kerstik. linask, ph.d. university of south florida ... · university of south florida children’s...

Alcohol -Induced Birth Defects and Folate Supplementation in

Mouse Model

KERSTI K. LINASK, Ph.D.

University of South Florida

Children’s Research Institute

Fetal Alcohol Spectrum Disorders (FASD) is a serious health and social

problem which affects individuals, families, and societies worldwide.

FASD is characterized by language and motor delays, cognitive delays

including intellectual disabilities, facial abnormalities, heart defects, and

vision and hearing problems

FASD afflicts millions of people worldwide – about one in every

100 births (May et al., 2009)

FASD Occurs World-wide …

� FASD can include Fetal Alcohol Syndrome

(FAS), Fetal Alcohol Effects, Alcohol-Related

Neurodevelopmental Disorder (ARND) and

Central Nervous System (CNS) problems.

� Fewer than 10% of FAS individuals are able

to take care of themselves and live on their

own. The annual total cost to society for the

care of children and adults with Fetal Alcohol

Syndrome is huge: in the US it is between 1.4

billion and 9.7 billion.

Statistics on Drinking…

Are alcohol dependent (alcoholics)

Are dependent in that they put themselves or

others at risk: binge drinking

women 4 drinks/occasion

men 5 drinks/occasion

On average 8 drinks are reported/occasion and

4 times per month

Those who drink moderately or do not drink

WHO: number 37 with 8.77 liters of pure alcohol consumption per capita every year

Statistics on Drinking - Eestis: Based on World Health Organization Global

Information on Alcohol and Health. Shown is alcohol per capita consumption based in

liters of pure alcohol drank per year between 1990-2010

The top 15 countries of alcohol consumption:

1. Belarus 14.37

2. Andorra 13.31

3. Lithuania 12.9

4. Czech Republic 12.69

5. Grenada 12.4

6. Austria 12.1

7. Ireland 11.92

8. France 11.7

9. Saint Lucia 11.6

10. Estonia 11.36

11. Luxembourg 11.36

12. Germany 11.21

13. Russian Federation 11.12

14. Slovakia 10.96

15. Portugal 10.84

Beer 4.68 liters (41.2%)

Wine 1.26 l (11.1%)

Spirits 4.18 l (36.8%

Other 1.24 l (10.9 %)

What is a Standard Drink?

From: Langman’s Essential Medical Embryology by T.W.Sadler, Lippincott,

Williams, and Wilkins, 2005

3 Weeks

Moore and Persaud. 2003

16 days Human Embryo

(from Visual embryo

website)

21 days

5 to 6 weeks:

pregnancy is usually

confirmed.

During Pregnancy ….

ChickFish Human

Vertebrate Embryos at Early Stages are Similar

Critical Periods in Human Prenatal Period

Gastrulation: IngressionHuman 17 days Mouse (Mu) 6.75 – 7.5 days

Hu 18 days Mu 7.50 – 8.0 days

Hu 20-21 days Mu 8-8.5 days

EP

MES

ECT

ME

S

EN

D

Neural Development

During gastrulation,

signaling, including

Wnt/beta-catenin, induces

the neural compartment in

the ectoderm layer

overlying the mesoderm

layer and this induction

occurs in an

anterior/posterior

progression initiating cell

specification and cell

differentiation down the

neural tube.Stiles and Jernigan, 2010

Congenital Heart Disease

� Congenital heart malformations, the most common of human birth defects, occur in ~ 1% of the population worldwide.

� Valve and associated structures continue to be the most common subtype of CHDs accounting for 25-30% of all cardiovascular malformations.

� Cardiac anomalies appear to arise from abnormal

� cell lineage decisions of early progenitor cells, disrupting

� normal patterning of morphogenetic pathways.

A recent recommendation suggested that children born with

cardiac defects also be tested for neural-related problems and

developmental disabilities (Marino et al., 2012).

This underscores that common developmental processes are

involved in both cardiac and neural development occurring at

the same time of development during gastrulation.

Alcohol (ethanol) consumption during pregnancy is

linked to congenital heart defects and to neural-related

problems that are associated with Fetal Alcohol

Syndrome (FAS).

