Effects of low doses of atrazine on the neurobehavioural development of mice

Effects of low doses of atrazine on the neurobehavioural development of mice

V. Belloni 1, E. AllevA 2, F. Dessì-Fulgheri 1,3, M. ZAccAroni 1 and D. sAntucci 2

1 Dipartimento di Biologia Animale e Genetica, Università di Firenze, Via Romana 17, I-50125 Firenze, Italy2 Sezione di Neuroscienze Comportamentali, Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Roma, Italy

Received 2 April 2007, accepted 18 July 2007

Recent studies suggest that atrazine (ATZ), a triazine herbicide widely used and frequently detected in ground and surface water, is able to affect estrogen production by inducing aromatase, the enzyme that converts androgen into estro-gen, an essential transformation occurring at the CNS level for maturation and expression of behaviour. In the present study we evaluated the effects of environ-mentally relevant doses of ATZ on somatic growth and early behavioural ontog-eny, a crucial stage in shaping future behaviour. For this purpose we observed mice born to mothers exposed to 1 or 100 µg/kg ATZ during pregnancy and lacta-tion. We studied, between postnatal day 2 to 15, the righting reflex, cliff aversion, forepaw grasping, auditory startle, eyelid and ear opening, and ultrasound vocali-zations. In both sexes ATZ effects were evident, on body weight at birth, on the maturation of righting and grasping reflexes, and on the rate of emission and the spectrographic characteristics of ultrasound vocalizations. Dosage level appeared to be particularly relevant because, in some cases, the lower ATZ exposure was more effective than the higher one in modifying behavioural response. The latter finding suggests that this compound, similarly to many others endocrine disrup-tors, does not follow a linear dose-response curve, and that, as a consequence, its effects should be studied carefully at very low doses. Our results, compatible with ATZ properties, suggest caution in the use of a chemical agent that may, even at low doses, interfere with brain development and differentiation, inducing alterations of the developmental trajectories of behaviours.

key worDs: atrazine, neonatal behaviour, vocalizations, mice, endocrine disrup-tors, aromatase.

3 Reference author: Marco Zaccaroni, Dipartimento di Biologia Animale e Genetica, Università di Firenze, Via Romana 17, I-50125 Firenze, Italy (Tel: +39-0552288225, Fax: 055-222565, E-mail: marco. [email protected]).

Ethology Ecology & Evolution 19: 309-322, 2007

310 V. Belloni et alii

Introduction . . . . . . . . . . . . . . . . . 310Material and methods . . . . . . . . . . . . . . 311Results . . . . . . . . . . . . . . . . . . 314

Gestational parameters . . . . . . . . . . . . . 314Somatic growth and sensorimotor development . . . . . . . 314Ultrasonic vocalization . . . . . . . . . . . . . 314

Discussion . . . . . . . . . . . . . . . . . 318Somatic growth . . . . . . . . . . . . . . . 318Neurobehavioural development . . . . . . . . . . . 318

Conclusions . . . . . . . . . . . . . . . . . 320Acknowledgments . . . . . . . . . . . . . . . 320References . . . . . . . . . . . . . . . . . 320

INTRODUCTION

An increasing number of environmental contaminants have been shown to interfere with the synthesis and action of natural hormones, consequently alter-ing the function of the endocrine system, and acting as endocrine disruptors (ED). Among them atrazine (ATZ), a triazine herbicide, widely used in agriculture, with an estimated 60-120 million pounds applied per year in the U.S. (thurmAn & cromwell 2000), is one of the most dispersed worldwide.

Due to its high mobility through soil, ATZ is also an important contaminant of aquatic ecosystems (clArk & goolsBy 2000, hAyes et al. 2002). In fact, ATZ is frequently detected in ground and surface water at levels in excess of the maximum contaminant level (MCL) of 0.1 µg L-1 imposed by the European Economic Council (hAmilton et al. 2003) and of 3 µg L-1 imposed by the U.S. Environmental Protection agency (U.S. EPA 2005). The extensive use of ATZ has made this compound a focus for environmental impact studies. Recent reports indicate that in humans acute die-tary exposures range from 0.234 to 0.857 µg/kg/day, and corresponding figures for chronic dietary exposure are 0.046 to 0.286 µg/kg/day, when all commodities with U.S. EPA tolerances and drinking water are considered (gAmmon et al. 2005).

