CHILDHOOD BEHAVIOR DISORDERS: NEUROPSYCHOLOGICAL DEFICITS IN NEONATAL DIETARY MANGANESE EXPOSURE

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CHILDHOOD BEHAVIOR DISORDERS
NEUROPSYCHOLOGICAL DEFICITS IN NEONATAL DIETARY
MANGANESE EXPOSURE 

• Francis M. Crinella
• Child Development Center
• Department of Pediatrics
• University of California, Irvine
•  

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DSM-IV SYMPTOMS OF ADHD

• INATTENTION
• CANT ATTEND TO DETAILS
• CANT SUSTAIN ATTENTION
• DOESNT LISTEN
• FAILS TO FINISH
• CANT ORGANIZE TASKS
• AVOIDS SCHOOLWORK
• LOSES THINGS
• EASILY DISTRACTED
• FORGETFUL

• HYPERACTIVITY/IMPULSIVITY
• FIDGETS
• CANT STAY SEATED
• RUN ABOUT AND CLIMBS
• CANT PLAY QUIETLY
• IS OFTEN ON THE GO
• TALKS TOO MUCH
• BLURTS OUT ANSWERS
• CANT WAIT TURN
• INTERRUPTS OR INTRUDES

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STUDIES ASSOCIATING HAIR MANGANESE Mn LEVELS
WITH ADHD Pihl, R.O. Parks, M. (1977). Hair
element content in learning disabled children.
Science, 198, 204-206. Collip, P.J., Chen, S.Y.
Maitinsky, S. (1983). Manganese in infant
formulas and learning disability. Annals of
Nutrition and Metabolism, 27, 488-494. Marlowe,
M. Bliss, L. (1993). Hair element
concentrations and young children's behavior at
school and home. Journal of Orthomolecular
Medicine, 9, 1-12. Cordova, E.J., Ericson, J.,
Swanson, J.M., Crinella, F.M. (1997). Head
hair manganese as a biomarker for ADHD.
Proceedings of the 15th Annual Conference on
Neurotoxicology.
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HEAD HAIR Mn LEVEL
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IS MN EXPOSURE AN ETIOLOGIC AGENT IN ADHD? 1.
CHILDREN WITH ADHD HAVE HIGH LEVELS OF HEAD HAIR
MN 2. MN IS A KNOWN NEUROTOXIN 3. MN TOXICITY
AFFECTS BRAIN DOPAMINE SYSTEMS 4. ADHD IS A
PRIMARILY DOPAMINERGIC DISORDER 5. BRAIN AREAS
AFFECTED BY MN TOXICITY HAVE EXTENSIVE ANATOMICAL
AND NEUROCHEMICAL OVERLAP WITH SYSTEMS SHOWN TO
BE DYSFUNCTIONAL IN ADHD
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BIOLOGICAL BASIS OF ADHD

• PSYCHOPHARMACOLOGY
• THE BEHAVIOR OF CHILDREN RECEIVING BENZEDRINE
  (Bradley, American Journal of Psychiatry, 1937)
• STIMULANT MEDICATIONS FOR THE TREATMENT OF ADHD
  (Wigal et al., 1999)
• MOLECULAR BIOLOGY
• CATECHOLAMINE HYPOTHESIS --GENETIC VARIATION IN
  NEUROTRANSMITTER FUNCTION (Wender, 1971)
• SUBSENSITIVE DOPAMINE HYPOTHESIS DRD4 GENE
  (LaHoste, Swanson, Wigal, et al, 1996)
• BRAIN IMAGING
• MBD (Clements, 1963 Crinella, 1972)
• QUANTITATIVE MRI IMAGING IN ADHDVOLUMETRIC
  DIFFERENCES (e.g., Castellanos, Giedd, et al.,
  1996 Aylward, Reiss et al., 1996)

