Nicotine, Tobacco and Brain Damage,Nicotine, Tobacco and Brain Damage,From the Fetus to the Adolescent:From the Fetus to the Adolescent:

Finding the Smoking GunFinding the Smoking Gun

Theodore Slotkin, Ph.D.Theodore Slotkin, Ph.D.

Dept. of Pharmacology & Cancer BiologyDept. of Pharmacology & Cancer Biology

Duke University Medical CenterDuke University Medical Center

Research Support: NIH DA14247 and the Philip Morris External Research Program

Carrie Nation (WCTU founder) - 1890s

These tobacco users transmit nervous diseases, epilepsy, weakened constitutions, depraved appetites and deformities of all kinds to their offspring.

The tobacco user can never be the father of a healthy child.

ETS exposure!

Recruiting Women Smokers - the Origin of the Problem

1932 - must be good for your

health!

1942 - it’s

patriotic to smoke!

1929 - avoid getting fat

1926 - don’t be left out!

1934 - cures depression and tiredness!

U.S. Annual Figures for Maternal Cigarette SmokingU.S. Annual Figures for Maternal Cigarette Smoking

•Spontaneous abortions: 19,000 - 141,000•Low Birthweight: 32,000 - 61,000•Neonatal ICU Admissions: 14,000 - 26,000•Perinatal Deaths: 1,900 - 4,800

•50-500% Increased Incidence of:•SIDS•Learning Disorders•ADHD•Disruptive Behaviors

Overall US Rate: 10-20% of all births

DiFranza et al, J. Fam. Pract. 1995

ETS exposure: part of the continuum of adverse effects

Fetal nicotine range ≈ 10-30% of active smoking

Maternal Cigarette Smoking

Effects on Fetal BrainGeneral Development

Hypoxia/IschemiaCO, HCNAnorexia

Maternal-Fetal UnitNicotine in Fetus

Perinatal Morbidity/MortalityGrowth RetardationBehavioral Anomalies

Risky Behaviors:Other drugs/alcohol

Prenatal CareSocioeconomic

Why Do We Need Animal Models?Why Do We Need Animal Models?

• Proof of cause-and-effect

• Rational approach to regulating ETS exposure (biomarker)

• Use of NRT in pregnancy

• Framework for understanding onset of addiction in adolescence

• Point to new outcomes to be studied in humans

Fetal Nicotine Exposure in RatsFetal Nicotine Exposure in Rats

Time

Blo

od

Lev

el

Time

Blo

od

Lev

el

Threshold forHypoxia/Ischemia

Injection Infusion

ETS exposure - inhalation chambers matching nicotine, CO, particulates, etc

Matching Blood Nicotine Levels: 6 mg/kg/day ≈ 1-2 packs; 2 mg/kg/day ≈ 0.5-1 packETS amniotic fluid nicotine ≈ active light smoker

Prenatal Nicotine Exposure: Neonatal CNS Cell Damage and Cell Deficits

Increased expression of p53Constitutive elevation of c-fos

100

120

140

160

180

per

cen

t co

ntr

ol

200

2 4 7 10 14

Postnatal Age (days)

ODC Activity

Forebrain (p < 0.01)Cerebellum (p < 0.01)

-40

-30

-20

-10

0

per

cen

t ch

ang

e fr

om

co

ntr

ol

10

2 4 7 10 14

Postnatal Age (days)

DNA Content

Forebrain (p < 0.01)Cerebellum (p < 0.01)

Prenatal Nicotine Exposure: Impaired Neonatal CNS Function

-20

-10

0

10

20

30

40

ForebrainP

erc

en

t R

ele

as

ed

Midbrain Hippo-campus

Norepinephrine Release By AcuteNicotine Challenge on Postnatal Day 30

ControlNicotine (p < 0.0005)

Fetal Treatment

50

60

70

80

90

100

110

120

130

140

0 10 20

Effects of Prenatal Nicotineon Norepinephrine Contentand Turnover in Forebrain

*

30

per

cen

t co

ntr

ol

40

Postnatal Age (days)