Gastrulation: E13 to E20

Cardiac progenitor cells Neural progenitor cells

Multiple axis, no cardiac tissueHeart anterior to head

Cardiabifida

Looping defects, wide hearts

Adverse effects are stage- and time- dependent

11-11.5 hrs / Early St.3

11.5 – 12 hrs / Mid St. 3

12 – 12.5 hrs / Late St. 3

to St. 4

Total embryos analyzed: 451

CONTROL

11 hrs after fertilization

(~ HH early St-3)

11.5 hrs after fertilization

(~ HH mid St-3)

By the end of the embryonic period,

week 8, the rudimentary structures

of the brain and central nervous

system are established and the

major compartments of the central

and peripheral nervous systems are

defined.

- Primitive patterning of

sensorimotor regions of the

neural compartment within the

neocortex is established (Bishop et

al., 2002);

- major compartments within

diencephalic and midbrain have

differentiated (Nakamura et al., 2005)

and &

- segmental organization of

hindbrain and spinal column are

specified (Gavalos et al., 2003).

Neural Development

Acute Ethanol Exposure on Day 9: Effects on Mouse Brain Development

High resolution

magnetic resonance

imaging study:

From: Parnell et al., 2013 Neurotox. Teratol. 39:77-83

Exposure: On day 9 (Hu D20)

of mouse gestation, pregnant

mouse given two i.p. injections

4 hrs apart of 2.9 g/kg/dose

ethanol (25% vol/vol) or of

Ringer’s solution.

Color-coding for significance:• yellow=slightly different p<0.05

• orange = more changes, p<0.01

• red = changes highly significant

p<0.005

Epidemiological studies suggest

that 54% of live-born children with

FAS have some kind of cardiac

anomaly. The malformations

include valvuloseptal defects,

stenosis of the pulmonary artery,

Tetralogy of Fallot, and d-

transposition of the great arteries.

Gastrulation: IngressionHuman 17 days Mouse (Mu) 6.75 – 7.5 days

Hu 18 days Mu 7.50 – 8.0 days

Hu 20-21 days Mu 8-8.5 days

EP

MES

ECT

MES

END

Neural Development

During gastrulation, signaling,

including Wnt/beta-catenin,

induces the neural

compartment in the ectoderm

layer overlying the mesoderm

layer and this induction occurs

in an anterior/posterior

progression initiating cell

specification and cell

differentiation down the neural

tube.

Stiles and Jernigan, 2010

Congenital Heart Disease

• Congenital heart malformations, the most common of human birth defects, occur in ~ 1% of the population worldwide.

• Valve and associated structures continue to be the most common subtype of CHDs accounting for 25-30% of all cardiovascular malformations.

• Cardiac anomalies appear to arise from abnormal

• cell lineage decisions of early progenitor cells, disrupting

• normal patterning of morphogenetic pathways.

A recent recommendation suggested that children born with cardiac

defects also be tested for neural-related problems and developmental

disabilities (Marino et al., 2012).

This underscores that common developmental processes are involved in

both cardiac and neural development occurring at the same time of

development during gastrulation.

Alcohol (ethanol) consumption during pregnancy is linked to

congenital heart defects and to neural-related problems that

are associated with Fetal Alcohol Syndrome (FAS).

Gastrulation: E13 to E20

Cardiac progenitor cells Neural progenitor cells

Multiple axis, no cardiac tissueHeart anterior to head

Cardiabifida

Looping defects, wide hearts

Adverse effects are stage- and time- dependent

11-11.5 hrs / Early St.3

11.5 – 12 hrs / Mid St. 3

12 – 12.5 hrs / Late St. 3

to St. 4

Total embryos analyzed: 451

CONTROL

11 hrs after fertilization

(~ HH early St-3)

11.5 hrs after fertilization

(~ HH mid St-3)

By the end of the embryonic period,

week 8, the rudimentary structures of

the brain and central nervous system

are established and the major

compartments of the central and

peripheral nervous systems are

defined.

- Primitive patterning of sensorimotor

regions of the neural compartment

within the neocortex is established

(Bishop et al., 2002);

- major compartments within

diencephalic and midbrain have

differentiated (Nakamura et al., 2005)

and …

- segmental organization of hindbrain

and spinal column are specified (Gavalos et al., 2003).

Neural Development

Acute Ethanol Exposure on Day 9: Effects on Mouse Brain Development

High resolution magnetic

resonance imaging study:

From: Parnell et al., 2013 Neurotox. Teratol. 39:77-83

Exposure: On day 9 (Hu D20) of

mouse gestation, pregnant mouse

given two i.p. injections 4 hrs

apart of 2.9 g/kg/dose ethanol

(25% vol/vol) or of Ringer’s

solution.