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BIOLOGICAL BASIS OF ADHD I. PSYCHOPHARMACOLOGY

• TREATMENT WITH CNS STIMULANTS
• BENZEDRINE (Bradley, 1937)
• DEXTROAMPHETAMINES (e.g., Dexedrine, Adderall)
• METHYLPHENIDATES (e.g., Ritalin, Concerta)
• EFFECTS (e.g., Effect of stimulant medication on
  children with attention deficit disorder a
  review of reviews, Swanson et al,, Exceptional
  Children, 1993)
• Improved classroom behavior
• Improved academic productivity
• Improved peer/adult interactions
• Less frequent oppositional conduct
• Reduced aggression

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BIOLOGICAL BASIS OF ADHD II MOLECULAR BIOLOGY

• DOPAMINE D4 RECEPTOR GENE POLYMORPHISM ASSOCIATED
  WITH ADHD (Lahoste, Swanson et al., 1996)
• ASSOCIATION OF THE DOPAMINE RECEPTOR D4 (DRD4)
  GENE WITH A REFINED PHENOTYPE OF ADHD (Swanson,
  Sunohara, Kennedy et al., 1998)

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BIOLOGICAL BASIS OF ADHD III STRUCTURAL IMAGING

• COGNITIVE NEUROSCIENCE OF ATTENTION DEFICIT
  HYPERACTIVITY DISORDER AND HYPERKINETIC DISORDER.
  Swanson, Castellanos, Murias, LaHoste,
  Kennedy, 1998.

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RECENT BRAIN IMAGING STUDIES IN ADHD
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BIOLOGICAL BASIS OF ADHD IV NEUROPSYCHOLOGICAL
EVIDENCE

• ADHD conceptualized as frontal lobe disorder
  (e.g., Douglas, 1980 Chelune et al., 1986)
• ADHD conceptualized as disorder of executive
  function (Pennington et al., 1990 Barkley et
  al., 1992 Crinella Yu, 2000)

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Brief Definitions of Executive Function

• Appropriate set maintenance to achieve a future
  goal (Pennington, Welsh Grossier, 1990)
• A process that alters the probability of
  subsequent responses to an event, thereby
  altering the probability of later consequences
  (Barkley, 1997).
• A process which enables the brain to function as
  many machines in one, setting and resetting
  itself dozens of times in the course of a day,
  now for one type of operation, now for another
  (Sperry, 1955)

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DISTINCT ANATOMICAL NETWORKS CARRY OUT SPECIFIC
ASPECTS OF ATTENTION

• ALERTING NETWORK
• LOCATION ARAS, ETC.
• FUNCTION ACHIEVE AND MAINTAIN STATE OF READINESS
• ORIENTING NETWORK
• LOCATIONS PARIETAL LOBE, SUPERIOR COLLICULUS
  PULVINAR
• FUNCTION REACT TO SENSORY STIMULI
• EXECUTIVE NETWORK
• LOCATION ANTERIOR CINGULATE DORSOLATERAL
  FRONTAL CORTEX BASAL GANGLIA
• FUNCTION PRIORITIZE OPERATION OF OTHER BRAIN
  AREAS

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• I believe that everything important in psychology
  can be investigated in essence through the
  continued experimental and theoretical analysis
  of the determiners of rat behavior at a choice
  point in a maze.
• Edward Chance Tolman Psychological Review
  (1938)

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BRAIN STRUCTURES COMPRISING THE RODENT EF SYSTEM

• SUPERIOR COLLICULUS
• MEDIAN RAPHE NUCLEI
• VENTRAL MESENCEPHALIC AREA
• SUBSTANTIA NIGRA
• PONTINE RETICULAR FORMATION
• CAUDATOPUTAMEN
• VENTRAL LATERAL THALAMUS
• GLOBUS PALLIDUS
• From Thompson, Crinella Yu, 1990

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BEHAVIORAL DEFICITS ASSOCIATED WITH LESIONS IN
THE RODENT EF SYSTEM

• Shifting cognitive sets
• Selective attention
• Procedural knowledge
• Planning behavioral sequences
• State control
• Inhibition of motor reactivity
• Response flexibility
• Transfer strategies
• Working memory
• From Thompson, Crinella Yu (1990)

