Effetti neurocomportamentali dell’esposizione a interferenti endocrini in roditori di laboratorio

Daniela Santucci, Igor Branchi, Virginia Belloni*, Enrico Alleva

Reparto di Neuroscienze comportamentali,Dipartimento di Biologia cellulare e Neuroscienze

Istituto Superiore di Sanità, Roma*Dipartimento di Biologia animale e Genetica, Università di Firenze, Firenze

Endocrine Disrupters

Agenti esogeni che interferiscono con la produzione, il rilascio, il trasporto, il metabolismo, il legame, l'azione o l'eliminazione di ormoni che, naturalmente presenti nell'organismo, sono responsabili del mantenimento dell'omeostasi e della regolazione dei processi di sviluppo

Alterano le funzioni dei sistemi endocrino, nervoso e immunitario tumore della mammella ed endometriosi, tumore della prostata e del testicolo, sviluppo sessuale anomalo, riduzione della fertilità maschile, alterazioni nelle funzioni tiroidee e pituitarie, immunosoppressione, effettineurocomportamentali

comportamento

sistema nervoso ormoni metabolismo

Comportamento come bioindicatoreWarner et al. (1) first suggested the use of behavioral studies to assess the impact of environmentally important agents. Studies of pharmacological agents had been carried out earlier.

The rationale for behavioral studies consists of three main points.-First, the behavior of an organism represents the final integrated result of a diversity of biochemical and physiological processes. Thus, a single behavioral parameter is generally more comprehensive than a physiological or biochemical parameter.-Second, behavioral patterns are known to be highly sensitive to changes in the steady state of an organism. This sensitivity is one of the key values for its use in exploring sublethal toxication. -Last, behavioral measurements can usually be made without directphysical harm to the organism.

Environ Health Perspect 104(Suppl 2):331-335 (1996)Workshop on Risk Assessment Methodology for Neurobehavioral Toxicity convened by the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC)

OUR STOLEN FUTUREAre We Threatening Our Fertility,

Intelligence and Survival? - A Scientific Detective Story

T. Colborn, D. Dumanoski, J.P. MeyersAbacus, London, 1997

Effetti su popolazioni animali:• alterazioni comportamento riproduttivo e socio-sessuale del gabbiano reale,

Grandi Laghi (Fry e coll., 1987, Rattner e coll, 1984) [ DDT, PCB]

• alta mortalità nidiate aquila reale, Grandi Laghi (Colborn, 1991) [ DDT, PCB]

• malformazioni organi sessuali alligatori, Lago Apopka (Guillette e coll, 1995) [ Dicofol ]

Effetti sull’ uomo:

• correlazione deficit neurocomportamentali in bambini e consumo di pesce contaminato da parte madri, Lago Michigan (Jacobson e coll, 1996) [ PCB ]

From: Clotfelter, Bell and Levering, Animal Behaviour 2004

Sviluppo e validazione metodologie

test di laboratorio standardizzati eco-etologicamente compatibili

analisi dettagliata di selezionati stadi ontogenetici durante lo sviluppo

analisi accurata dei profili etologici

appropriati strumenti statistici

TOXICOLOGICAL APPROACH vs EDCs PARADIGMS

Chronic exposure

The effects may be latent, sometimes not observable foryears after the exposure

No “lowest dose” with no adverse effect (compounds thatmimic biological molecules will by definition exceed a

treshold level)

EDCs in the environment occur in conjunction with othercompounds (additive or synergistic?)

Crews et al., 2000

MULTI TIER-STRATEGY

Different levels of behavioural testing characterised by an increasing complexity of the responses scored

- the first-tiers consist in the assessment of simple behavioural and functional responses, such as reflexes, postures, motor coordination, sensory abilities, etc.

- the higher-tiers represent a more complex behavioural approach, including testing of learning and memory, attention and anxiety

a) SENSORY AND MOTORto exploit the development at specified postnatal ages of several reflexes and early responses to standard sets of stimuli

b) EMOTIONAL REACTIVITYto investigate ontogeny of emotionality

c) SPONTANEOUS ACTIVITY and EXPLORATORY ACTIVITYto measure general activity, exploratory activity and the display of specific behavioral patterns

d) SOCIAL INTERACTIONSto evaluate social behavior

e) LEARNING AND MEMORYto investigate cognitive functions

BEHAVIORAL ENDPOINTS

- developmental batteries have been designed to include a number of endpoints that are representative of various components of neural and behavioral development in the first postnatal weeks. This experimental paradigm is thought to assess both reflexes and simple behavioral responses

- it is also essential to perform a parallel assessment ofsomatic developmental endpoints, including at least body weight gain and time of eye opening, ear opening, and incisor eruption

SENSORY AND MOTOR DEVELOPMENT

attività sperimentale

PCB

PBDE

Bisfenolo A

Atrazina

ATRAZINA: 2 cloro-4 etilammino-6 isopropilammino-1.3.5 triazine

Membro clorinato della famiglia delle triazine, è un erbicida pre-e post-emergenza, che agisce interferendo con il processo fotosintetico ed è attualmente uno dei piùampiamente utilizzati in agricoltura ed in selvicoltura (Friedmann 2002).