Color-coding for significance:• yellow=slightly different p<0.05

• orange = more changes, p<0.01

• red = changes highly significant

p<0.005

Epidemiological studies suggest that

54% of live-born children with FAS have

some kind of cardiac anomaly. The

malformations include valvuloseptal

defects, stenosis of the pulmonary

artery, Tetralogy of Fallot, and d-

transposition of the great arteries.



(from Visual embryo

website)3 Weeks

49% of pregnancies are unintended! Walker, D., et al., Fetal Alcohol Spectrum Disorders Prevention: An exploratory

study of women's use of, attitudes toward, and knowledge about alcohol. J Amer Acad Nurse Practitioners, 2005. 17(5): p. 187-193.

Gastrulation (between embryonic days E 13 to E 20) should be considered a high risk

period of pregnancy, as a woman most likely is not yet unaware of her pregnancy and

is not taking precautionary measures to reduce exposure risks to the embryo, as for

example, by not taking certain drugs, not drinking alcohol, and not smoking.

The mother also may not have started folic acid dietary supplementation known to

decrease risk of birth defects.

Early cardiac

progenitor

cells begin to

differentiate in

the heart

fields.

21 days

During gastrulation, Wnt/beta-

catenin signaling and the

induction of the heart

compartment occur in an

anterior/posterior

progression initiating cardiac

cell specification and cell

sorting across the

bilateral regions

of the ventral mesoderm layer.

Linask et al., 1992; 1997;

2003

Heart Organogenesis

Environmental exposures

induce similar cardiac defects

in vertebrate animal models as

in the human depending upon

developmental timing of

exposure and dose.

Defining the effects of alcohol on heart development

in the mouse model.

• Pregnant mice were exposed

acutely to an accepted binge

drinking level of EtOH (two i.p. injections of 2.9 g EtOH/kg maternal

weight, given 4 hrs apart).

• Exposure targeted ED 6.75

(gastrulation).

METHODOLOGY:

ED6.75

ED15.5

On ED 15.5 of gestation doppler echocardiography was used to

determine effects of alcohol on embryonic mouse heart

function in control and ethanol exposed embryos.

A binge drinking exposure of ethanol on ED 6.75 resulted in

abnormal fetal cardiac function and valve defects.

Arrhythmia Atrioventricular Valve Regurgitation

Semilunar Valve Regurgitation Semilunar Valve Stenosis

Blue signal is active Wnt/β-catenin signaling using the TopGal transgenic line.

Question: Does folic acid when provided early in

gestation on morning after conception reverse the

adverse effects of EtOH on mouse heart

development,?

Serrano, M., Brinez, P., Han, M., Lastra-Vicente, R., and K.K. Linask. 2010. Fetal Alcohol

Syndrome: Cardiac birth defects and prevention with folate. Amer. J. Obst. Gyn. 203(1):75.e7-

75.e15.

Folic Acid Supplementation

Mouse Model: Dietary folate supplementation (10

mg/kg or 6.2 mg/kg) was started on morning of

vaginal plug detection.

Only the high dose of folate provided complete protection

of cardiac defects.

A combination of folate and myo-inositol is more effective

in embryonic protection than folate alone.

Microarray Analysis of Cardiac Gene Expression after Hcys or Li Environmental Exposure and Folate Protection:

46,000 genes analyzed- partial list shown

Total RNA extracted

cDNA

Hybridization to Affymetrix

Gene Chips

Right ventricle

and

outflow tract

isolated

Gender-Associated Changes: Gene Ontology of Specific Biological Processes

Fatty acid β-oxidation

Organic acid catabolic proc

Carboxylic acid catab proc

ATP biosynthesis

Fatty acid catab processes

Lipid oxidation

Fatty acid metabolic proc

Coenzyme metabolic proc

Energy coupled proton trans

Electron transport chain

Cofactor metabolic proc

The bioinformatic

analyses indicated

lipid metabolism is

most affected and the

misexpression can

have a gender bias for

specific genes.