Recent studies of the effects of exposure to water-borne ATZ contamination on frogs revealed that ATZ is able, at low, environmentally relevant concentrations, to affect developmental and reproductive processes, retarding development and caus-ing hermaphroditism in exposed males (cArr et al. 2003; hAyes et al. 2002, but see also coADy et al. 2005). In addition, several studies implicated the CNS as the pri-mary target site of ATZ’s effects on reproductive function (cooper et al. 1999, stok-er et al. 2000, roDrigueZ et al. 2005).

ATZ increases estradiol circulating levels in rodents (stevens et al. 1994, elDriDge & wetZel 1999) and is associated with estrogen-like activity and estrogen-dependent effects in fish (moore & wAring 1998, spAno et al. 2004), amphibians (hAyes et al. 2002, cArr et al. 2003, Du preeZ et al. 2005), reptiles (keller & mcclel-lAn-green 2004), and birds (mAtsushitA et al. 2006). ATZ also increases estrogen-sensitive reproductive cancers in rodents (elDriDge & wetZel 1999) and has been associated with similar estrogen-sensitive reproductive cancers in humans (kettles et al. 1997, mAclennAn et al. 2003). The availability of estradiol is due to the con-version of androgen into estrogen by aromatase cytochrome enzyme P450, produced by the CYP19 gene (simpson et al. 1994). The ability of ATZ to modulate aromatase activity has been demonstrated in reptiles (keller & mcclellAn-green 2004) and human cells (sAnDerson 2001, FAn et al. 2007). Moreover, it should be noted that the

311Effects of atrazine on behaviour

disrupting effects of ATZ may result from a combination of androgen depletion and increased estrogen availability. These activities may result in an alteration of some developmental trajectories of behaviour: it is well known that sexual steroids, testo-sterone and estradiol in particular, at CNS level, are essential both for the maturation and adult expression of sexual behavioural patterns (ArnolD & gorski 1984).

Recent studies also revealed that ATZ is able to alter brain dopaminergic sys-tems of rats (roDrigueZ et al. 2005) and somatostatine expression in hypothalamic and extrahypothalamic areas of mouse brain (giusi et al. 2006). Collectively, these studies support the view that ATZ can produce detrimental effects in systems criti-cal for the mediation of movement as well as for cognition and executive functions.

Appropriate behavioural tests represent a valuable index for establishing the properties of EDs by revealing alterations to selected behavioural endpoints (AllevA et al. 1998).

The aim of the present work was to study possible neurodevelopmental effects of perinatal exposure to low, environmentally relevant doses of ATZ in mouse pups.

Specifically, we considered somatic parameters and a wide range of reflexes that together provide a complete picture of the level of sensorimotor maturation (Fox 1965). In addition, we recorded and analyzed ultrasonic vocalizations (USVs) emitted by the pups under brief isolation distress. Newborn rodents isolated from dam and littermates emit ultrasounds which are considered a measure of “separa-tion distress” (hoFer & shAir 1978) and generally the rate of ultrasound calling fol-lows an ontogenetic profile, increasing during the first week of life, reaching a peak around postnatal day 8, after which it start to decrease (elwooD & keeling 1982). The pattern of USVs has proved to be a reliable, sensitive indicator of changes in the normal ontogenetic course brought about by a variety of perinatal treatments (sAntucci et al. 1993, BrAnchi et al. 2001).

For this purpose we administered orally ATZ to mouse CD-1 dams, during ges-tation and lactation and measured somatic growth, reflex development and USVs in their offspring.