*50

Content: CON > NIC, p < 0.01; NIC x Age, p < 0.01Turnover: CON > NIC, p < 0.01; NIC x Age, p < 0.01

60

Content

*

***

*

Turnover *

*

*

50

75

100

125

0 10 20 30 40

per

cen

t co

ntr

ol

50

ANOVA:CON > NIC, p < 0.0001NIC age, p < 0.0005

*

Postnatal Age (days)

Effects of Prenatal Nicotine Exposure on

Hippocampus [3H]Hemicholinium-3 Binding

**

*

* *

Effects on CNS Development Belowthe Threshold for Growth Impairment

-40

-20

0

20

40

per

cen

t ch

ang

e

60

2 10

ODC

7Postnatal Age (days)

Biomarkers

10 20

DNA

42 7

NETurnover

42 6050

60

70

80

90

100

110

120

130

140

0 10 20

Body and Tissue Weights

30 40

per

cen

t co

ntr

ol

50Postnatal Age (days)

60 70

BodyForebrainBrainstemCerebellum

Neurotransmitter Signals Control Cell FateNeurotransmitter Signals Control Cell Fate

NerveTerminal

Receptors

SignalingCascades Nucleus

Gene Transcription

Replicate Differentiate Grow Die Learn

The same neurotransmitter may be used for multiple decisions

Prenatal Nicotine Preempts the Natural Trophic Role of ACh by Stimulating ACh Receptors

0

20

40

60

80

100

120

0 10 20 30 40 50

Forebrain: Ratio of Choline Uptake toCholine Acetyltransferase Activity

*

CON

rati

o

NIC

Postnatal Age (days)

*CON > NIC, p < 0.01

NIC x Age interaction, p < 0.01

100

110

120

130

140

150

6 mg/kg/day 2 mg/kg/day

per

cen

t co

ntr

ol

*CON < NIC, p < 0.01

[3H]Nicotine Binding to Receptorsin Fetal Brain on Gestational Day 18

*

Nicotine Inhibits DNA Synthesis in Developing Brain Through Effects on Nicotinic Receptors

•2 µg to CNS - same effect

•blocked by nAChR antagonists

•effect mediated by nAChRs in culture

-80

-60

-40

-20

0

20

GD18 PN3 PN10

BrainstemForebrainCerebellum

*

pe

rce

nt

ch

an

ge

*

CON > NIC, p < 0.001Nic x Region interaction, p < 0.01

Age

* * * *

*

Effects During Neural Tube Stagebut -- apparent subsequent recovery!

Hippocampus in Young Adulthood afterLow-Dose Prenatal Nicotine Exposure in vivo

dentate gyrus

reduced cell size

increased cell packing density

increased numbers of glial cells

Prenatal Nicotine Exposure in Rhesus Monkeys:nAChR Upregulation Enhanced by Supposed Ameliorative Therapies

Choline or Vitamin C double the nicotine concentration in amniotic fluid!

Perinatal ETS exposure in Rhesus Monkeys:nAChR Upregulation ≈ Nicotine

Nicotine is a Neuroteratogen

• Cell damage and deficits in cell number

• Impaired synaptic activity

• Receptor-Mediated, therefore low threshold

• Affects Cell Replication/Differentiation Switchover

• Initiates the Program for Cell Death

• Morphological changes subtle but detectable in adulthood

Does altered neurochemistry account for functional deficits?

Catecholamine Surge at BirthLagercrantz & Slotkin, Sci. Amer.,1986

0 10 20 30 40 50

Catecholamines (nM)

Infants at Birth

Pheochromocytoma

Women During Delivery

Heavy Exercise

Sauna

Adults at Rest

Interference with surge - cardiorespiratory collapse

Source90% Adrenal10% SNS

Prenatal Nicotine Exposure Compromises the Neonatal Response to Hypoxia

0

5

10

15

20

25

30

Per

cen

t M

ort

alit

y60 minutes

Mortality During 5% O2

Exposure on Postnatal Day 1

Control

75 minutes

p < 0.007

p < 0.004

Nicotine

0

10

20

30

40

50

NE

Per

cen

tag

e S

ecre

ted

Epi Total

*

Adrenal Catecholamine SecretionCaused by 5% O

2 for 75 minutes

**

ANOVA: CON > NIC (p < 0.004)