Table 2. Gender -Associated Changes: Gene Ontology (GO) Specific Biological Processes

GO ID Description hcys_f1_

vs_cont_f

1

hcys_f1_

vs_fol_f1

lith_f1_vs

_cont_f1

lith_f1_vs

_fol_f1

lith_f1_vs

_lith_fol_

f1

lith_f1_vs

_lith_fol_

f2

fol_f1_vs_

cont_f1

hcys_m1

_vs_cont

_m1

hcys_m1

_vs_fol_

m1

hcys_m1

_vs_hcys

_fol_m1

hcys_m1

_vs_hcys

_fol_m2

lith_m1_

vs_cont_

m1

lith_m1_

vs_fol_m

1

fol_m1_vs

_cont_m1

Number

of

instances

GO:0007186 G-protein coupled receptor signaling pathwayNS INCONSISTENTUP UP NS NS NS UP UP NS UP UP UP DOWN 9

GO:0006754 ATP biosynthetic process UP UP NS UP NS NS DOWN NS DOWN DOWN DOWN NS NS UP 8

GO:0003012 muscle system process UP UP UP UP NS NS DOWN NS NS NS DOWN NS NS UP 7

GO:0006635 fatty acid beta-oxidation NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 7

GO:0007155 cell adhesion NS DOWN NS NS DOWN NS NS UP NS UP UP NS DOWN UP 7

GO:0007218 neuropeptide signaling pathway UP DOWN UP NS NS NS UP NS NS NS UP NS UP DOWN 7

GO:0009201 ribonucleoside triphosphate biosynthetic processUP UP NS NS NS NS DOWN NS DOWN DOWN DOWN NS NS UP 7

GO:0009206 purine ribonucleoside triphosphate biosynthetic processUP UP NS NS NS NS DOWN NS DOWN DOWN DOWN NS NS UP 7

GO:0015985 energy coupled proton transport, down electrochemical gradientUP UP NS UP NS NS DOWN NS NS DOWN DOWN NS NS UP 7

GO:0015986 ATP synthesis coupled proton transportUP UP NS UP NS NS DOWN NS NS DOWN DOWN NS NS UP 7

GO:0016054 organic acid catabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 7

GO:0022610 biological adhesion NS DOWN NS NS DOWN NS NS UP NS UP UP NS DOWN UP 7

GO:0046395 carboxylic acid catabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 7

GO:0055114 oxidation-reduction process UP UP NS NS NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 7

GO:0071804 cellular potassium ion transport NS UP UP UP NS NS DOWN UP UP NS NS UP NS NS 7

GO:0071805 potassium ion transmembrane transportNS UP UP UP NS NS DOWN UP UP NS NS UP NS NS 7

GO:0006082 organic acid metabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 6

GO:0006412 translation UP NS NS NS NS NS NS NS DOWN DOWN DOWN NS DOWN UP 6

GO:0006457 protein folding NS NS NS NS NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 6

GO:0006818 hydrogen transport UP UP NS UP NS NS DOWN NS NS DOWN DOWN NS NS NS 6

GO:0006936 muscle contraction UP UP NS UP NS NS DOWN NS NS NS DOWN NS NS UP 6

GO:0009062 fatty acid catabolic process NS NS NS NS NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 6

GO:0009145 purine nucleoside triphosphate biosynthetic processUP UP NS NS NS NS NS NS DOWN DOWN DOWN NS NS UP 6

GO:0019395 fatty acid oxidation NS NS NS NS NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 6

GO:0019752 carboxylic acid metabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 6

GO:0021545 cranial nerve development NS DOWN NS NS NS DOWN DOWN UP NS UP UP NS NS NS 6

GO:0030198 extracellular matrix organization NS DOWN UP NS NS NS UP NS NS UP UP NS NS UP 6

GO:0032787 monocarboxylic acid metabolic processNS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 6

GO:0034440 lipid oxidation NS NS NS NS NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 6

GO:0043062 extracellular structure organization NS DOWN UP NS NS NS UP NS NS UP UP NS NS UP 6

GO:0043436 oxoacid metabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 6

GO:0044281 small molecule metabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 6

GO:0044282 small molecule catabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 6

GO:0044712 single-organism catabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS DOWN NS 6

GO:0060349 bone morphogenesis NS DOWN UP NS NS NS UP UP UP NS UP NS NS NS 6

GO:0061053 somite development NS DOWN NS NS NS NS NS UP UP UP UP NS UP NS 6

GO:0072329 monocarboxylic acid catabolic processNS NS NS NS NS NS NS DOWN DOWN DOWN DOWN DOWN DOWN NS 6

GO:0001973 adenosine receptor signaling pathwayNS NS NS NS NS NS DOWN UP UP NS NS UP UP NS 5

GO:0006091 generation of precursor metabolites and energyUP UP NS NS NS NS NS NS DOWN DOWN DOWN NS NS NS 5

GO:0006310 DNA recombination NS NS NS NS NS NS NS DOWN DOWN DOWN DOWN DOWN NS NS 5

