Prenatal exposure to the pesticide metam sodium induces sensorimotor and neurobehavioral abnormalities in mice offspring

Journal Pre-proof Prenatal exposure to the pesticide metam sodium induces sensorimotor and neurobehavioral abnormalities in mice offspring Nour-eddine Kaikai, Saadia Ba-M’hamed, Mohamed Bennis, Abderrazzak Ghanima

PII:

S1382-6689(19)30184-X

DOI:

https://doi.org/10.1016/j.etap.2019.103309

Reference:

ENVTOX 103309

To appear in:

Environmental Toxicology and Pharmacology

Received Date:

15 April 2019

Revised Date:

22 November 2019

Accepted Date:

26 November 2019

Please cite this article as: Kaikai N-eddine, Ba-M’hamed S, Bennis M, Ghanima A, Prenatal exposure to the pesticide metam sodium induces sensorimotor and neurobehavioral abnormalities in mice offspring, Environmental Toxicology and Pharmacology (2019), doi: https://doi.org/10.1016/j.etap.2019.103309

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

1

Prenatal exposure to the pesticide metam sodium induces sensorimotor and neurobehavioral abnormalities in mice offspring

Nour-eddine Kaikai1,2, Saadia Ba-M’hamed1, Mohamed Bennis1, Abderrazzak Ghanima2,* 1

Laboratory of Pharmacology, Neurobiology and Behavior (URAC-37), Cadi Ayyad University,

Laboratory of Bioorganic and Macromolecular Chemistry. Cadi Ayyad University, Faculty of

ro

2

of

Marrakech, Morocco.

author: Abderrazzak Ghanima

re

*Corresponding

-p

Sciences and Techniques, Marrakech, Morocco.

FAX: (+212) 524 43 31 70.

lP

Phone: (+212) 524 43 34 04; (212) 661 250 618.

ur na

e-mail: [email protected] ; [email protected]

Jo

The key findings of our study following prenatal exposure to the pesticide MS are as follow: 

MS affected reproduction and fertility parameters (it reduced deliverance, viability and lactation indices in treated dams);



It delayed significantly the ontogeny of innate reflexes and sensorimotor performances in offspring;



It induced depression-like behavior in mice at their adulthood;



It impaired short- and long-term memory in adult mice prenatally treated.

2

Abstract The present study has investigated developmental neurotoxicity of Metam sodium (MS), from gestational day 6 and throughout the gestation period until delivery. Therefore, mated female mice were orally exposed on a daily basis to 0 (control), 50, 100 or 150 mg of MS/kg of body

of

weight and their standard fertility and reproductive parameters were assessed. The offspring

ro

were examined for their sensorimotor development, depression and cognitive performance. Our results showed that MS exposure during pregnancy led to one case of mortality, two cases of

-p

abortion and disturbed fertility and reproductive parameters in pregnant dams. In offspring, MS induced an overall delay in innate reflexes and sensorimotor performances. Furthermore, all

re

prenatally treated animals showed an increased level of depression-like behavior as well as a

lP

pronounced cognitive impairment in adulthood. These results demonstrated that prenatally exposure to MS causes a long-lasting developmental neurotoxicity and alters a wide range of

ur na

behavioral functions in mice.

Keywords: Metam sodium, developmental neurotoxicity, reproduction, fertility, depression,

Jo

learning and memory.

3

1. Introduction Prenatal exposure to environmental toxicants has been shown to exert adverse effects on the developing nervous system (Rice and Barone, 2000). These effects often outlast the duration of exposure to such toxicants, and some changes are known to be permanent leading to developmental neurotoxicity (Dobbing and Smart, 1974). The developmental neurotoxicity due to pesticides have been fueled by numerous epidemiological observations as well as

of

experimental studies, showing that human or animal models exposed prenatally or during early

ro

postnatal life are more sensitive and suffer from various neurological deficits such as cognitive and psychomotor alterations (Ribas-Fito et al., 2003; Perera et al., 2005; Gonzalez-Casanova et

-p

al., 2018).