MATERIAL AND METhODS

Animals and breeding procedure

Mice of an outbred Swiss-derived strain (CD-1) were purchased from a commercial breed-er (harlan, Italy). Upon arrival at the laboratory, the animals were kept in an air-conditioned room (temperature 21 ± 1 °C, relative humidity 60 ± 10%; lights on from 20.30 to 8.30 hr), in 42 × 27 × 14 cm Plexiglas cages with a metal top and sawdust (Scobis 1, Mucedola, Settimo Milanese, Italy) as bedding. Pellet food (Enriched standard diet purchased from Mucedola, Settimo Milanese) and tap water were continuously available. Adequate measures were taken to minimize pain or discomfort during of all phases of the animal experimentation.

Breeding pairs were formed and housed in 33 × 13 × 14 cm Plexiglas cages. The females were inspected daily for the presence of a vaginal plug (Gestation Day 0).

On gestation day (GD) 12, males were removed and females housed singly in Plexiglas cages. Subsequently, GD 14 females were randomly assigned to one of three treatment groups: atrazine high dose (ATh; n = 11) which received 100 µg/kg/day of atrazine: atrazine low dose (ATL; n = 10) which received 1 µg/kg/day of atrazine; control (n = 11), which received the vehicle (corn oil).

ATZ (2-chloro-4-ethylamino-6-isopropylamino-s-triazine, Sigma Aldrich, CAS# 1912-24-9, 98.4% purity) was dissolved in corn oil and administered daily to female mice by self-


administration drinking from a modified syringe. The amount of solution given to the dams was 0.002 ml/g of body weight. Each self-administration procedure was observed by the experimenter and any attempt by pups to drink the solution from the syringe was avoided. Furthermore, the ingestion by the female mice of all the oil present in the modified syringe was controlled at the end of each treatment procedure.

The procedure was not stressful since the animals were previously trained to drink from this apparatus 0.1 ml of corn oil during a 5 min period every day.

The treatment ran from GD 14 to postnatal day (PND) 21 because it is at these critical developmental stages that ATZ, acting on the hypothalamic-pituitary axis, can directly or indi-rectly affect the developmental organizational events of the central nervous system (ArnolD & gorski 1984). The pups were not treated directly and therefore were only exposed by transpla-cental transfer (ATZ crosses placenta; ugAZio et al. 1991) or via lactation (ATZ or its metabo-lites are present in milk of dams treated with ATZ; stoker quoted in rooney et al. 2003, U.S. EPA 2001).

On PND 2, litters were culled to 8 per litter, 4 males and 4 females and all pups for each litter were marked for individual identification.

Experiments were carried out in accordance with NIh Guide for the Care and Use of Laboratory Animals and strictly following recommendations reported in European Communi-ties Council Directive of 24 November 1986 (86/609/EEC).

Behavioural testing

Two male and two female pups from 32 different litters were used for postnatal assess-ment of somatic and neurobehavioural development. A total of 128 mice were subjected to behavioural testing between 2 and 15 days of age.

Assessment of somatic growth and sensorimotor development

Four sensorimotor responses (indicative of neurobehavioural maturation) and two measures of somatic growth were recorded on PND 2, 4, and 9, and daily from PND 12 to PND 15. All tests were conducted between 11.00 and 14.00 by an experimenter blind to the treatment group.

Specifically, the following reflexes and responses were scored (Fox 1965): Righting: when pup is placed on its back, it instantly turns over to rest with all its feet

on the ground; Cliff aversion: when pup is placed with snout and forepaws over the edge of a flat sur-

face, it immediately turns around and crawls away from the edge; Forepaw grasp: when the shaft of a toothpick is stroked across the inside of the forepaw,

the hand is flexed to grasp the stick; Auditory startle: a loud snap of the fingers close to the pup results in an immediate star-

tle response.Assessment of somatic growth entailed measurements of eyelid and ear opening. The following scores were used for each variables mentioned: 0, no response; 1, uncer-

tain; 2, incomplete; 3, full. On PND 2, 4, 9, and 12, at the end of each test pups were weighed to the nearest 0.01

gr (Mettler PK-300 balance correcting for body movements).