GO:0006631 fatty acid metabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS NS NS 5

GO:0006732 coenzyme metabolic process NS NS DOWN NS NS NS NS DOWN DOWN DOWN DOWN NS NS NS 5

GO:0006941 striated muscle contraction UP UP NS UP NS NS DOWN NS NS NS NS NS NS UP 5

GO:0009108 coenzyme biosynthetic process DOWN NS DOWN DOWN DOWN NS NS NS NS NS DOWN NS NS NS 5

GO:0009142 nucleoside triphosphate biosynthetic processUP UP NS NS NS NS NS NS DOWN DOWN DOWN NS NS NS 5

GO:0015672 monovalent inorganic cation transportUP UP NS UP NS NS DOWN NS NS NS NS NS NS UP 5

GO:0022900 electron transport chain UP UP NS NS NS NS NS NS DOWN DOWN DOWN NS NS NS 5

GO:0030317 sperm motility UP UP NS NS DOWN DOWN DOWN NS NS NS NS NS NS NS 5

GO:0033539 fatty acid beta-oxidation using acyl-CoA dehydrogenaseNS NS NS NS NS NS NS DOWN DOWN DOWN DOWN DOWN NS NS 5

GO:0035282 segmentation NS DOWN NS NS NS NS NS UP UP UP UP NS NS NS 5

GO:0044710 single-organism metabolic process NS NS NS DOWN NS NS NS NS DOWN DOWN DOWN NS DOWN NS 5

GO:0051186 cofactor metabolic process NS NS NS DOWN NS NS NS DOWN DOWN DOWN DOWN NS NS NS 5

GO:0055085 transmembrane transport UP UP NS UP NS NS DOWN NS NS NS NS UP NS NS 5


vs_cont_f

1

hcys_f1_

vs_fol_f1

lith_f1_vs

_cont_f1

lith_f1_vs

_fol_f1

lith_f1_vs

_lith_fol_

f1

lith_f1_vs

_lith_fol_

f2

fol_f1_vs_

cont_f1

hcys_m1

_vs_cont

_m1

hcys_m1

_vs_fol_

m1

hcys_m1

_vs_hcys

_fol_m1

hcys_m1

_vs_hcys

_fol_m2

lith_m1_

vs_cont_

m1

lith_m1_

vs_fol_m

1

fol_m1_vs

_cont_m1

Number

of

instances
























































(from Visual embryo

website)3 Weeks

49% of pregnancies are unintended! Walker, D., et al., Fetal Alcohol Spectrum Disorders Prevention:

An exploratory study of women's use of, attitudes toward, and knowledge about alcohol. J Amer Acad Nurse Practitioners, 2005. 17(5): p. 187-193.

Gastrulation (between embryonic days E 13 to E 20) should be considered

a high risk period of pregnancy, as a woman most likely is not yet unaware

of her pregnancy and is not taking precautionary measures to reduce

exposure risks to the embryo, as for example, by not taking certain drugs,

not drinking alcohol, and not smoking.

The mother also may not have started folic acid dietary supplementation

known to decrease risk of birth defects.

Early cardiac

progenitor

cells begin to

differentiate in

the heart

fields.

21 days

During gastrulation,

Wnt/beta-catenin signaling

and the induction of the

heart compartment occur in

an anterior/posterior

progression initiating

cardiac cell specification

and cell sorting across the

bilateral regions

of the ventral mesoderm

layer.

Linask et al., 1992; 1997;

2003

Heart Organogenesis

Environmental exposures

induce similar cardiac defects

in vertebrate animal models as

in the human depending upon

developmental timing of

exposure and dose.

Defining the effects of alcohol on heart development

in the mouse model.

• Pregnant mice were exposed

acutely to an accepted binge

drinking level of EtOH (two i.p. injections of 2.9 g EtOH/kg maternal

weight, given 4 hrs apart).

• Exposure targeted ED 6.75

(gastrulation).

METHODOLOGY:

ED6.75

ED15.5

On ED 15.5 of gestation doppler echocardiography

was used to determine effects of alcohol on

embryonic mouse heart function in control and

ethanol exposed embryos.

A binge drinking exposure of ethanol on ED 6.75

resulted in abnormal fetal cardiac function and valve

defects.

Arrhythmia Atrioventricular Valve Regurgitation

Semilunar Valve Regurgitation Semilunar Valve Stenosis

Blue signal is active Wnt/β-catenin signaling using the TopGal transgenic line.