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• CAN AN ANIMAL MODEL OF ADHD BE INDUCED BY
  NEONATAL MN EXPOSURE?
• ADHD IS A DISORDER OF EXECUTIVE FUNCTION
• Selective attention
• Shifting mental sets
• Response inhibition
• Preparatory set
• Working memory
• EXECUTIVE FUNCTION DEFICITS ARE CAUSED BY LESIONS
  TO EF SYSTEM
• Substantia nigra
• Caudate nucleus
• Putamen
• Globus pallidus
• SAME STRUCTURES ARE DAMAGED BY MN NEUROTOXICITY
• SAME STRUCTURES ARE IDENTIFIED IN IMAGING STUDIES
  OF ADHD

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MECHANISMS OF Mn-INDUCED NEUROTOXICITY I

• ? Autoxidation of dopamine
• ? Catalysis of toxic catecholamines, e.g.,
  6-hydroxydopamine
• ? Free radicals, e.g., O2. and OH
• Mn2 oxidation ? Mn3, ? ? Lipid peroxidation of
  membranes

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MECHANISMS OF Mn-INDUCED NEUROTOXICITY II

• ? NMDA excitotoxic process
• ? Aberrant neuronal sprouting
• ? Compensatory imbalances among basal ganglia
  nuclei
• caudate
• putamen
• globus pallidus
• ? Calcium metabolism, ? synaptic transmission
• ? O-methyl transferase activity, ? homovanillic
  acid

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MECHANISMS OF Mn-INDUCED NEUROTOXICITY III

• ? iNOS mRNA, ? nitric oxide (NO), ? apoptosis
• ? glutamate/aspartate transporter (GLAST) mRNA,
• ? ? excitotoxic response in basal ganglia
  astrocytes
• ? metallothionein (MT-I) mRNA, ? sequestration of
  oxidants by metallothionein (MT-I)
• ? expression of transferrin receptor, ? influx of
  iron from blood to CSF, ? iron-induced oxidative
  stress in sensitive brain regions.

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RESULTS OF Mn-INDUCED NEUROTOXICITY

• Major feature of Mn is its ready transformation
  into several oxidative states
• 2H O2. Mn2 ? H2O2 Mn3
• Neurotoxic effect of Mn stems from aberrations of
  its regulatory role
• Chemical constituents of particular brain regions
  favor formation of higher valency Mn--lesions
  tend to occur in these areas
• substantia nigra
• globus pallidus
• putamen

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Observations on Mn in infants and children

• Manganese in head hair of children with ADHD may
  be the result of soy-based infant formulas
  (Collip et al., 1983)
• Term infants fed soy formula have significantly
  higher blood Mn than breast-fed infants
  (Kirchgessner et al., 1981 Lonnerdal, 1994)
• High, positive retention of Mn from formula, but
  not breast milk in preterm infants (Atkinson et
  al.)

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INFANT DIETARY MN INTAKE
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HYPOTHESES

• Since Mn is well absorbed from infant diets, and
  absorbed Mn is retained by the body, it will
  accumulate in brain, resulting in
• 1. Depleted striatal DA
• 2. Neuromotor delay
• 3. Executive function deficits

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• SUBJECTS male Sprague-Dawley rats
• TREATMENTS
• POST NATAL DAYS 1- 21
• ALL ANIMALS BREAST FED
• AND GAVAGED DAILY
• Control--0 ?g/d
• Low group--50 ?g/d
• Medium group--250 ?g/d
• High group--500 ?g/d
• POST NATAL DAYS 22-50 AND 55-65
• (Animals fed commercial chow ad lib)
• POSTNATAL DAYS 50 - 64
• Behavioral testing

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Study 1 Experimental design
Tissue Mn and Fe (AAS)
d1 d6 d10 d14 d20
d35 d58
d60
Control (0) 50 ug Mn/d 250 ug Mn/d 500 ug Mn/d
Passive Avoidance (d35)
Righting (d6)
Digging latency running time (d58)
Homing (d10)
Passive Avoidance (60-64)
Other measurements Hb and Wt
Mn levels chosen to reflect Mn levels in infant
formulas
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Mn Uptake in Suckling Rats
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Concentrations of Mn in tissues of rats killed at
day 14, 21 and 35
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Striatal Dopamine in Animals Killed at d35