Ultrasonic calls

Evaluation of pup vocalizations was performed in a sound-attenuating chamber (Ampli-silence, Rossobassomero, Italy) between 10.00 and 15.00 hr, i.e. during the red-light period.


On PND 4, 9 and 12, after 15 min of habituation to the experimental room, pups were tested for 2 min. We used the same males and females previously used for Fox’s battery of tests. The pups’ axillary temperature was recorded by a temperature probe (BAT-12 Physitemp Instrument Inc., Clifton, USA) and then they were individually introduced in a glass cylinder (5 cm diameter, 5 cm height) kept at constant temperature (24 ± 1 °C) in a temperature con-trolled incubator (Elmed Ginevri, Rome, Italy).

To avoid recording ultrasonic calls (USV) induced by handling during transfer of the subject from the home cage to the experimental apparatus, vocalization recording started 10 sec after the subject was placed in the glass container.

At the end of each test the beakers were cleaned to avoid contamination by any odour and the weight (± 0.01 g; Mettler, PK-300 balance set for automatic compensations of varia-tion due to movements during weighing) of the subject was recorded; then the pup was trans-ferred to a clean cage. After all four pups from a given litter were tested, they were moved back to the cage of the mother.

Bat detectors (US 30 Bat Detector; Ultra Sound Advice, London, UK) converted USV to audible sounds so that it was possible to count the number of ultrasounds produced. Calls were manually recorded.

At PND 9, ultrasonic calls were recorded for the first minute using a Bruel & Kjael (B&K, Denmark) microphone model 4135 (preamplifier B&K, 2633), suspended 1 cm above the beaker. Vocalizations were filtered (tunable band-pass Khron-hite filter 3500 set at 35 to 95 khz), amplified (B&K measuring amplifier 2610), and recorded on a Racal Store 4DS tape-recorder (tape Ampex 7971ESR1) by a direct-mode recording procedure (tape speed 76.2 cm/sec).

Recording were replayed to an audible output of the tape recorder upon reduction of the tape speed to 9.5 cm/sec. The analyses were performed on a PC (Dell 466 DMT Dimen-sion). Vocalizations were digitized (sampling frequency = 48 khz) and analyzed with the Avisoft-SASLab Pro 4.2 R. Specht 1993 package of Avisoft Bioacoustic. The analytical param-eters were: Frequency resolution: FFT-Length = 256, frame (%) = 100, window = hamming, bandwidth = 1125 hz, resolution = 750 hz overlap = 50%. Temporal resolution: 1/bandwidth = 0.889 msec, fix resolution = 0.6667 msec.

At PND 4, 9, 12 the number of the vocalizations were measured for 2 min; for the spec-trographic analysis, at the first minute of the evaluation period on PND 9, that is the peak day in the ontogenetic profile of USV calling (BrAnchi et al. 2001), the following features of ultra-sounds were tested for significant differences: pulse duration, interpulse duration, and peak frequency of the vocalization.

Statistical analysis

The relevant gestational parameters were submitted to one-way analyses of variance (ANOVA) in order to test the treatment effects.

Mixed models of ANOVA were performed on body weight, axillary temperature, number and spectrographic parameters of USV; in these models treatment and sex were considered as grouping factors and litter as random blocking factor.

In addition, in the analysis of axillary temperature and number of vocalizations, PNDs were considered as a repeated measures factor. Since the age × treatment interaction was signifi-cant, indicating that the treatment effect depends on the animals’ age, as a second step, in order to evaluate the treatment effect, we analyzed the simple main effect for each age separately.

Post-hoc comparisons were performed by the least significant difference (LSD) pairwise multiple comparison test, with the Bonferroni ‘s correction when the main effect of treatment was carried out separately at the three age.

The individual scores data on somatic and neurobehavioural development were simpli-fied to a single value; we chose the first day of full response as an index of the score profile. Then, data were analyzed with the Kruskal-Wallis test to evaluate the treatment effect. In a second step, in order to evaluate sex × treatment interaction, the analysis was applied sepa-


rately to males and females. When significant differences were found, multiple comparisons were conducted by Dunn’s test.