Introdotta nel 1958 come erbicida per il controllo di infestantiappartenenti al gruppo delle dicotiledoni e utilizzata prevalentemente

nelle coltivazioni di mais, sorgo e canna da zucchero. E' un erbicida ad adsorbimento radicale e, in misura minore, fogliare. Viene trasportato

per via xilematica e agisce a livello del fotosistema II, inibendo il trasporto di elettroni.

Il suo impiego è stato vietato su tutto il territorio nazionale dal 1992 (con Decreto del Ministero della Sanità del 18 marzo 1992, n.705/910) dopo diversi episodi di contaminazione di falde idriche, ma viene ancora prodotta ed utilizzata in molti paesi Europei e negli Stati Uniti (più di 38 milioni di Kg venduti ogni anno).

stabile a ph ambientale

incolore

solubilità non molto elevata

elevata persistenza ambientale

decadimento provocato da esposizione alla luce e microorganismi

DECADIMENTO:• provocato da esposizione alla luce e microorganismi

• al suolo di circa 100 giorni alla temperatura di 20°C

• maggiore è la profondità maggiore è il tempo di dimezzamento

(Navarro et al. 2004)

LIMITI DI CAUTELA:

• Organizzazione Mondiale Sanità: 3 μg/lt

• Legislazione italiana: 0,1 μg/lt

Atrazine Concentration(μg/L)

Rain water 0.01– 0.4

Groundwater 0.01– 6

Tap water 0.01-5

Effetti potenziali:

Ermafroditismo in anfibi (Hayes 2002, 2003); diminuzione

peso corporeo (Narotsky et al 2001, Cummings et al.2000);

inibizione spermatogenesi; aumento triiodotironina

(Friedmann 2002, Stoker et al. 2002); anomalie sistema

riproduttivo; interazione con processi metabolici (Cooper et

al. 1996, Laws et al 2000).

Comportamento

TRATTAMENTO- 2 cloro-4 etilammino-6 isopropilammino-1.3.5 triazine

Dal 14°giorno di gestazione (GD) al 21°giorno postnatale. Compostisomministrati giornalmente per via orale

TEST COMPORTAMENTALE

- scale di fox (PND 2.4.9.12-15)- analisi vocalizzazioni (PND 4.9.12)- open-field e novelty-test (PND 16)- social interaction-test (PND 31)- passive avoidance (PND 60)

DOSE- 1 µg/kg/day (ATB)

- 100 µg/kg/day (ATA)

ANIMALETopo ceppo Swiss-CD-1 (maschi e femmine)

Disegno Sperimentale

- analisi parametri riproduttivi (sex ratio. peso. n°piccoli per nidiata. durata gestazione)

NEUROBEHAVIORAL ENDPOINTS OF POSTNATAL SENSORY AND MOTOR DEVELOPMENT

a) Righting reflex, pup returns to its feet when placed on its back;

b) Cliff aversion, pup withdraws from the edge of a flat surface when its snout and forepaws are placed over the cliff;

c) Forelimb and hindlimb stick grasp reflex, pup grasps a toothpick when it is touched to the palm of each paw;

d) Strong and weak tactile stimulation tests, a headturning response istriggered by the application of tactile stimuli in the perioral area on both sides of the head;

e) Pole grasping, pup grips a wooden pencil with its forepaws;

NEUROBEHAVIORAL ENDPOINTS OF POSTNATAL SENSORY AND MOTOR DEVELOPMENT

f) Level or vertical screen test, pup holds onto a wire mesh (5 x 5 mm) when dragged across it horizontally or vertically by the tail;

g) Screen climbing test, pup dimbs up the vertical screen using both fore- and hindpaws;

h) Auditory startle response, pup shows a whole-body startle response when a loud clap of the hands occurs less then 10 cm away;

g) Vibrissa placing reflex, pup places its forepaw on an object stroked across its vibrissae;