Significant difference between control and low
Mn exposure
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Mn Exposure Affects Serotonin-Dopamine Balance
Plt0.03
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Behavior Assessment Homing Test (Day10)

• 20 min separation from home cage
• Nest clean (sawdust) bedding placed on opposite
  ends
• Time recorded for both of the pups forelimbs to
  cross the goal line (120 sec)

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Results of Homing Test
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PASSIVE AVOIDANCE TEST
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Results of Passive Avoidance Test at d32
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Study 2 Experimental design
Tissue Mn and Fe Assays (d14. d21. d35) Striatal
DA /5HT Assays (d35, d65)
Breast Supplemental Feeding
Regular Rat Chow
Control (0) 50 ug Mn/d 250 ug Mn/d 500 ug Mn/d
Passive Avoidance (d60-64)
Digging latency (d58)
d21
Other measurements Hb and Wt
Mn levels chosen to reflect Mn levels in infant
formulas
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Results of Burrowing Detour Test d55
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Results of Passive Avoidance Test, d63
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STRIATAL DOPAMINE LEVELS AT d65
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Study 3 Experimental design
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Mn-Fe Interactions

• Fe deficiency
• Most common nutritional deficiency world-wide
• Deficiency directly impairs behavioral and
  intellectual performance

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Manganese-Iron Interactions

• Effect of Mn on Fe
• High Mn decreases Fe absorption
• Effect of Fe on Mn
• Fe deficiency increases the absorption of Fe
  several-fold

Up-regulation of Fe absorption causes
several-fold increase in Mn absorption
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STRAIGHT RUNWAY LATENCY
0 ugMn/d 250 ugMn/d
500 ugMn/d
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PASSIVE AVOIDANCE LEARNING d64
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STUDY 4 NONHUMAN PRIMATE MODELS

• ADVANTAGES OVER RODENT MODEL
• Maturity of brain development at birth
• Prolonged period of postnatal brain development
• Complexity of behavioral repertoire
• Assessments similar to humans

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Study Design

• Subjects Male newborn rhesus monkeys
• Treatment Exclusively formula fed freom 0-4
  months of age
• Groups (n 8)
• Cows milk based infant formula, 0.03 µg Mn /ml
• Soy based infant formula, 0.3 µg Mn/ml
• Soy Mn soy based infant formula with added
  manganese, 1 µg Mn/ml

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Behavior testing schedule
dopamine drug challenge
impulsivity
delayed
nonmatch
to sample
CPT
position reversal
Formula feeding
diurnal activity
Gross motor maturation
1
2
1
4
7
8
1
0
1
1
1
3
15
16
9
1
2
3
4
5
6
0
17
18
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Gross Motor Maturation
Control
Soy
Soy Mn
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Gross motor maturationBehavior changes
0-3 changes/30 sec (median was 3/30 sec)
4 or more changes/30 sec
140
.01
120
120
.01
100
100
80
80
number of intervals (of max 150 )
number of intervals (of max 150 )
60
60
40
40
20
20
0
0
55
Duration of Wake and Sleep Periods48 h actimeter
monitoring period
WAKE
PAM actimeters
1200
1000
.01
.01
800
minutes
600
400
200
0
SLEEP
1200
1000
.01
.01
800
600
400
200
0
4 months
8 months
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Amount of activity
WAKE
120
100
80
Number of counts/ 2 min
60
40
20
0
SLEEP
14
12
10
.01
8
6
4
2
0
4 months
8 months
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WGTA
One-way mirror
Door on pulley
Sliding test board
58
Delayed nonmatch to sample
Test board 1
Test board 2
.12
.
.1
.
.08
Percent
.
.06
.
.04
.
.02
0
Balks-no sample choice made
59
Position reversals
Test board
sessions to criterion for learning
.05
6
60
Food reward
Test board
Sliding opaque cover
MOTOR IMPULSIVITY TEST
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Impulsivity-response inhibition
average number of trials (of 40) on which the
monkey responded at each interval
25
22.5
20
17.5
15
.04
12.5
10
7.5
.03
number of trials
5
2.5
0
0 1-6 7 balk
interval
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CANTABFixed intervaldopamine challengeContinuou
s performance test
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Dopamine drug challenge
Fixed interval responding
100
75
50
25
.01
0
0.1 mg/kg
0.2 mg/kg
Change in response rate from vehicle injection
amphetamine
-25
.0.3 mg/kg
-------apomorphine-----
-50
-75
.02
-100
haloperidol
haloperidol apomorphine
-125
-150
apomorphine, dopamine agonist,? response
rate haloperidol, dopamine antagonist, ? response
rate
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Social Interaction Study