ControlNicotine

Same effect in controls with adrenalectomy or adrenergic receptor blockade

Subjects matched for:Subjects matched for:age, parity, SES, Page, parity, SES, PaaOO22, , PPaaCOCO22, pH, pHaa

0

1000

2000

3000

4000

5000

6000

7000

8000

Norepinephrine Epinephrine

Catecholamines in Umbilical ArterialBlood at Delivery, Before the First BreathANOVA: Non-smoker > Smoker, p < 0.05

Non-smoker (21)Smoker (20)

0

100

200

300

400

500

600

700

800

No

rep

ine

ph

rin

e (

pg

/ml)

Measure

Ep

ine

ph

rin

e (

pg

/ml)

Directresponse

Reflex response

Increasing Age

Normal Development: Direct response replaced by reflex

Directresponse

Reflex response

Increasing Age

Nicotine Treatment: accelerated adrenomedullary development

VulnerableWindow

Receptor imbalancescardiorespiratory

inhibition during hypoxia

Nicotine is a NeuroteratogenNicotine is a Neuroteratogen

• Causal relationship between smoking, SIDS, ADHD, etc

• Effects shared by ETS in a dose-response continuum

• Caution in using NRT in pregnancy (is it even effective?)

• formulation and pharmacokinetic issues

• Interaction with OTC nutritional supplements

Brain development continues into adolescence

How does the adolescent brain respond to nicotine?

Adolescent Smoking

Nearly all smokers begin as adolescents

75% become daily smokers by 20 y.o.

Higher daily consumption, lower quit rate

Female > Male

Affective and Cognitive Components

Vulnerable subset: loss of autonomy with a few cigsalso - greater withdrawal problems

Relationship to maternal smoking during pregnancy?

Brain Development Continues into Adolescence:So Does the Developmental Neurotoxicity of Nicotine

-20

-10

0

10

20

30

40

during Rx 1 month post Rx

Midbrain Nicotinic Receptor Upregulation

adolescentadult

% c

ha

ng

e f

rom

co

ntr

ol

adolescent > adult, p < 0.001

*

*

-60

-40

-20

0

20

60 75 60 50 60 45 80

Hippocampus — Adolescent Females

% c

ha

ng

e f

rom

co

ntr

ol

* * *

* *

*

*

Synaptic ActivityMarkers

membraneprotein

p53mRNA

HC-3Binding

NEturnover

DamageMarkers

6 mg/kg/day: adolescent rat 25 ng/ml; nonpregnant adult 100 ng/ml

PN30 PN47

posttreatment

Nicotine

50 60 75+

Significant effects at 2.5 ng/ml ≈2 cigs per day or ETS!!!

Effects with little as 2 days of exposure

Adolescent Nicotine Effects - Low Threshold!!0.6 mg/kg/day ≈ 2.5 ng/ml = 2 cigs/day ≈ ETS

Other targets: Serotonin - mood & appetite

ImmuneCardiac autonomic

effect @ 2 days

same results for

intermittent exposure -

daily injections

*

Adolescent Nicotine Effects

Greater Sensitivity of ACh and Serotonin systems• enhanced onset of nAChR upregulation and greater persistence• persistent deficiency in synaptic activity - ACh and Serotonin• exquisite sensitivity - down to level of ‘chipper’ or ETS

Cell damage• loss of synaptic function• brain areas involved in learning and memory, mood

Sex selectivity: effects on females > males (also true for adolescent smokers)

Conclusion: There is a biological basis for the susceptibility of the adolescent

brain to nicotine addiction

Does Prenatal Nicotine Exposure Alter theResponse to Nicotine in Adolescence?