Question: Does folic acid when provided

early in gestation on morning after conception

reverse the adverse effects of EtOH on

mouse heart development,?

Serrano, M., Brinez, P., Han, M., Lastra-Vicente, R., and K.K. Linask. 2010. Fetal

Alcohol Syndrome: Cardiac birth defects and prevention with folate. Amer. J. Obst.

Gyn. 203(1):75.e7-75.e15.

Folic Acid Supplementation

Mouse Model: Dietary folate supplementation

(10 mg/kg or 6.2 mg/kg) was started on

morning of vaginal plug detection.

Only the high dose of folate provided complete

protection of cardiac defects.

A combination of folate and myo-inositol is more

effective in embryonic protection than folate

alone.

Microarray Analysis of Cardiac Gene Expression after Hcys or Li Environmental Exposure and Folate Protection:

46,000 genes analyzed- partial list shown

Total RNA extracted

cDNA

Hybridization to Affymetrix

Gene Chips

Right

ventricle and

outflow tract

isolated

Gender-Associated Changes: Gene Ontology of Specific Biological Processes

Fatty acid β-oxidation

Organic acid catabolic proc

Carboxylic acid catab proc

ATP biosynthesis

Fatty acid catab processes

Lipid oxidation

Fatty acid metabolic proc

Coenzyme metabolic proc

Energy coupled proton trans

Electron transport chain

Cofactor metabolic proc

The bioinformatic

analyses indicated

lipid metabolism is

most affected and

the misexpression

can have a gender

bias for specific

genes.

Table 2. Gender -Associated Changes: Gene Ontology (GO) Specific Biological Processes


vs_cont_f

1

hcys_f1_

vs_fol_f1

lith_f1_vs

_cont_f1

lith_f1_vs

_fol_f1

lith_f1_vs

_lith_fol_

f1

lith_f1_vs

_lith_fol_

f2

fol_f1_vs_

cont_f1

hcys_m1

_vs_cont

_m1

hcys_m1

_vs_fol_

m1

hcys_m1

_vs_hcys

_fol_m1

hcys_m1

_vs_hcys

_fol_m2

lith_m1_

vs_cont_

m1

lith_m1_

vs_fol_m

1

fol_m1_vs

_cont_m1

Number

of

instances























































vs_cont_f

1

hcys_f1_

vs_fol_f1

lith_f1_vs

_cont_f1

lith_f1_vs

_fol_f1

lith_f1_vs

_lith_fol_

f1

lith_f1_vs

_lith_fol_

f2

fol_f1_vs_

cont_f1

hcys_m1

_vs_cont

_m1

hcys_m1

_vs_fol_

m1

hcys_m1

_vs_hcys

_fol_m1

hcys_m1

_vs_hcys

_fol_m2

lith_m1_

vs_cont_

m1

lith_m1_

vs_fol_m

1

fol_m1_vs

_cont_m1

Number

of

instances






















































Genes Appearing in Multiple Gene Ontology Categories

Pertaining to Lipid Metabolism

Analyzing genes that appear in all of the classes as (i) cellular processes of fatty

acid oxidation, (ii) fatty acid beta oxidation, (iii) lipid oxidation or (iv) fatty acid

beta oxidation using acyl CoA dehydrogenase, in both Li- and HCy-exposed heart

tissue, the genes appearing in all classes included:

acyl CoA dehydrogenase 11 (Acad11)

acyl CoA dehydrogenase long chain (Acadl)

acyl CoA dehydrogenase medium chain (Acadm)

acyl CoA dehydrogenase short chain (Acads)

acyl CoA dehydrogenase very long chain (Acadvl) and

electron transferring flavoprotein dehydrogenase (Etfdh)

Notably all of the protein products of these genes localize to the mitochondria.

Lipids essentially serve three critical cell functions: energy storage, structural

components of membranes, and lipid modifications for activity of cell signaling

factors.

Alcohol alters gene expression similarly to that of elevated HCy

exposure: RT-PCR Analyses of the ED 15.5 mouse heart

Alcohol Exposure Homocysteine

Exposure

Importantly, our RT-PCR data

analyzing the alcohol-exposed

fetal heart indicate the fatty

acid metabolism- and folate-

related genes are upregulated

within the embryonic and fetal

heart tissues, seemingly to

overcome folate metabolism-

related deficiency.

Folate protects gene

expression by maintaining

expression close to control

levels.