re

Metam sodium (MS) is a widely used biocide with methyl-bis-dithiocarbamate structure (Pruett et al., 2001). According to Environmental Protection Agency (EPA), MS is active against all

lP

living matter in the soil and therefore acts as a fungicide, herbicide, insecticide as well as nematicide simultaneously (EPA, 2004). MS is widely used because it is cost effective (Stevens

ur na

and Freeman, 2018), and in consequence, hundreds of thousands of persons are exposed to it or its major breakdown product, methyl isothiocyanate, at levels greater than recommended by the EPA (Pruett et al., 2009). Despite its apparent low toxicity, MS poisoning incidents indicated that this compound is responsible of a variety of toxicity signs occurring at very low

Jo

concentrations, including burns, eye irritation, headache, nausea, breathing difficulty, and vomiting (O’Malley et al., 2004). Furthermore, many published reports have well documented the human exposure to MS following transport accidents as well as normal agricultural use, and evidenced its capacity to exert neurotoxic effects including cases of depression, anxiety, pain, memory loss and a reduction in leg strength and motor activity (Bowler et al., 1994 ; Goldman et al., 1994 ; EPA, 2004). Current data are more focused on teratogenic effects showing that oral

4

exposure to MS during pregnancy caused birth defects in the nervous system, fetal death and a drastically reduction in the number of surviving fetuses at both high and lowest tested doses in animal models of rats, rabbits as well as fishes (Hodge, 1993; Tinston, 1993; Haendel et al., 2004; Tilton et al., 2006). However, these data are of limited value in assessing the toxicity of this pesticide on the brain and behavior at postnatal age. In this regard, we have designed our present study, to investigate the developmental and neuro-behavioral consequences of prenatal

Jo

ur na

lP

re

-p

ro

of

exposure to the MS pesticide.

5

2. Material and methods 2.1.Animals Male and female Swiss mice (8–10 weeks old) from the animal husbandry of the Faculty of Sciences, Cadi Ayyad University, Marrakesh, Morocco were used. The animals were housed in Plexiglas cages (30 cm × 15 cm × 12 cm) under standard conditions of temperature (22±2°C) and photoperiod 12h/12h (light on at 08:00 h) with access to food and water ad libitum. All

of

procedures were performed in accordance with approved institutional protocols and guidelines of

ro

European Council Directive (EU2010/63). All efforts were made to minimize any animal

suffering. The study was approved by the Council Committee of Research Laboratories of the

-p

Faculty of Sciences, Cadi Ayyad University, Marrakesh.

re

2.2. Pesticide

In this study animals were treated with metam sodium nematicide in its liquid commercial form,

lP

Nemaprop (containing 510 g/l of MS), supplied by Taminco company (USA), with linear formula C2H4NNaS2, molecular weight of 129.19 g/mol, melting point 97 – 102 °C and a density

ur na

of 1.21 g/cm3.

2.3. Mating procedure and treatment Virgin female mice (N=25) were mated with males (2:1) in each cage overnight. The onset of

Jo

pregnancy was confirmed by the observation of a vaginal plug. Confirmation of positive spermplug corresponded to gestational day 0 (GD0). After mating, the females certified to be pregnant were randomly assigned to the following four groups: a control group (N=5) received tap water (0 mg/kg of MS) and treated groups with 50 (N=6), 100 (N=7) or 150 mg/kg (N=7) of MS dissolved in tap water. These doses are sublethal and were determined from the results of our prior conducted toxicity study (unpublished data). All groups were treated orally by gavage from GD6 until delivery (Figure 1).

6

2.4. Maternal observation Pregnant mice were observed daily during the whole gestational period for symptoms of poisoning (e.g. mortality, morbidity, abortion or premature delivery) and the body weight evolution was recorded daily from GD6 until delivery. Moreover, different parameters of fertility and reproduction were calculated as previously described by Ait-bali et al. (2016), briefly:  Pregnancy index = (number of pregnant females / number of all females with a positive

of

vaginal plug) x 100

ro

 Deliverance index = (number of females delivering / number of pregnant females) x 100  Live-birth index = (number of offspring born alive / number of descendants delivered) x 100

-p

 Viability index = (number of living descendants on the fourth day of lactation / number of

pups born alive) x 100

re

 Lactation index = (number of living pups on day 21 / number of pups born alive) x 100

lP

2.5. Evaluation of the offspring development following prenatal exposure to MS At the delivery day (P0), the offspring of each litter was counted and checked for apparent

ur na

morphological changes. Litter size as well as the body weight, were monitored continuously until P21. Meanwhile, a battery of behavioral tests was performed for all animals in order to evaluate reflex development and neuromotor parameters (Figure 1). 2.5.1. Surface righting reflex test

Jo

This test was conducted to evaluate the muscular and vestibular maturation in pups at P5, 7 and 9 (Fox, 1965). It consists on placing each pup on its back on a flat surface and record the time needed to get back on its all four paws. Each pup underwent one trial with a maximum of 60s for each trial.

