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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
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