embryo implants

birthGD22

weaningPN21

Nicotine

PN30 PN47

posttreatment

Nicotine

50 60 75+

4 Groups: Vehicle/Vehicle Nicotine/Vehicle

Vehicle/Nicotine Nicotine/Nicotine

-20

0

20

40

60

80

Prenatal Adolescent Prenatal+Adolescent

nAChR Binding in Midbrain

PN45PN50PN60PN75

Per

cent

cha

nge

from

con

trol

ANOVA: Rx, p < 0.0001; Rx x Age, p < 0.0001

*

*

*

**

*

Control vs. Adolescent, p < 0.0001Prenatal vs. Adolescent, p < 0.0001

Prenatal vs. [Prenatal+Adolescent], p < 0.002Adolescent vs. [Prenatal+Adolescent], p < 0.0001

-20

-10

0

10

20

30

40

Norepinephrine Release By AcuteNicotine Challenge on Postnatal Day 30

ControlNicotine (p < 0.0005)

Forebrain

Pe

rcen

t R

ele

ase

d

Midbrain Hippo-campus

Fetal Treatment

Prenatal Nicotine Blunts the Cholinergic Responseto Nicotine in Adolescence

Additional: Enhanced neural damage/loss

Withdrawal synaptic activity

Effects of Prenatal Nicotine Exposure on Adolescent NicotineSelf-Administration — Withdrawal Triggers Elevated Consumption

0

2

4

6

8

10

12

14

16

18

Infusionsper

Session

1 2 3 4 Nic Phase No Access Reinstatement MAOI Phase

Week

Nicotine Self-Administration after Prenatal Nicotine Exposure

Prenatal Nicotine

Control

p<0.05

p<0.05

Prenatal ± Adolescent Nicotine: A Model for the Effects of Maternal Smoking During Pregnancy

On Subsequent Smoking in Offspring

Animal Studies:• adolescent brain shows high sensitivity to nicotine stimulation

• profound neurochemical effects associated with withdrawal in adolescence

• prenatal nicotine produces ACh, 5HT deficits that emerge in adolescence

• prenatal nicotine desensitizes nAChR, necessitating high doses for stimulation

• prenatal nicotine worsens withdrawal from adolescent nicotine

Human Studies:Jacobsen, Slotkin, Westerveld,

Mencl & Pugh, 2006

which suggested

How Prenatal Nicotine Exposure Predisposes the Brainto Nicotine Addiction in Adolescence

Prenatal Nicotine ACh/Serotonin DysfunctionEmerging in Adolescence

MoodReward

Learning & Memory

Effects Relievedby Nicotine Intake

BUTRelatively InsensitiveHigh Doses Required

Higher Susceptibilityof Adolescent Brain

CNS Damageto Pathways Mediating

Mood, Reward,Learning & Memory

Profound Loss ofSynaptic Activityon Withdrawal -

cognitive deficits,depression make

quitting difficult

Permanent Change?

Reproduction

Adolescent smoking is the “cause”

of smoking during pregnancy

How Tobacco Companies Target AdolescentsDurant et al, Amer. J. Public Health, 1997

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MTV VH1 CMT BET

Per

cen

t

By Network

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Rap Rock AdultContemp

Country R&B

Pe

rce

nt

By Genre / Target Group

Pre-TeenTeen Adult

Targeting Women — Taking Aim at MinoritiesCurrent Ads in Women’s Magazines

Medical Bias in Considering Tobacco a "Drug of Abuse"

0

100

200

300

400

500

Drug Abuse Alcohol Tobacco

US Annual Deaths (thousands)

McGinnis & Foege, 1999

0

20

40

60

80

100

per

cen

t o

f su

bst

ance

abu

se p

ages

Drug Abuse Alcohol Tobacco

Ginzel, 1985

Medical Textbook Coverageof Substance Abuse

Pediatricians advise adolescents about tobacco <2% of visits

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20

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60

80

'90 '91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02

Rev

iew

s'9

0-'9

7

Rev

iew

s'9

8-'0

2

Annual Publications — Fetal or Neonatal Development

CocaineNicotine

No.

of

Pub

licat

ions

Source: Medline