• Method-videotape of dyadic interaction
• Familiar same group, unfamiliar same group,
  unfamiliar opposite group
• Social buffering
• Used previously to compare field cage with
  nursery reared males

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Dyadic social interaction
Low
2.25
16
2
14
1.75
12
1.5
.003
.002
.01
1.25
10
initiations/session
.03
duration
.003
.006
.09
.01
.03
1
.06
8
0.75
6
0.5
4
0.25
2
0
0
Rough play
cling
Chase play
Rough play
Chase play
ratio of rough play to total play initiations
0.8
0.7
.05
0.6
0.5
Control
Low
0.4
ratio
High
0.3
0.2
0.1
0
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dyadic interactions during round robin
socialization (16 sessions)
40
.01
35
30
.03
25
control
number of occurrences
Soy
20
.06
.003
SoyMn
.003
15
.003
10
5
0
Chase play
Rough play
cling
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Age and formula effects onCSF catecholamine
metabolites
5HIAA
HVA
500
160
450
control
140
400
low mn
120
350
hi mn
100
300
Cell Mean
250
Cell Mean
80
200
60
150
40
100
20
50
0
0
3 10 12
3 10 12
Months of age
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Relationship between CSF catecholamine
metabolites and dyadic interaction at 4 months of
age
HVA 4 months of age
5HIAA 4 months of age
450
400
350
300
250
200
Total duration of chase play
Frequency of cling behavior
150
100
50
0
-50
160
180
200
220
240
260
280
300
320
340
360
R2 0.271 P .02
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Relationship between CSF catecholamine
metabolites and impulsivity
5HIAA- 10 months of age
HVA- 10 months of age
45
45
40
40
35
35
30
30
25
25
Early responses
20
20
15
15
10
10
5
5
10
20
30
40
50
60
70
80
90
100
150
200
250
300
350
400
450
500
550
R2 0.19
R2 0.156
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Relationship between CNS catecholamine
metabolites and response to apomorphine
5HIAA-10 months of age
HVA-10 months of age
200
200
150
150
100
100
50
50
.change in response rate
0
0
-50
-50
-100
-100
-150
-150
150
200
250
300
350
400
450
500
550
10
20
30
40
50
60
70
80
90
100
R2 0.248 P .03
R2 0.173
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Prenatal Manganese Absorption Linked to Childhood
Behavioral Disinhibition Jonathon E. Ericson,
Francis M. Crinella, K. Alison Clarke-Stewart,
Virginia D. Allhusen, Tony Chan, andRichard T.
RobertsonUNIVERSITY OF CALIFORNIA, IRVINE
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THE TOOTH FAIRY STUDY

• Participants 27 children (11 boys) from the
  NICHD Study of Early Child Care and Youth
  Development
• Procedures
• Shed molars collected from 11 to 13 year old 400
  children 27 teeth randomly selected
• Measures of childrens behavioral disinhibition
  collected at ages 3 to 9 years.
• Tooth samples cross-sectioned by a diamond blade
  IMS analyses performed using the CAMECA IMS 1270,
  a high-resolution, high-sensitivity ion
  microprobe.
• Concentration of manganese in the molar cusp tip,
  an area near the dentine/enamel junction, formed
  at approximately the 20th gestational week, used
  as an indication of prenatal Mn absorption