PESO

PND2PND2 PND4PND4 PND9PND9 PND12PND12

ControlloControllo (11)(11) 1.91±0.04 2.90±0.05 6.07±0.07 7.80±0.08

ATB (10)ATB (10) 1.76±0.03* 2.92±0.05 5.96±0.08 7.78±0.11

ATA (11)ATA (11) 1.76±0.02* 2.90±0.04 6.01±0.07 7.90±0.08

0,0

0,5

1,0

1,5

2,0

2,5

Maschi Femmine

Peso

(gr)

Controllo ATB ATA

*

*

*

*

RightingRighting CliffCliffaversionaversion

ForepawForepawgraspgrasp** # ** # §§

Auditory Auditory startlestartle

EyesEyesopeningopening

EarsEarsopeningopening

MalesMales

Controllo(11)

4.2 ± 0.4 4.2 ± 0.4 7.5 ± 0.5 11.4 ± 0.4 14.9 ± 0.1 14.3 ± 0.1

ATB (10)

4.6 ± 0.7 4.1 ± 0.5 5.2 ± 0.5 11.2 ± 0.4 14.6 ± 0.2 14.0 ± 0.1

ATA (11)

6.3 ± 0.7 3.5 ± 0.2 5.2 ± 0.5 11.2 ± 0.4 14.9 ± 0.1 14.3 ± 0.1

AA * #* #

FemalesFemales

Controllo(11)

4.7 ± 0.7 4.4 ± 0.4 6.0 ± 0.6 11.7 ± 0.3 14.9 ± 0.1 14.3 ± 0.1

ATB (10)

5.0± 0.7 4.2 ± 0.4 6.8 ± 0.6 10.4 ± 0.4 14.9 ± 0.1 14.2 ± 0.1

ATA (11)

6.8 ± 0.7 4.3 ± 0.5 6.6 ± 0.6 10.7 ± 0.4 15.0 ± 0.0 14.3 ± 0.1

CRESCITA SOMATICA E SVILUPPO NEUROCOMPORTAMENTALE (pnd 2CRESCITA SOMATICA E SVILUPPO NEUROCOMPORTAMENTALE (pnd 2--18)18)

* effetto significativo (p<0.05). ** effetto significativo (p<0.01). A effetto marginalmente significativo (0.5< p<0.1); # controllo vs ATB. § controllo vs ATA. δ ATB vs ATA. I dati rappresentano il giorno di rispostra adulta (medie ± S.E).

ULTRASONICVOCALISATION

1

3

4

5

6

1) microphone (B&K, 4135)2) preamplifier (B&K, 2633)3) tunable band-pass filter, K-H 3500) 4) measuring amplifier (B&K, 2610)

2

4) Tape-Recorder (Racal Store 4DS)

5) Personal computer with "Duetto" software(developed by Dr. Gianni Pavan, University of Venice)

ADVANTAGES of ANALYSING ULTRASONIC VOCALISATION RATE in INFANT RODENTS

i) UVZs are one the few responses produced by very young mice that can be quantitatively analysed and be elicited by quantifiable stimuli;

ii) mainly, UVZs are produced from birth to PND 14-15, thus allowing longitudinal neurobehavioural analysis during very early postnatal ontogeny;

iii) this technique has the advantage of minimal-animal-handling request.

ONTOGENETIC PROFILE of ULTRASONIC VOCALISATION PRODUCTION*

1 5 7 9post-natal day

3 11 13

50

100

0

mea

n nu

mbe

r of c

alls

/1 m

in

*CS-1 miceElwood & Keeling, 1982

21 msec

CONSTANT FREQUENCY MODULATED FREQUENCY

FREQUENCY STEPS COMPOSITE

50

100

kHz

50

100

kHz

TEMPERATURA CORPOREA

MaschiMaschi PND4PND4 PND9PND9**

PND12PND12

ControlloControllo (11)(11) 31.5±0.27 34.1±0.24 35.5±0.29

ATB (10)ATB (10)30.9±0.38 32.4±0.23* 35.8±0.34

ATA (11)ATA (11) 32.0±0.33 33.5±0.25 36.1±0.32

FemmineFemmine

Controllo (11)Controllo (11) 31.0±0.26 34.2±0.28 35.9±0.33

ATB (10)ATB (10) 31.6±0.35 32.7±0.19* 35.9±0.33

ATA (11)ATA (11) 31.8±0.34 33.5±0.26 35.9±0.35

Effetti esposizione prenatale all’atrazina sulla temperatura corporea in topi neonati (PND4-12). I dati sono espressi come medie±es. * p<0.5, ** p<0.01