All statistical tests were carried out using SPSS and StatView computer software. Dif-ferences were considered significant when P < 0.05.

During the experiment some animal and some data were lost. This was made evident in the attribution of the degrees of freedom.

RESULTS

Gestational parameters

No differences were observed in dam body weight gain during gestation [F(2,29) = 0.24, P > 0.2] gestation days [F(2,29) = 0.48, P > 0.2], proportion of suc-cessful deliveries, litter size [F(2,29) = 0.64, P > 0.2] and sex-ratio [F(2,29) = 1.59, P > 0.2] among the treatment groups (see Table 1).

Somatic growth and sensorimotor development

A clear reduction in body weight was observed in pups exposed to both ATZ doses as early as on PND 2 ([F(2,28) = 4.67, P < 0.02]; see Table 2). No differences in body weight were observed starting from PND 4. Finally, no effects of sex were found on postnatal body weight gain, except for the fact that males were signifi-cantly heavier than females (see Table 2).

ATZ-treated pups showed a delay in the righting reflex [day of emergence of adult-like response χ2(2) = 9.26, P < 0.02 ] (see Table 3A). Moreover, when consider-ing only male pups, acceleration in the emergence of grasping was also observed in ATZ-treated groups ([χ2(2) = 11.1, P < 0.01]; see Table 3B).

Cliff aversion, auditory startle, eye and ear opening were not affected by ATZ treatment in either sex (see Table 3A).

Ultrasonic vocalization

As expected, axillary temperature increased over days in all groups [F(2,22) = 69.2, P < 0.001]. however, there was a significant interaction between age and treat-

Table 1.

Gestational length and litter characteristics of the atrazine-treated mothers and their control (number of litters).

Control(11)

ATL(10)

ATh(11)

Gestational days 19.90±0.51 19.70±0.61 19.91±0.49

Litter size 11.54±1.12 12.50±0.69 12.72±0.33

Number males 5.18±0.71 6.60±0.58 6.72±0.59

Number females 6.36±0.72 5.80±0.59 6.00±0.63

ATL, low dose atrazine; ATh, high dose atrazine. Data are expressed as mean ± SE.


Table 2.

Body weight of the pups in the atrazine-treated groups and their controls (number of pups).

Control(44)

ATL(40)

ATh(44)

PND 2 1.91±0.04 1.76±0.03* 1.76±0.02*

PND 4 2.90±0.05 2.92±0.05 2.90±0.04

PND 9 6.07±0.07 5.96±0.08 6.01±0.07

PND 12 7.80±0.08 7.78±0.11 7.90±0.08

ATL, low dose atrazine; ATh, high dose atrazine. * P < 0.05. Data are expressed as mean ± SE.

Table 3A.

Assessment of somatic growth and neurobehavioural development (day of maturation) of atrazine-treated pups and their controls from PND 2 to PND 15 (number of pups).

Day of full response

RightingCliff

aversionForepaw

graspAuditory

startleEyes

openingEars

opening

Control (40) 4.60±0.48 4.30±0.28 6.77±0.43 11.59±0.22 14.92±0.07 14.32±0.08

ATL (35) 4.82±0.50 4.17±0.33 6.00±0.42 10.75±0.30 14.77±0.08 14.11±0.08

ATh (40) 6.60±0.48*§ 3.92±0.27 5.95±0.41 10.96±0.28 14.97±0.04 14.32±0.07

ATL, low dose atrazine; ATh, high dose atrazine. * P < 0.05; § ATh vs control. Data are expressed as mean ± SE.

Table 3B.

Assessment of somatic growth and neurobehavioural development (day of maturation) of atrazine-treated pups and their controls from PND 2 to PND 15 (number of pups).