RA LA

RVLV

Infer. Vena Cava

Umbilical Artery

Ductus Venosus

PLACENTA

HEARTLIVER

Umbilical Vein

Cross-Talk between Utero-Placental and Fetal Circulation

When genes are knock-out only in the placenta, heart defects can arise. If those

placental genes are re-expressed, the cardiac defects are prevented.(Barak et al., 1999; Adams et al., 2000)

Heart-Placenta Axis: IUGR and Other Effects

Oil Red O staining for neutral lipids (triglycerides, diacylglycerols, and cholesterol esters)

Organization of the labyrinth layer is significantly altered with alcohol exposure

and lipid synthesis is decreased. Folate supplementation returns cell organization and

lipid metabolism nearer to control levels.

Heart-Placenta Axis: IUGR and Ethanol Effects

NMHC-IIA has a unique, but necessary

role in placentation. Function is unknown.

If knocked out, embryonic lethality occurs.

NMHC-IIB appears to be involved in

cell motility. If knocked out, embryos

develop, but die from cardiac defects at

mid-gestation.

Han, et al., 2012. Am J Obstet Gynecol 207, 140 e147-119.

Changes in Fatty Acid Metabolism within the Embryo:

An emerging common theme in...

• Alcohol abuse

• Diabetes

• Obesity

• Lithium exposure

• Elevated homocysteine (folate deficiency)

• ...and they all relate to a higher risk of birth defects

Genes involved in fatty acid metabolism can be epigenetically regulated by specific

methylation patterns within promoter and intragenic regions.

These patterns can be transmitted from one generation to another.

In conclusion…

How much alcohol is safe to drink during

pregnancy?

• No known amount of alcohol is safe for a

growing embryo and fetus.

In the United States 1 in 5 of over half a million women

who report drinking alcohol during pregnancy also admit

to binge drinking. It is known that even low levels of

alcohol (ethanol; EtOH) prenatally can produce human

birth defects.

Severity of defects depends on dose and timing of

alcohol intake and seemingly gender of fetus.

2008 2009 2010 2011 2012 2013

EU (28

countries)5469434 5412572 5411129 5266162 5231177

5075691 (e

p)

EU (27

countries)5425681 5367995 5367768 5224965 5189406

5035752 (e

p)

Euro area

(18

countries)

3492593 3435284 3442269 3363927 33195573244394 (e

p)

Euro area

(17

countries)

3468196 3413240 3422488 3345102 32996603223798 (e

p)

Belgium 127205 127198 130100 128705 128051 125606 (p)

Bulgaria 77712 80956 75513 70846 69121 66578

Czech

Republic119570 118348 117153 108673 108576 106751

Denmark 65038 62818 63411 58998 57916 55873

Germany 682514 665126 677947 662685 673544 685000 (e)

Estonia 16028 15763 15825 14679 14056 13531

Ireland 75173 75554 75174 74033 72225 68930 (p)

Greece 118302 117933 114766 106428 100371 94146 (p)

Spain 518503 493717 485252 470553 453348 424494 (p)

France 829311 825564 833654 824263 821844 810820 (e)

Live births are the births of

children that showed any

sign of life (total births minus

stillbirths).Additional information on Eurostat web

page

Live Births In Estonia and Other EU Countries:

What could drinking

during pregnancy mean

for Estonia in relation to

livebirths?

If one considers FASD occurs about one in

every 100 live births (May et al., 2009),

the prevalence of FASD in Estonia

between 2008-2013 (6 years) would be

expected to be ~899 individuals, with

most having varying degrees of

developmental disabilities, with ~54%

(495) possibly having some type of cardiac

defect, or many more being among the

stillbirths.

Mis tähendaks alkahooli joomine raseduse ajal

Eesti ühiskonnale?

Genes Appearing in Multiple Gene Ontology Categories

Pertaining to Lipid Metabolism

Analyzing genes that appear in all of the classes as (i) cellular processes of

fatty acid oxidation, (ii) fatty acid beta oxidation, (iii) lipid oxidation or

(iv) fatty acid beta oxidation using acyl CoA dehydrogenase, in both Li-

and HCy-exposed heart tissue, the genes appearing in all classes included:

acyl CoA dehydrogenase 11 (Acad11)

acyl CoA dehydrogenase long chain (Acadl)

acyl CoA dehydrogenase medium chain (Acadm)

acyl CoA dehydrogenase short chain (Acads)

acyl CoA dehydrogenase very long chain (Acadvl) and

electron transferring flavoprotein dehydrogenase (Etfdh)

Notably all of the protein products of these genes localize to the

mitochondria.