28

32

36

4 9 12Giorno postnatale

°C

Controllo ATB ATA

**

VOCALIZZAZIONI (rate)

MaschiMaschi PND4PND4 PND9PND9 PND12PND12ControlloControllo (11)(11) 157±15 266±20 97± 18

ATB (10)ATB (10) 172± 18 223±30 98±19

ATA (11) 132± 14 276±19 123±25

FemmineFemmineControllo (11)Controllo (11) 165±19 234±22 126± 22

ATB (10)ATB (10) 166± 16 192±29 85±23

ATA (11)ATA (11) 136± 20 227±24 121±22

0

100

200

300

4 9 12Giorno postnatale

Num

ero

Controllo ATB ATA

TotaleTotale Controllo (6 )Controllo (6 ) ATB (7 )ATB (7 ) ATA (7 )ATA (7 )

Durata (ms) 45.0±2.0 45.0±2.0 47.0±3.0

Interpulse (s) 0.68±0.09 1.57±0.63 0.61±0.08Picco di frequenza (kHz) * 55.0±0.8 55.9±1.1 58.7±0.7

FemmineFemmine

Durata (ms) 44.0±2.5 44.0±3.7 47.0±4.9

Interpulse (s) 0.81±0.15 1.50±0.44 0.65±0.11

Picco di frequenza (kHz) 55.2±1.3 54.0±1.8 59.5±1.0

Maschi Maschi

Durata (ms) 46.0±3.4 45.0±2.6 47.0±3.3

Interpulse (s) 0.62±0.09 1.76±1.20 0.62±0.12

Picco di frequenza (kHz) 54.9±0.9 57.8±1.2 58.0±0.8

CARATTERISTICHE SONOGRAFICHE DELLE EMISSIONI ULTRASONICHE (pnd CARATTERISTICHE SONOGRAFICHE DELLE EMISSIONI ULTRASONICHE (pnd 9)9)

Durata

0

18

36

54

Maschi Femmine

ms

Picco di frequenza

0

20

40

60

Maschi Femmine

Freq

uenz

a (k

Hz)

*

Interpulse

0

1

2

3

Maschi Femmine

sec

CON ATB ATA

Trattamento Sesso Trattamento x Sesso

Durata (ms) F= 0,17 ns

F= 0,81 ns

F= 0,44 ns

Interpulse (s) F= 1,78 ns

F= 0,00 ns

F= 0,59 ns

Picco di frequenza (kHz)F= 2,11

nsF= 0,59

ns F= 3,80 p<0,05

OPEN FIELD TEST (10 min)

- thigmotaxis (time spent in contact with the walls)

- crossing

- grooming (e)

- exploratory sniffing

- jumping

- immobility (f)

- rearing (b), wall rearing

- urination and defecation

NOVELTY TEST (5 min)

- Sniff object

EFFETTI DELL’ESPOSIZIONE PERINATALE ALL’ATRAZINA SUL COMPORTAMENTO NON SOCIALE DEL TOPO IMMATURO

OPEN FIELD TEST

-rearing (wall rearing and non-supported)

- grooming

- exploratory sniffing

- freezing

- lying still (without freezing)

- thigmotaxis (time spent in contact with the walls)

- stereotypes (i.e., increase to above a zero or very low control level of acts such as face washing, gnawing, circling, jamping, head scanning, and focused sniffing)

- digging and push digging (if the floor is covered with bedding material)

-urination and defecation

NOVEL OBJECT

-latency to the first approach

-frequency and duration

TrattamentoTrattamento(df=2,29)

BloccoBlocco(df=2,58)

Trattamento x BloccoTrattamento x Blocco(df=4,58)

F p F p F p

Crossing0,06 ns 3,52 <0,04 3,06 <0,02

Wall rear (f)0,18 ns 2,63 ns 2,75 <0,04

Wall rear (d)0,06 ns 3,12 <0,05 2,27 ns

Explore (f)0,79 ns 17,3 <0,00 2,77 <0,04

Explore (d)3,76 <0,04 # 22,6 <0,00 1,19 ns

Rearing (f)0,75 ns 5,51 <0,01 0,88 ns

Rearing (d)0,28 ns 4,44 <0,02 1,03 ns

Grooming (f)0,32 ns 1,94 ns 1,82 ns

Grooming (d)1,13 ns 1,19 ns 0,94 ns

TWO-WAY ANOVA APPLICATA ALLE MISURE COMPORTAMENTALI REGISTRATENELL’OPEN FIELD TEST NEL TOPO IMMATURO (PND 16)

df: gradi di libertà; n.s: non significativo; (f): frequenza; (d): durata. # ATB vs CON