Day of full response

RightingCliff

aversionForepaw

graspAuditory

startleEyes

openingEars

opening

Males

Control (20) 4.20±0.40 4.20±0.40 7.50±0.53 11.45±0.37 14.90±0.10 14.35±0.13

ATL (17) 4.65±0.73 4.12±0.49 5.18±0.53** 11.18±0.42 14.65±0.17 14.05±0.13

ATh (20) 6.35±0.69 3.50±0.20 5.25±0.50** 11.25±0.40 14.95±0.08 14.30±0.10

Females

Control (20) 4.70±0.67 4.40±0.37 6.05±0.65 11.72±0.27 14.95±0.11 14.30±0.01

ATL (18) 5.00±0.71 4.22±0.45 6.77±0.60 10.38±0.43 14.89±0.08 14.17±0.09

ATh (20) 6.85±0.68 4.35±0.51 6.65±0.62 10.71±0.41 15.00±0.00 14.35±0.11

ATL, low dose atrazine; ATh, high dose atrazine. ** P < 0.01. Data are expressed as mean ± SE.


ment [F(4,22) = 3.30, P < 0.05], low dose ATZ-treated pups (ATL) showing lower axillary temperature on PND 9 when compared with other groups ([F(2,28) = 7.67, P < 0.01]; see Table 4).

The expected ontogenetical profile of USV was observed, mean rate being lower on PND4, increasing on PND 9 and decreasing on PND 12 [F(2,56) = 47.11, P < 0.001].

Table 4.

Body temperature of atrazine-treated pups and their controls at three different ages (PND) (number of pups).

Control ATL ATh

Males

PND 431.54±0.27

(17)30.96±0.38

(13)32.02±0.33

(18)

PND 934.15±0.24

(22)32.42±0.23**

(19)33.50±0.25

(22)

PND 1235.47±0.29

(22)35.77±0.34

(19)36.09±0.32

(22)

Females

PND 431.03±0.26

(16)31.60±0.35

(16)31.80±0.34

(16)

PND 934.16±0.28

(22)32.66±0.19**

(20)33.56±0.26

(22)

PND 1235.87±0.33

(22)35.94±0.33

(20)35.91±0.35

(22)

ATL, low dose atrazine; ATh, high dose atrazine. ** P < 0.01. Data are expressed as mean ± SE.

Table 5.

Number of USV emitted by atrazine-treated pups and their controls at three different ages (PND) (number of pups).

Control(22)

ATL(19)

ATh(22)

Males

PND 4 157±15 172±18 132±14

PND 9 266±20 223±30 276±19

PND 12 97±18 98±19 123±25

Females (22) (20) (22)

PND 4 165±19 166±16 136±20

PND 9 234±22 192±29 227±24

PND 12 126±22 85±23 121±22

ATL, low dose atrazine; ATh, high dose atrazine. Data are expressed as mean ± SE.


A significant interaction between age and sex [F(2,56) = 3.85, P < 0.05] was found. Specifically, there was a tendency towards a higher number of calls in males than in females on PND 9 (see Table 5). Moreover, on PND 9, the number of USVs showed a tendency to decrease, although the drop was not statistically significant, in ATL pups compared to high dose ATZ-treated pups (ATh) and the control group (see Table 5).

When considering the spectrographic characteristic of the sound, while no dif-ferences were observed in duration, nevertheless the USV mean interpulse showed a tendency to be longer in ATL compared to both ATh and control groups, this main effect of treatment just failed to reach statistical significance [F(2,13) = 3.38, P = 0.07; see Table 6]. Moreover, the USV interpulse, showed a significant difference between sexes [F(1,13) = 8.30, P < 0.05] and a significant sex × treatment interac-tion [F(2,13) = 4.56, P < 0.05], indicating that the treatment effect depends on the pup’s sex (see Table 6).

Instead, for the peak frequency a sex × treatment interaction [F(2,17) = 3.80, P < 0.05] was evident: peak frequency was differently affected by ATZ administration in males and females (see Table 6). Precisely, although in both sexes the treatment

Table 6.

Spectrographic analysis of the USVs emitted by atrazine-treated pups and their controls on PND 9 (number of pups).