7

2.5.2. Cliff avoidance test This test was used to assess the coordination of limbs as well as muscular strength and ability to avoid the fall during P6. Each pup was placed on a table edge with the forepaws and nose over the edge. The latency to get back and turn away from the edge (avoidance of fall) was measured. The animals were tested for a maximum of 60s. 2.5.3. Body stimulation test

of

The body stimulation test was assessed in order to evaluate the tactile sensibility in offspring. It

ro

was conducted as described by Arakawa and Erzurumlu (2015) at P7, P9 and P12. Briefly,

offspring were poked manually 5 times on a total of 7 points along the body (top of the head,

-p

medial back, both forepaws and hind paws as well as the base part of the tail) using a 0.5 mm

re

diameter stainless rod. Offspring were then scored for their response over the total of stimulated points as follow: 1 point was attributed in the presence of a response such as withdrawal or

lP

twitching, 0 point for the absence of a response. 2.5.4. Negative geotaxis test

ur na

The negative geotaxis test was conducted to examine the orientation reflex in response to gravitational stimulation at P5,7 and 9. The pups were placed head down on a 45° inclined plane and the time taken by the animal to rotate 180° was recorded. The animals were tested for a

Jo

maximum duration of 60s per trial.

2.6. Evaluation of the effect of prenatal exposure to MS on behavior in adulthood

Male animals, previously separated from female littermates, were individually submitted to a neurobehavioral evaluation on P60 consisting of locomotor activity, depressive-like behavior as well as learning and memory (Figure 1). The behavior of all mice during the tests was recorded and analyzed using Ethovision XT Noldus8.5 video tracking program (Noldus, Netherlands) connected to a video camera (JVC) or the video-tracking software (Debut video capture

8

software, NHC) connected to a video camera (Samsung SCO-2080-R). All devices were thoroughly cleaned with alcohol and dried before each animal's passage, in order to avoid any influence of the odor on the results. 2.6.1. Open field test In order to evaluate locomotor and exploratory activity in mice prenatally exposed to MS, we performed the open field test. The experimental device consisted of a black square field (50 x 50

of

x 50cm) illuminated by a 75W lamp placed in porthole diffusing light and located at 200 cm

ro

from the device, allowing the center of the arena to be under a dim light of 100 lx. During each session, mice were placed individually in the center of the device field and left to explore for a

-p

duration of 10 min. The parameters recorded in this test were the total distance travelled and the

re

velocity of each animal. 2.6.2. Tail suspension test

lP

The tail suspension test was performed to examine depression-like behavior in treated mice. The test consisted of suspending each mouse by the tail with adhesive tape 50 cm above the floor

ur na

during 6 min. The immobility time was recorded for each animal. 2.6.3. Splash test

The Splash test is widely used to evaluate the motivational deficits and self-care difficulties as

Jo

symptoms of depression in rodents (David et al., 2009). A 10% sucrose solution was squirted all over the dorsal coat of the mice in their home cage. The time spent grooming was recorded for a period of 5 min after the sucrose application. 2.6.4. Y-maze test The spontaneous alternation test is widely used to evaluate spatial working memory in rodents by making use of their natural exploratory instinct (Douglas, 1989). The Y-maze is composed of

9

three arms of the same dimensions (10 cm wide, 41 cm long and 25 cm high) interconnected at 120°. At the beginning of each session, mice were placed individually at the end of one of the three arms and left to explore freely for 8 min. A correct alternation was considered when mice visited all three arms without going into the same arm twice during a single row. Then, the percentage of spontaneous alternation for each mouse was calculated as described by Chen et al. (2016): % alternation= [(number of alternations) / (total arm entries - 2)] x 100.

of

2.6.5. Step-through passive avoidance test

ro

Passive avoidance test is a rapid one-trial learning where animals learn to avoid an environment in which an aversive stimulus was previously delivered (Michalak and Biala, 2017). The test was