Wall rearing

0

25

50

75

100

0-200 200-400 400-600

Dur

ata

(s)

Con Atrl Atrh

Crossing

0

25

50

75

100

0-200 200-400 400-600

Fre

quen

za

Explore

0

30

60

90

120

0-200 200-400 400-600

Dur

ata

(s)

Self grooming

0

50

100

150

0-200 200-400 400-600

Dur

ata

(s)

Explore

0

10

20

30

0-200 200-400 400-600

Fre

quen

za

Wall rearing

0

25

50

75

100

0-200 200-400 400-600

Dur

ata

(s)

Crossing

0

25

50

75

100

0-200 200-400 400-600

Fre

quen

za

Explore

0

30

60

90

120

0-200 200-400 400-600

Dur

ata

(s)

Se

Dur

ata

(s)

lf grooming

0

50

100

150

0-200 200-400 400-600

Explore

0

10

20

30

0-200 200-400 400-600

Fre

quen

za

*

*

*

Con, controllo; Atrl,, atrazina bassa dose; Atrh, atrazina alta dose * p< 0.5

MASCHI FEMMINE

COMPORTAMENTO ESPLORATIVO VERSO NOVEL OBJECT (pnd 16)

0

70

140

210

280

Late

nza

Controllo ATB ATA

0

5

10

15

Dur

ata

(s)

* *

0

2

4

6

Freq

uenz

a

*

EFFETTI DELL’ESPOSIZIONE PERINATALE ALL’ATRAZINA SUL COMPORTAMENTO SOCIALE DEL TOPO IMMATURO

valutazione profilo comportamento sociale (PND 31):

- dipendente dall’età- sessualemente dimorfico

SOCIAL

Sniffing: area anogenitale, muso o corpo del copmpagno

Follow: seguire il partner

Mutual circle: i partners si annusano reciprocamente nell’area anogenitale

Squire: seguire il partner mantenendo un contatto con il muso

Push under, push past, crawl over, crawl under, allogrooming

NON-SOCIAL

Exploring: rearing, sniffing the air

Digging, self-grooming

SOCIAL BEHAVIOR- the investigation of social behavior in animals presents several concerns that are not usually present when studying individual subjects

- the assessment of social behavior profile at successive developmental phases is important since it is markedly age-dependent

- sexually dimorphic

- two kinds of social interactions during development:

Automated data collection is impractical at best and usually necessitate the use of observational (i.e., ethological) methodologies.

a) mother-offspring b) between individuals of same age

COMPORTAMENTI AGGRESSIVI

Aggressive grooming (allogrooming persistente e vigoroso),

Attack (d), offensive up-right (a) (animale assume posizione

verticale e fronteggia in modo aggressivo il compagno);

COMPORTAMENTI DIFENSIVI

Defensive up right (animale assume posizione verticale e respinge attacco

del partner con le zampe anteriori);

Crouch (c) (postura assunta da un’animale impaurito),

Withdraw (movimenti di allontanamento dal compagno).

Fattore 1esplorazione

sociale

Fattore2esplorazione non-sociale

Fattore 3up-right postures

Fattore 4a /difensivo

Fattore 5agonistico

Fattore 6

Squire 0.9Genital sni ff 0.9Follow 0.7Allogrooming 0.5

Exploring 0.9Wall rearing 0.9Sniff 0.7

Up rightoffensive 0.9Up right defensive 0.9

Withdraw 0.9Aggressive groom 0.9

Attack 0.9Jump 0.8

Self grooming 0.8Nosesniff 0.7

22.1. 39.4 .61.91 3

.Varianza % 22. 17. 12 5 10.0 8.5 7.7Varianza cumulativa % 51.9 70.4 78.1

Extraction Method: Principal Component Analysis. Rotation Method: Varimax with Kaiser Normalization

Maschi

Femmine

Trattamento Inrtervallo Trattamento x Inter vallodf= 2,26 df= 2,52 df= 4,52

F p F p F pfattore 1: esplorazione sociale 6.26 0.006 # 5.92 0.011 2.36 nsfattore 2: : esplorazione non sociale 3.60 0. 042§ 47.6 0.000 1.71 nsfattore 3: up right postures 1.13 ns 0.11 ns 1.05 nsfattore 4: aggressivo- difensivo 3.10 ns 1.81 ns 0.57 ns

df gradi di libertà; livello di significatività p <0.5. # ATB vs Controllo § ATA vs Controllo,