Control(24)

ATL(28)

ATh(28)

Alls

Duration (msec)44.89±2.10

(28)44.75±2.21

(28)47.07±2.90

(24)

Interpulse (sec)0.71±0.08

(28)1.04±0.24

(27)0.64±0.08

(22)

Peak frequency (khz)55.0±0.80

(28)55.89±1.12

(28)58.74±0.68

(24)

Males


(14)45.03±2.64

(14)47.39±3.33

(12)


(14)0.55±0.06

(13)0.62±0.11

(12)


(14)57.81±1.18

(14)57.98±0.78

(12)

Females


(14)44.48±3.65

(14)46.76±4.91

(12)


(14)1.49±0.44

(14)0.65±0.11

(10)


(14)53.98±1.81

(14)59.51±1.09

(12)

ATL, low dose atrazine, 1 µg/kg; ATh, high dose atrazine, 100 µg/kg. Data are expressed as mean ± SE.


induced an increase of frequency, in males it was apparent in both ATZ-treated groups compared to the control group, while in females it was apparent in just the ATh compared to both the ATL and control groups (see Table 6).

DISCUSSION

In the present study we tested the short-term effects of exposure to environmentally-relevant doses of ATZ given during the perinatal period and lactation on the somatic growth and behavioural ontogeny of male and female mice. Our find-ings show that the exposure to ATZ, even at a very low dosage during the fetal and suckling period, is able to interfere with some aspects of the somatic growth of pups in a reversible manner, and more clearly with their neurobehavioural development.

Somatic growth

In utero exposure to both ATZ doses affected the intrauterine development of fetuses as indicated by the significant reduction of body weight at birth. In con-trast with previous studies, this effect is not related with an alteration in the ges-tational length and dam’s body weight or with effects on reproductive parameters (cummings et al. 2000, nArotsky et al. 2001). however these discrepancies with lit-erature data could be due to the higher dosage administered in those studies. More-over, from PND 4, a rapid compensatory mechanism redressed differences in body weight between treated and control groups. Other important indexes of maturation, such as eye and ear opening, were not affected by ATZ treatment.

Neurobehavioural development

Whereas postnatal somatic growth was only limitedly affected by our treat-ment (on PND 2), neurobehavioural development, as described by Fox (1965), was affected differently in both sexes and in respect to treatment dosage. In both sexes, the higher dose of ATZ delayed the righting reflex, normally strong and stereotyped by 3 days of age, when the mouse is more efficient at feeding and locating the nip-ple (Fox 1965). Otherwise, in male pups both ATZ doses accelerate the maturation of the forepaw grasping reflex. In fact, motor coordination normally is poorly devel-oped until the second week of age (Fox 1965).

Thus, since reflex ontogeny and the start of manipulative and locomotor abili-ties seem to involve several systems and different maturational periods, the disturb-ing effects of ATZ could be due to a different period of sensibility to ATZ of these systems.

In the case of grasping, the effects of ATZ on the differentiation of the under-lying neuronal circuits are seen only in males, probably due to the different hormo-nal milieu in males and females pups during the perinatal period.

Aromatase induction has been demonstrated as the mechanistic basis of ATZ action in reptiles (keller & mcclellAn-green 2004) and in human cells (sAnDer-son et al. 2001) and this is a possible mechanism for the effects observed in the present study.


In males the classical masculinization/defeminization hypothesis (ArnolD & gorski 1984) requires an aromatase action on an androgen substratum at the CNS level. The early effect of ATZ on behaviour could be related to the presence of aromatase, and consequent estrogen increase. On the other hand, the female brain is thought to develop normally in the absence of perinatal contact with estrogen (ArnolD & gorski 1984). however, recently AmAteAu et al. (2006) showed that aro-matase is active in the female rat brain in the neonatal periods, and weninger et al. (1993) showed that after 5th day of age rat ovaries are able to produce estrogen, which implies early aromatase action. These findings support the hypothesis that ATZ could affect both male and female behaviour by increasing estrogen production by aromatase modulation. Another possibility, not necessarily alternative, is related to the effects of ATZ on dopaminergic systems and somatostatine expression report-ed by roDrigueZ et al. (2005) and giusi et al. (2006), respectively.