-p

conducted in a two-chambered device containing a bright and a dark Plexiglas-compartments of

re

the same size, separated by a gate door allowing the passage of the mice. The dark compartment was related to an electrical shocker (Panlab, Spain).

lP

On the first day, mice were allowed to explore freely both compartments for a duration of 3 min. Animals whose latency to dark compartment during this session exceeded 60s were excluded

ur na

from the test. On the following day, mice were placed in the bright compartment facing the gate door. After a brief period of 10s, the door separating the chambers was raised. As soon as the animal completely entered the dark compartment, the door was closed and a single electric foot shock of 0.2mA was immediately delivered for 1s. After the shock exposure, the animal was

Jo

removed back to its home cage. The retention of the avoidance performance was tested two hours after the shock exposure and 24 hours later (the day 3). During the retention session, the same procedure was repeated but without electric foot shock delivery. The latency of the mice to reach the dark compartment was measured. Lower latencies indicate that mice were unable to keep in memory the foot shock previously received. The retention session was ended once the animal entered completely the dark compartment or after a maximum duration of 300s.

10

2.7. Statistical analysis Statistical analysis was performed using SigmaPlot 12.5 for Windows. Data were tested either by one-way analysis of variance (ANOVA) or two-way ANOVA with or without repeated measures followed by Holm-sidak’s post hoc for multiple comparisons. The results were presented as

Jo

ur na

lP

re

-p

ro

of

mean ± S.E.M, and the difference was considered statistically significant at P< 0.05.

11

3. Results 3.1.Effect of MS on fertility and reproduction parameters of pregnant mice Dams from the four groups were observed during pregnancy and lactation, and gestational data were recorded. During the exposure period to MS, no signs of toxicity were observed for all doses used, except for two cases of abortion and one case of mortality at 150 mg/kg. Our results also showed a delay in gestation period in treated dams with the higher dose (t= 3.08, P<0.05)

of

(Table 1).

ro

Regarding reproduction parameters, our results showed a reduced deliverance index in females treated with the highest dose (57.14 % vs 100% for all other groups). The data analysis did not

-p

show any significant difference in live-birth index between treated and control groups [F(3,22)=

re

0.41, P > 0.05]. In contrast, the viability index, as well as lactation index were reduced in all treated animals, especially in those treated with 50 mg/kg for both indices (t=3.05, P< 0.05; t=

lP

3.95, P< 0.01), and with 150 mg/kg for lactation index (t= 4.87, P< 0.01) compared to control group. The number of pups did not differ between groups [F(3,22)= 0.37, P>0.05] (Table 1).

ur na

As for the body weight gain of pregnant females, the two-way repeated measures ANOVA was performed taking into account the treatment and gestation day as main factors. It showed a significant effect of the factor gestation day [F(2,71)= 658.61, P<0.001], while the factor treatment

Jo

as well as the interaction treatment x gestation day had no effect [F(3,71)= 0.39, P>0.05; F(6,71)= 1.09, P>0.05; respectively] (Table 1). Concerning the offspring body weight, the data analysis was performed using two-way repeated measures ANOVA with the factors, treatment and age. The result revealed a main effect of the factor age [F(3,159) = 756.72, P< 0.001]. However, no effect of the factor treatment as well as the interaction treatment x age [F(3,159)= 0.63, P > 0.05; F(9,159)= 0.39, P > 0.05; respectively]. It is to

12

mention that although not significant, the offspring of treated females exhibited a low bodyweight at birth in comparison with control group (Table 1). 3.2.Effect of prenatal exposure to MS on sensorimotor development 3.2.1. Surface righting reflex test The surface righting reflex was altered in treated mice. Indeed, a two-way repeated measures ANOVA demonstrated a significant effect of age [F(2,119)= 236.78, P< 0.001], treatment [F(3,119)=

of

27.94, P< 0.001] as well as the interaction age x treatment [F(6,119)= 3.87, P< 0.01]. The post hoc

ro

analysis showed a significantly reduced righting reflex response in pups prenatally exposed to MS especially to 100 and 150 mg/kg doses, at P5 (t= 6.19, P< 0.001; t= 7.03, P< 0.001,