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Tooth Enamel Biomarker

• Tooth enamel layers, like tree rings, provide a
  temporal record of mineral absorption
• Absorbed minerals, as reflected in the tooth
  enamel record, may be associated with
  embryogenetic variations
• Depending on corresponding embryological
  developments in CNS, Mn absorption, as reflected
  in tooth enamel record, may be associated with
  specific variation in behavioral outcomes

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Human Tooth Enamel

• Each tooth develops over a particular period
• Tissue laid down as incremental growth rings
• Oldest enamel at incisal tip
• Forming tissue is in equilibrium with blood
• Mature enamel is a metabolic isolate
• Mn stable in calcium hydroxyapatite

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Methods

• NICHD Early Childhood Study has measured
  behaviors of 1340 subjects for 11 years in 10
  sites distributed nationwide
• Subjects were healthy at birth
• Collected deciduous teeth of 400 subjects
• Randomly selected 27 teeth from 7 sites
• Enamel biomarker analysis of Mn at 20th week by
  ion microprobe mass spectrometer
• Statistical analysis of Mn concentration and
  behavioral measures

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Analytical Measurements

• ion microprobe mass spectrometer (ims)
• 10 - 35 um spot resolution
• auger sputter sample
• measurement of Mn concentration
• detection lt30 ppb
• 90 accuracy

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Behavior Battery

• Data base of NICHD Early Childhood Study
• Administered Age 3, Grade 1 and Grade 3
• Teachers, mothers, and standardize tests of
  subjects
• 21 behavior measures (disinhibition, intelligence
  and depression) over 5 years
• Same subjects maintain position

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Correlations Between Childhood Behavior and Mn
Deposited in Tooth Enamel at 20 weeks Gestation I
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Correlations Between Childhood Behavior and Mn
Deposited in Tooth Enamel at 20 weeks Gestation
II
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RESULTS

• Children with higher Mn scored higher on
  measures of disinhibition
• gt Play with Forbidden Toy (36 mo.)
• gt Impulsive errors on CPT (54 mo.)
• gt Impulsive errors on Stroop Test (54 mo.)
• gt Externalizing and attentional problems reported
  by teachers and mothers (1st and 3rd grades)
• gt Disruptive disorders (ADHD, hyperactivity/
• impulsivity, inattention reported by teachers
  (1st and 3rd grades)

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• MULTIPLE REGRESSION ANALYSIS
• (Predicting Mn Level With Behavioral Measures)
• CPT (54 months)
• Stroop (54 months)
• CBCL Inattention (1st grade)
• DBD3 HYPERACTIVITY (3RD GRADE)
• R .79 R2 0.62 df 4, 26 P lt .001
• Adding socioeconomic confounds did not increase
  significance
• Mothers education
• Income
• Ethnicity
• (F of change .13, p .97)

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Conclusion

• A link was demonstrated between prenatal Mn
  absorption, as reflected in Mn in tooth enamel
  tracing back to the 20th gestational week, and
  measures of behavioral disinhibition in later
  childhood
• Source(s) of absorbed Mn are unknown

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CONTRIBUTORS  

• University of California, Irvine
• Aleksandra Chicz-DeMet
• Louis Le
• Mike Parker
• Jonathon E. Ericson
• K. Alison Clarke-Stewart
• Virginia D. Allhusen
• Tony Chan
• Richard T. Robertson
• University of California, Davis
• Bo Lonnerdal
• Mari Golub
• Winyoo Chowanadisai
• Stacey Germann
• Casey Hogrefe
• University of California, San Francisco
• Trinh Tran

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SPONSORS  

• The Violence Research Foundation, Inc.
• San Clemente, California
• The John T. Wacker Foundation
• Dallas, Texas
• The Rehabilitation Center for Brain Dysfunction,
  Inc.
• Costa Mesa, California
• The John B. Zurlo Living Trust
• Los Angeles, California