When the effect of ATZ exposition on USV was analyzed, a different profile emerged in the treatment groups. In particular, on PND 9, there was a tendency for the number of vocalizations to be reduced by the low dose of treatment (ATL) in both sexes. This effect is consistent with previous studies describing inhibitory effects on ultrasonic vocalizations in rat exposed in perinatal period to environmen-tal polluter such as estrogenic or androgenic chemicals (pocock et al. 2002).

Contrary to expectation, in the same group of treatment (ATL) we found a reduced body temperature. In fact, it has been reported that thermal factors are activators of pup ultrasound calling and many studies reported a correlation between decreased environmental temperature and increased number of ultra-sonic vocalizations (elwooD & keeling 1982). Such a discordant effect could be related to a neurobehavioural deficit that assumes particular interest considering how infant USVs promote in a strain-specific way, maternal response and behav-iour (BrAnchi et al. 2004). Therefore, observed changes in USV could alter crucial mother-pup interactions in different ways.

Moreover, it should be noted that ATZ affected sound characteristics in both sexes, even if they showed different windows of sensibility to treatment dosage. In fact, the USV interpulse, that was sexually dimorphic in the control group, was longer in females exposed to the lower dose. Otherwise, although we observed a similar tendency in the peak frequency, females were sensible only to the higher dose, while males were sensible to both doses.

Again, considering the underlying hormonal mechanisms, this tendency could reflect the induction of aromatase activity by ATZ and the increased synthesis of estrogen at the expenses of androgen.

however, an alternative explanation may concern the relevant role of neuro-transmitters in the modulation of USV. For example, dopaminergic modulation of infant rodent USV has been investigated. In particular, previous studies showed how the cholinergic system plays a role in inhibiting USV emissions by rodent pups (kehoe et al. 2001, BrAnchi et al. 2004) and the GABAergic system is deep-ly implicated in modulating the USV of isolated pups (winslow & insel 1991). In this respect, recent studies reported disturbing effects of atrazine action on systems such as GABAergic circuits (shAFer et al. 1999), dopaminergic pathways (roDrigu-eZ et al. 2005) and somastotaninergic neurons (giusi et al. 2006) able to mediate movements.

Finally, we cannot exclude the possibility that hormonal alterations in dams, induced by ATZ, may result in an altered maternal cares, and consequently in altered pup behaviour.


CONCLUSIONS

The present findings increase the evidence that not all effects of ATZ exposure follow a monotonic dose-response curve. In fact, previous studies showed a non-linear dose-response to ATZ administration describing lower doses as more potent than higher doses in measured effects (hAyes et al. 2002) In particular, low-dose effects of ATZ may be mediated by endocrine-signaling pathways, evolved to act as powerful amplifiers, with the result that large changes can occur in response to extremely low concentrations (welshons et al. 2003).

Our results increase the evidence that developmental exposure to non-toxic, environmentally-relevant doses of an ED can produce subtle functional alterations, only detectable in rodent pups by detailed behavioural analysis, more efficiently than by means of current indices of somatic growth.

In conclusion, our present findings confirm that behavioural end-points repre-sent a useful tool for the study of alterations produced by early exposure to agents reportedly interacting with the developmental action of the endocrine system. This is compatible with the ATZ properties while suggesting caution in the use of a man-made chemical that, at the studied doses, significantly influences the development of the brain during a critical and vulnerable period, resulting in an alteration of developmental trajectories of behaviour.

ACKNOWLEDGMENTS

We gratefully acknowledge the advice of Flavia Chiarotti for statistical analysis. This study was supported by Istituto Superiore di Sanità – Istituto Superiore Prevenzione e Sicu-rezza sul Lavoro (ISS-ISPESL) “Multicentric Study on the Relationship between Endocrine Pathology and Occupational and Environmental Exposure to Environmental Pollutants”.

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Effects of low doses of atrazine on the neurobehavioural development of mice

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