-p

respectively) and P7 (t= 2.85, P< 0.05; t= 5.22, P< 0.001, respectively), but not at P9 (P>0.05).

re

Significant increase in righting reflex time was also detected in both groups of 100 and 150 mg/kg vs 50 mg/kg at P5 (t= 4.23, P< 0.001; t= 5.08, P< 0.001, respectively) and only in the

3.2.2. Cliff avoidance test

lP

group of 150 mg/kg vs 50 mg/kg at P7 (t= 3.04, P< 0.05) (Fig. 2A).

ur na

The cliff avoidance ability was altered in prenatally treated mice [F(3,39)= 15.71, P<0.001]. The multiple comparisons indicated that animals treated with the doses, 50, 100 and 150 mg/kg of MS took more significant time to avoid the cliff at P6 (t= 5.32, P<0.001; t= 4.53, P<0.001; t=

Jo

6.35, P<0.001, respectively) compared to control group (Fig. 2B). 3.2.3. Body stimulation test

The results showed that MS exposure during pregnancy altered normal twitching responses in offspring. The data analysis with two-way repeated measures ANOVA revealed a main effect of the factor age [F(2,119)= 17.86, P< 0.001], treatment [F(3,119)= 40.40, P< 0.001] as well as the interaction between these two factors [F(6,119)= 2.45, P< 0.05]. The post hoc comparisons showed a significant increase of tactile responses in mice prenatally treated with both 100 and 150 mg/kg

13

at P7 (t= 4.81, P< 0.001; t= 7.69, P< 0.001, respectively), and only with the higher dose at P9 (t= 5.53, P< 0.001) as well as P12 (t= 6.25, P< 0.001) (Fig. 2C). Moreover, significant differences were also detected at P7 between animals treated with 100 mg/kg and 150 mg/kg vs those treated with 50 mg/kg (t= 5.05, P< 0.001; t= 7.93, P< 0.001, respectively), and 150 mg/kg vs those treated with 100 mg/kg (t= 2.88, P< 0.05), and at P9 as well as P12 between 150 mg/kg vs both 50 mg/kg and 100 mg/kg treated mice (t= 5.29, P< 0.001; t= 3.60, P< 0.01; t= 5.53, P<

of

0.001; t= 4.57, P< 0.001, respectively).

ro

3.2.4. Negative geotaxis

The prenatal exposure to MS impaired negative geotaxis reflex in offspring. Indeed, the two-way

-p

repeated measures ANOVA indicated a significant effect of the factors age [F(1,79)= 22.40, P<

re

0.01] and treatment [F(3,79)= 20.58, P< 0.001]. However, no significant effect of the interaction age x treatment was detected [F(3,79)= 2.46,P>0.05]. The post hoc analysis showed a significant

lP

higher latency in prenatally treated mice with 50, 100 as well as 150 mg/kg to accomplish a 180° turn at P10 (t= 3.97, P< 0.01; t= 6.45, P< 0.001; t= 3.51, P< 0.01, respectively) and only those

ur na

treated with 100 and 150 mg/kg of MS at P12 (t= 3.62, P< 0.01; t= 4.30, P< 0.001, respectively) (Fig. 2D). Significant differences were also detected in 100 vs 50 mg/kg (t= 3.48, P< 0.05) as well as 150 vs 100 mg/kg treated mice at P10 (t= 2.94, P< 0.05). 3.3.Effect of prenatal exposure to MS on behavior in adulthood

Jo

3.3.1. Locomotor activity

In this study, locomotor activity was mainly assessed using open field test. The analysis of the recorded data failed to show any significant difference in the total distance traveled and the moving velocity in MS prenatally treated groups [F(3,45)= 2.06, P>0.05; F(3,45)= 2.41, P>0.05; respectively] compared to control group (Fig. 3A,B).

14

3.3.2. Depression-like behavior In order to evaluate the integrity of this function, prenatally exposed animals to MS underwent tail suspension test and splash test during adulthood. In tail suspension test, there was a statistically significant difference in immobility time among groups [F(3,44)= 7.41, P< 0.001]. The post hoc analysis showed a dose dependent increase of the immobility time in prenatally exposed animals especially in those treated with 100 mg/kg (t= 2.90, P< 0.05) and 150 mg/kg (t= 4.34,

of

P< 0.001) (Fig. 4A). Moreover, a difference between 150 and 50 mg/kg treated mice (t= 3.29,

ro

P< 0.05) was also detected.