Trattamento Inrtervallo Trattamento x Inter vallodf= 2,26 df= 2,52 df=4,52

F p F p F pfattore 1: esplorazione sociale 0.21 ns 1.65 ns 1.24 nsfattore 2: esplorazionenon sociale 0,05 ns 64.8 0.000 0.17 nsfattore 3: up right postures 1.23 ns 1.06 ns 3.82 nsfattore 4: aggressivo- difensivo 3.35 ns 0.81 ns 0.83 ns

df gradi di libertà; livello di significatività

Esplorazione sociale

0

5

10

15

20

0-5 12-17 25-30

Freq

uenz

aCONATLATH

Maschi

Esplorazione sociale

0

5

10

15

20

0-5 12-17 25-30

Freq

uenz

a

Femmine

Esplorazione non sociale

0

25

50

75

100

125

0-5 12-17 25-30

Freq

uenz

a

Esplorazione non sociale

0

25

50

75

100

125

0-5 12-17 25-30

Freq

uenz

a

**

Medie ed Errore Standard della somma delle frequenze dei comportamenti che caratterizzano il fattore CON,controllo; ATL, atrazina bassa dose, ATH, atrazina alta dose. *p<0.5

EFFETTI DELLEFFETTI DELL’’ ESPOSIZIONE PERINATALE ALLESPOSIZIONE PERINATALE ALL’’ ATRAZINA SU ATRAZINA SU RISPOSTA IN PASSIVE AVOIDANCE NEL TOPO ADULTORISPOSTA IN PASSIVE AVOIDANCE NEL TOPO ADULTO

• test ha inizio il 60°PND, caratterizzato da 2 fasi intervallate da un periodo di 24 ore

- 1°giorno learning session: multi trials test (max 5 prove)

- 2°giorno retention test: unico trial (retest)

pF p F

§

x Sesso Trattamento Sesso Trattamento

df= 2,106 df= 1,106 df=4,52

4.5Latenza learning session 3.04 <0.04 <0.04

F p

0.19 ns

0

40

80

120

Trial1 Trial2 Trial3 Trial4 Trial5

Late

nza

(s)

CON ATB ATA

*

0

20

40

60

Retest

Late

nza

(s)

0

40

80

120

Trial1 Trial2 Trial3 Trial4 Trial5

Late

nza

(s)

0

40

80

120

Trial1 Trial2 Trial3 Trial4 Trial5

Late

nza

(s)

0

20

40

60

80

Femmine Maschi

Late

nza

(s)

Femmine Maschi

•Conclusioni (atrazina)

•no effetto su parametri riproduttivi e sviluppo morfologico

riduzione peso alla nascita effetto sullo sviluppo intrauterino del feto

• ritardo righting (capacità di coordinamento motorio ed orientamento)

accelerazione forepaw, grasping, auditory startle (comparsa di capacità sensoriali)

≠ in maschi e femmine (diverso substrato ormonale)

trattamento con atrazina influenza il profilo di vocalizzazione ultrasonica nel topo neonato:

al PND9 numero vocalizzazioni e temperatura ridotti in ATB

• lievi ma significative alterazioni caratteristiche sonografiche

alterazione in termini di deficit

competenza/relazione madre-prole

trattamento con atrazina influenza il comportamento non sociale:

crossing, exploring in entrambi i sessi profilo di abituazione alterato

• aumento durata e frequenza exploring object ma non latenza

• ≠ fra maschi e femmine nella risposta alle due dosi

trattamento con atrazina influenza il comportamento sociale:

• effetto a lungo termine ATB e ATA

• nei maschi una femminilizzazione della componente investigativa e affiliativa

• l’ipotesi di un’azione (estrogenica indiretta?) dell’atrazina in fase organizazzionale del SNC

• interazione con processi di sintesi e funzionalità dei neurotrasmettitori osservata sia in maschio che in femmina di ratto (Rodriguez et al. 2005; Canonaco et al. 2006)

• interagisce con attività aromatasi inducendo sintesi estrogeni a spese degli androgeni (Sanderson 2000)

• atrazina interferisce con i percorsi ontogenetici durante fasi sensibili dello sviluppo

• Effetti esposizioni a basse dosi, nel range delle dosi a cui uomini e animali non umani sono esposti nell’ambiente (NOAEL, no observed adverse effect level, (1.8 mg/kg/gg); LOAEL, lowest observed adverse effect (3.6 mg/kg/gg)

• comportamento rappresenta l’end-point di sistemi integrati essendo quindi un buon biomarcatore di alterazioni anche lievi in uno di tali sistemi

• nella valutazione degli effetti osservati è importante considerare non solo lo sviluppo del comportamento ma anche il ruolo del comportamento nello sviluppo (Laviola & Terranova 1998).

