

14

12

1

08

06

04

02

0

lowast

Con N1 times 10

minus7

5 times 10minus7

Casp

ase 3

120573-a

ctin

mRN

A ex

pres

sion

(x-fo

ld o

f veh

icle

)

N + 120573-car N + 120573-car

(b)







(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)







5 Conclusions






Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















carotene in cultured mouse embryos exposed to nicotine

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