Regarding the splash test, data analysis indicated a statistically significant effect of treatment on

-p

the time of grooming [F(3,41)= 11.15, P< 0.001]. The multiple comparisons clearly showed that

re

treated mice exhibited a significantly reduced time of grooming, specifically in those prenatally treated with 100 mg/kg (t= 5.36, P< 0.001) and 150 mg/kg (t= 3.65, P< 0.01) as compared to the

significant (t= 3.70, P< 0.01).

lP

control group (Fig. 4B). The difference between 100 and 50 mg/kg treated mice was also

ur na

3.3.3. Learning and memory

In order to illustrate the effect of prenatal exposure to MS on learning and memory during adulthood, we have conducted the step-through passive avoidance test and Y-maze test. Overall, the passive avoidance test indicated that MS exposure impaired short- and long-term memory.

Jo

Indeed, the two-way ANOVA revealed a significant impact of the factor treatment [F(3,55)= 26.64, P< 0.001], and retention time [F(1,55)= 7.26, P< 0.05], while the interaction treatment x retention time was not significant [F(3,55)= 0.53,P>0.05]. Multiple comparisons showed a significant decrease in the latency to enter the dark compartment in treated groups with 50, 100 and 150 mg/kg after 2h (t= 4.78, P< 0.001; t= 3.69, P< 0.01; t= 4.71, P< 0.001, respectively) as well as

15

24h from the shock administration (t= 6.32, P< 0.001; t= 5.25, P< 0.001; t= 5.72, P< 0.001, respectively) (Fig. 5). The recordings from Y-maze test indicated that treated mice displayed no difference in the spontaneous alternation from non-exposed animals [F(3,44)= 0.25, P>0.05]. Similarly, the data analysis revealed no significant difference between treated animals and control group regarding

Jo

ur na

lP

re

-p

ro

of

the number of arm entries [F(3,44)= 1.04, P>0.05] (Fig. 6A, B).

16

4. Discussion The existing developmental studies on MS have only focused on its teratogenic sequelae in offspring following pregnancy exposure. To our knowledge, no study has investigated the developmental neurotoxicity of MS. The current study was designed in this regard using several paradigms aiming to assess in prenatally exposed offspring, the ontogeny of sensorimotor functions as well as behavioral neurotoxicity.

of

In the present work, the daily exposure of pregnant mice to MS did not bring on any observable

ro

clinical signs at all three experimented doses. However, two cases of abortion and one case of mortality have occurred especially in females treated with the highest dose. These observations

-p

are in accordance with EPA’s report showing that oral exposure of female rats to MS from gestational day 6, induced a pregnancy loss especially at high doses (EPA, 2004). According to

re

this report, the MS metabolite carbon disulfide would be the origin of abortions. Moreover,

lP

accidently exposed women to MS breakdown product methyl isocyanate have also showed several spontaneous abortions (Dhara and Dhara, 2002; EPA, 2004). However, the one case of

ur na

mortality detected in our study could not be related to MS exposure since no mortality cases were noticed in anterior developmental studies (EPA, 2004). Our results also indicated a tendency to a delay in gestation period especially in females treated with the highest dose. The increase in gestation length was also found by Jacobsen et al. (2010) in rats orally exposed by

Jo

gavage to a mixture of pesticides containing fungicides and dithiocarbamates. Regarding reproduction parameters, except for the live-birth index, our results showed a reduced deliverance as well as viability and lactation indices in treated females. The offspring of treated females exhibited a low birth body weight. Our results join in part those of Short et al. (1976) who have demonstrated that oral exposure to DTC led to a very poor survival and growth in pups especially those prenatally treated with the highest dose. The lipophilic properties of DTC allow

17

them to pass through cell membranes and physiological barriers including fetal-placental barrier (Frank et al., 1995; Rath et al., 2011). In line with this, prenatal exposure to MS or to its breakdown product has been shown to decrease the placental weight as well as mean food consumption in dams during the treatment period (EPA, 2005). Moreover, experimental studies in rodents have shown that reduced food consumption throughout gestation induced changes in placental weight and morphology (Gonzalez et al., 2016). Accordingly, reduced placental weight

of

in experimental animals has been linked to a decreased birth body weight in offspring along,

ro

with postnatal abnormalities in physiological functions and an increased disease risk in later

life (Fowden et al., 2008; Gonzalez et al., 2016). These MS induced effects on placental weight

-p

and food consumption in dams could be the cause of low birth body weight in offspring of the

re

present study.