Appropriate behavioural tests and adequate statistical tools may help to establish the ED properties of a given compound by pointing out short-, medium-, and long-term alterations in selected behavioural endpoints

Eco-etho-toxicology provides powerful tools for monitoring changes or alterations in behavioural patterns or endpoints that are rare or difficult to observe under natural circumstances

Conclusions coming from laboratory data are more easily interpreted if validated in nature as is the case for the field observations for potential adverse effects of EDCs

Complement to epidemiological data

Contribute to the rational designing of further studies

Francesco Dessì Fulgheri

Massimo Pandolfi

Marcello Canonaco

Elena de Felip

Elsa Traina

Antonella Olivieri

Emanuela Testai

PCBs are mixtures of synthetic organic chemicals with the same basic chemical structure and similar physical properties ranging from oily liquids to waxy solids. Due to their non-flammability, chemical stability, high boilin point and electrical insulating properties, PCBs were used in hundreds of industrial and commercial applications including electrical, heat transfer, and hydraulic equipment; as plasticizers in paints, plastics and rubber products; in pigments, dyes and carbonless copy paper and many other applications. More than 1.5 billion pounds of PCBs were manufactured in the United States prior to cessation of production in 1977.

Welcome to the PCB Home Page at EPA

From: T. Colborn, D. Dumanoski, J.P. Meyers (1997), Our Stolen Future: Are We Threatening Our Fertility, Intelligence and Survival?

2,2',6,6' - dichlorobiphenyl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

PCB 54

3,3',4,4'- dichlorobiphenyl

PCB 77

Policlorobifenili, PCBs

Cl Cl

Cl

Cl Cl

2,2' - dichlorobiphenyl

Di-ortho-PCB

Cl Cl

ClCl

Cl

Cl

2,3',4,4',5 - dichlorobiphenyl

Mono-ortho-PCB

3,3',4,4'- dichlorobiphenyl

Co-planar-PCB

PERSISTENTIUBIQUITARILIPOFILICI

Experimental Design (1)

TREATMENT- post natal-day 2 and 4, subcutaneously

DOSES- 0.8, 8 mg/Kg

ANIMAL- CD-1 Swiss outbred mice (males and females )

TREATMENT- 3,3’,4,4’ or 2,2’,6,6 tetrachlorobiphenyl

2,2',6,6' - tetrachlorobiphenyl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

3,3',4,4'- tetrachlorobiphenyl

PCB 54

PCB 77

NEUROBEHAVIOURAL TESTS- sensorimotor development (pnd 0-12, Fox scale)- ultrasonic vocalization (pnd 5, 8, and 11)- spontaneous activity (open-field, pnd 19)- passive avoidance task (pnd 20-21)- social interactions (pnd 40)- aggressive behaviour (pnd 90)

- NGF and testosterone levels (day 90)- Chat activity in the hippocampus (day 90)

SOCIAL INTERACTIONS (juveniles, day 41 )

Sniffing: anogenital, head, or the snout of the partner

Follow: following the partner around the cage

Mutual circle: partners mutually sniffing each other's anogenital region

Social inactivity: lying flat or standing still (with eyes closed or open) while maintaining close physical contact with the partner

Push under: pushing the snout or the whole anterior part of the body under the partner's body

Crawl over: crawling over the partner's back

NON-SOCIAL

Exploring: moving around the cage, rearing, sniffing the air

Digging: digging in the sawdust, pushing and kicking it around

Self-grooming: wiping, licking, combing, or scratching any part of its own body

AGGRESSIVE BEHAVIOUR (adults, day 90)

Attack: number of fighting episodes initiated by the experimental mouse

Aggressive grooming: violent grooming of the animal on the back of the partner

Rattling: species-specific rattle-snake like movement of the tail

Offensive upright posture: mouse stood on its hind legs and faced theopponent aggressively

Defensive upright posture: animal's standing on its hind legs pushing the aggressive opponent with its forepaws

Submission posture: animal lies on the ventral part of the body

Freezing: refers to the animal's physical immobility

0

0.4

0.8

1.2

1.6

2.0

COMPOSITE

0

4

8

12

16

FREQUENCY STEPS

ultr

asou

nds/

min

0

4

8

12

16

SHORT

0

4

8

12

16MODULATED FREQUENCY

T

0

0.4

0.8

1.2

1.6

2.0CONSTANT FREQUENCY

Vehicle

Pcb77 0.8 mg/Kg

Pcb54 0.8 mg/KgPcb54 8 mg/Kg

Pcb77 8 mg/Kg

ultr

asou

nds/

min

ultr

asou

nds/

min

ULTRASONIC VOCALISATIONS FOLLOWING PERINATAL EXPOSURE TO PCB (day 8)