Lipids essentially serve three critical cell functions: energy storage,

structural components of membranes, and lipid modifications for

activity of cell signaling factors.

Alcohol alters gene expression similarly to that of

elevated HCy exposure: RT-PCR Analyses of the ED

15.5 mouse heart

Alcohol Exposure Homocysteine

Exposure

Importantly, our RT-PCR

data analyzing the alcohol-

exposed fetal heart indicate

the fatty acid metabolism-

and folate-related genes are

upregulated within the

embryonic and fetal heart

tissues, seemingly to

overcome folate metabolism-

related deficiency.

Folate protects gene

expression by

maintaining expression

close to control levels.

RA LA

RVLV

Infer. Vena Cava

Umbilical Artery

Ductus Venosus

PLACENTA

HEARTLIVER

Umbilical Vein

Cross-Talk between Utero-Placental and Fetal Circulation

When genes are knock-out only in the placenta, heart defects can arise. If

those placental genes are re-expressed, the cardiac defects are prevented.(Barak et al., 1999; Adams et al., 2000)

Heart-Placenta Axis: IUGR and Other Effects

Oil Red O staining for neutral lipids (triglycerides, diacylglycerols, and cholesterol esters)

Organization of the labyrinth layer is significantly altered with alcohol exposure

and lipid synthesis is decreased. Folate supplementation returns cell

organization and lipid metabolism nearer to control levels.

Heart-Placenta Axis: IUGR and Ethanol Effects

NMHC-IIA has a unique, but necessary

role in placentation. Function is

unknown. If knocked out, embryonic

lethality occurs.

NMHC-IIB appears to be involved in

cell motility. If knocked out, embryos

develop, but die from cardiac defects at

mid-gestation.

Han, et al., 2012. Am J Obstet Gynecol 207, 140 e147-119.

Changes in Fatty Acid Metabolism within the

Embryo: An emerging common theme in...

� Alcohol abuse

� Diabetes

� Obesity

� Lithium exposure

� Elevated homocysteine (folate deficiency)

� ...and they all relate to a higher risk of birth

defectsGenes involved in fatty acid metabolism can be epigenetically regulated

by specific methylation patterns within promoter and intragenic regions.

These patterns can be transmitted from one generation to another.

In conclusion/

How much alcohol is safe to drink during

pregnancy?

� No known amount of alcohol is safe for a

growing embryo and fetus.

In the United States 1 in 5 of over half a million

women who report drinking alcohol during pregnancy

also admit to binge drinking. It is known that even low

levels of alcohol (ethanol; EtOH) prenatally can

produce human birth defects.

Severity of defects depends on dose and timing of

alcohol intake and seemingly gender of fetus.

2008 2009 2010 2011 2012 2013

EU (28

countries

)

5469434 5412572 5411129 5266162 52311775075691 (

ep)

EU (27

countries

)

5425681 5367995 5367768 5224965 51894065035752 (

ep)

Euro

area (18

countries

)

3492593 3435284 3442269 3363927 33195573244394 (

ep)

Euro

area (17

countries

)

3468196 3413240 3422488 3345102 32996603223798 (

ep)

Belgium 127205 127198 130100 128705 128051125606 (p

)

Bulgaria 77712 80956 75513 70846 69121 66578

Czech

Republic119570 118348 117153 108673 108576 106751

Denmark 65038 62818 63411 58998 57916 55873

Germany 682514 665126 677947 662685 673544685000 (e

)

Estonia 16028 15763 15825 14679 14056 13531

Ireland 75173 75554 75174 74033 72225 68930 (p)

Greece 118302 117933 114766 106428 100371 94146 (p)

Spain 518503 493717 485252 470553 453348424494 (p

)

France 829311 825564 833654 824263 821844810820 (e

)

Live births are the births of

children that showed any

sign of life (total births

minus stillbirths).Additional information on Eurostat web

page

Live Births In Estonia and Other EU Countries:

What could drinking

during pregnancy mean

for Estonia in relation to

livebirths?

If one considers FASD occurs about

one in every 100 live births (May et al.,

2009), the prevalence of FASD in

Estonia between 2008-2013 (6 years)

would be expected to be ~899

individuals, with most having varying

degrees of developmental disabilities,

with ~54% (495) possibly having

some type of cardiac defect, or many

more being among the stillbirths.

Mis tähendaks alkahooli joomine raseduse

ajal Eesti ühiskonnale?

kerstik. linask, ph.d. university of south florida ... · university of south florida children’s...

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