On the other hand, numerous DTC compounds including mancozeb, maneb as well as MS are

lP

often reported as endocrine disruptors (Cecconi et al., 2007; Pruett et al., 2009; Axelstad et al., 2011). Therefore, DTC could act via different mechanisms all the way from influencing hormone

ur na

synthesis to interfering with them at receptor level (Bisson and Hontela, 2002). Many investigations have reported the relationships between exposures to chemicals during pregnancy, particularly endocrine disrupters, and abortions as well as impaired lactation in dams, and low birth weight as well as higher mortality rates in offspring (Sullivan, 1993; Rauch et al., 2012;

Jo

Manservisi et al., 2015; Birks et al., 2016). Thus, the endocrine disruption ability of MS could also explain the observed impairment of some reproduction and fertility parameters in the present study.

Postnatal development of pre-exposed offspring Unlike the so-called precocial animal species, because of the set-up of basic motor functions at birth, the altricial species among of which the mouse, have their organism in general and their

18

nervous system in particular, immature at birth (Fox, 1966). As a result, the development of the mouse is essentially postnatal, and the maturation of nerve structures is rapid during the first three weeks of life. It is almost complete at weaning (Himwich, 1962a and 1962b), and will gradually lead to the repertory of the adult. In this context, a battery of noninvasive behavioral tests was carried out to check the degree of sensorimotor maturation of young mice, at dates spread over the first 3 weeks of life. The surface righting reflex is a complex coordinated action

of

that requires reflexive response of different muscles including those of the neck, trunk, and

ro

limbs. From our results, it appeared that prenatally treated offspring showed a delayed

development of this reflex during the first two days of test and began to improve markedly

-p

around P9. The impairment observed in the achievement of the righting task indicates a probable alteration of proprioceptive sensation and vestibular function important for head and body

re

orientation (Aruga et al., 1998; Secher et al., 2006; Santillán et al., 2010). The negative geotaxis

lP

on the other hand requires an orienting response and a movement expressed in opposition to cues of a gravitational vector (Kreider and Blumberg, 2005). Similarly, our findings showed that MS

ur na

exposure altered the performance of the mice during the required task. The alteration of the negative geotaxis success rate was reported as deficits accompanying a delay in mouse cerebellum development as well as in spinal motor control (Aruga et al., 1998; Santillán et al., 2010). Concerning the avoidance of the fall, the recorded failure of offspring to avoid the fall

Jo

could be associated to motor, arousal, or cognitive dysfunction (Yoshida et al., 1998). The cognitive dysfunction for instance may reflect the fact that animals could not understand the risk involved with placement at a high location (Yoshida et al., 1998). In body stimulation test, pups were required to detect a contact of the rod and exhibit a body reaction to the tactile stimulus. Tactile sensation is processed within the somatosensory cortex. Our results showed that prenatally treated offspring especially by the highest dose (150 mg/kg) displayed a high response

19

level during the test. The hypersensitivity to tactile stimulation exhibited by treated animals could be related to changes at one or more sensory processing stages, ranging from peripheral receptors in the skin, spinal synapses through the brain’s perceptual system processes (Blakemore et al., 2006). In addition to this, it has been reported that exposure to dithiocarbamates promoted degeneration of GABAergic neurons (Soleo et al., 1996; Negga et al., 2012). In line with this, a decrease of GABA neurotransmitter concentrations in the

of

somatosensory cortex has been hypothesized to play a role in tactile hypersensitivity (Sapey‐

ro

Triomphe et al., 2019). Adult behavior

-p

Recent data demonstrate that developmental exposure to neurotoxic substances, e.g. pesticides,

re

can have long-lasting consequences and alter CNS function in a way that does not compromise the growth and viability of the fetus, but causes severe neural and/or behavioral changes (Ait-bali

lP

et al.; 2016; Gallegos et al., 2016; Li et al., 2016). In this regard, we have tested our animals during adulthood for their locomotor activity, depression as well as learning and memory.

ur na

Animals exposed prenatally to MS did not show any disturbance in the locomotor activity during adulthood. Since the dopaminergic system is strongly involved in neuromotor system (GiménezLlort et al., 1997), the absence of any significant effect on locomotor activity could be explained, according to Barlow et al. (2003), by the fact that MS does not affect dopamine levels within the

Jo

brain, while other DTC compounds such as ethylene-bis-dithiocarbamates and diethyldithiocarbamates increased dopamine accumulation particularly in the striatum of mice (Barlow et al., 2003).