100

120

140

0

20

40

60

80

wall rearing

0

10

20

30

40

50

60

rearing

0

5

10

15

20

25

30

35

40

45

50

0

25

50

75

100

125

150

175

200

225

wall rearing

rearing

dura

tion

(sec

)

freq

uenc

y

dura

tion

(sec

)

freq

uenc

y

control

pcb 54 (0.8 mg/Kg)pcb 54 (8 mg/Kg) pcb 77 (8 mg/Kg)

pcb 77 (0.8 mg/Kg)

OPEN FIELD (juveniles)

0

20

40

60

80

100

120

140

160grooming

0

2

4

6

8

10

12

0

5

10

15

20

25

30

35jumpingcrossing

0

250

300

350

400

450

grooming

control

pcb 54 (0.8 mg/Kg)

dura

tion

(sec

)

freq

uenc

y

freq

uenc

y

freq

uenc

y

pcb 54 (8 mg/Kg) pcb 77 (8 mg/Kg)

pcb 77 (0.8 mg/Kg)

OPEN FIELD (juveniles)

*

control

pcb 54 (0.8 mg/Kg)pcb 54 (8 mg/Kg) pcb 77 (8 mg/Kg)

pcb 77 (0.8 mg/Kg)

EXP

TRANSITIONS BETWEEN BEHAVIOURAL ITEMS (juveniles)

1.0<RR<2.0 0.5<RR<1.0

DIG SEL

EXP

DIG SEL

EXP

DIG SEL

EXP

DIG SEL

High dose vs Control

PCB [54]

Low dose vs Control

Low dose vs Control

High dose vs Control

PCB [77]

Faster Slower

0

75

150

225

0

100

200

300

Offensive (total) Defensive (total)

Vehicle

Pcb77 0.8 mg/Kg

Pcb54 0.8 mg/Kg Pcb54 8 mg/Kg

Pcb77 8 mg/Kg

***

*

AGGRESSIVE BEHAVIOR (adults)

0

100

200

300

400

ControlPCB 54 (0.8 mg/kg)PCB 54 (8 mg/kg)PCB 77 (0.8 mg/kg)PCB 77 (8 mg/kg)

nmol

/hr/m

g of

pro

tein

Chat activity in the hippocampus

*

NEUROBIOLOGICAL MARKERS (adults)

POLYBROMINATED DIPHENYL ETHERS

Brominated flame retardants (BFRs) are a group of chemicals added to many products,including computers, TVs and household textiles, in order to reduce fire risk. Most concern has been expressed over polybrominated biphenyls (PBB), which have similar properties to PCBs, and whose use is fairly restricted, polybrominated diphenyl ethers (PBDEs). Because of their lipophilic characteristics and their persistence, PBDEs have become ubiquitous environmental contaminants.

Organohalogen compounds in human milk in Sweden

TREATMENTFrom post-natal day (PND) 1 to PND 21. Compounds have been administered daily to lactating mice by ‘non-gavage’ administration.

BEHAVIOURAL TESTS- open-field (PND 24)

DOSES- PBDE 9918, and 36 mg/kg/day

- Aroclor 1254 10 mg/kg/day

ANIMALCD-1 Swiss outbred mice (male and female)

HORMONAL ESSAYS- total and free thyroxine (PND 26)

NEUROCHEMICAL ANALYSIS- ChAT activity (PND 26)

Experimental Design

EYE OPENINGINCISOR ERUPTION HAIR GROWTH

SCREEN CLIMBING POLE GRASPING

2 6 8 10 12 14 16 18 204 2 6 8 10 12 14 16 18 204

SWIFT RIGHTING SLOW RIGHTINGGRASPING

2 6 8 10 12 14 16 18 204

SENSORI-MOTOR DEVELOPMENT

post-natal day

perc

enta

ge o

f adu

lt-lik

e re

s pon

s e

100

50

0

100

50

0

100

50

0

PBDE 99 LOW

PBDE 99 HIGH

AROCLOR 1254

PBDE 99 MEDIUM

VEHICLE*

* = main effect of treatment [treatment, Kruskal-Wallis, H (4)

= 8.91, p = 0.0194].

0

25

75

100

VEH A1254 LOW MED HIGH

THIGMOTAXIS (PND 60)

**

* = significant difference from controls, p < 0.05

an index of emotionality%

tim

e sp

ent n

ear

the

wal

ls