Several lines of evidence suggest that pesticide exposure underlie some psychiatric disorders such as depression (Lima et al., 2009; Joo and Roh, 2016; Koh et al., 2017). In this regard, we have evaluated depressive behavior in prenatally MS treated offspring, and the results showed

20

clearly a depression-like behavior during adulthood. These findings join those of previous studies obtained following long-term oral exposure in rats to other DTC compounds such as zineb, ferbam, maneb, mancozeb, as well as their metabolite ethylene-thiourea, at high dose levels (Ivanova-Chemishanska, 1969; Graham et al., 1975; Worthing and Walker, 1983). Our findings are also in accordance with previous clinical investigations showing that exposure to several fungicides (e.g. maneb, ziram) and fumigants (e.g. methyl bromide), is strongly

of

associated with depression among male applicators (Beard et al., 2014). As MS is known to alter

ro

hypothalamic catecholamines due to its ability to suppress the activity of dopamine-beta-

hydroxylase necessary for synthesis of noradrenaline (Goldman et al., 1994), and since Tamaki

-p

and Hidaka (1976) have reported that rats treated with dopamine-beta-hydroxylase inhibitors developed depression-like behavior, thus, the effect of MS on this enzyme could be associated

re

with depression-like behavior developed in adult mice.

lP

Behavioral impairments indicative of cognitive changes have been associated with exposure to a number of chemicals. Indeed, developmental exposure to MS caused a significant impairment in

ur na

short- and long-term memory assessed by step through passive avoidance test, while working memory during spontaneous alternation task seemed to remain intact. The effect of DTC compounds on learning and memory evaluated by the passive avoidance test has been previously studied by Palfai and Walsh (1980), showing that intraperitoneal administration of diethyl-dithio-

Jo

carbamate at doses superior to 300 mg/kg in mice significantly impaired the retention in passive avoidance test. According to these authors, the recorded memory impairment was attributed to reduced noradrenalin levels following suppression of dopamine-beta-hydroxylase activity. It is noteworthy to mention that from an anatomical point of view, the retention in passive avoidance task relies heavily on the dorsal hippocampus (Lorenzini et al., 1996; Fanselow and Dong, 2010; Vanz et al., 2018), where it uses a sequence of molecular events very remindful of those of long-

21

term potentiation (Izquierdo and Medina, 1995; 1997; Bernabeu et al., 1997). It is also intensely modulated by the basolateral nucleus of the amygdala where it uses other sequences of molecular events (for a review see: Izquierdo et al., 2006). In rodents, significant retention deficits following dorsal hippocampal lesion have been reported in early studies (Best and Orr, 1973; Cogan and Reeves, 1979). In contrast, working memory evaluated by Y-maze paradigm differs a lot from inhibitory memory in passive avoidance task. On one hand, it is due to its essentially

of

“executive” function since it stores information for a very short period of time and leaves no

ro

long-lasting record of any kind (Baddeley, 1997; Camina and Güell, 2017). On the other hand, it is often reported that working memory is mainly settled in the prefrontal cortex that presents a

-p

strong neural activity during processing of the tasks relied on this type of memory (Baddeley, 1992; Goldman-Rakic, 2000; De Saint Blanquat et al., 2010). Furthermore, the fact that our

re

treatment induced cognitive impairment in the passive avoidance, but not in Y-maze testing join

lP

the result found in previous studies (Lukawski et al., 2008; Dela Pena et al., 2014). Thus, the differences in the neurobiological processes involved in the two tests could explain the

ur na

discrepancies in the observed effect of MS on learning and memory. It should be emphasized that from mechanistic point of view, the DTC compounds are not specific in action, and it is difficult to identify a single mechanism for their neurotoxic effects (Bjørling-Poulsen et al., 2008). However, several studies have demonstrated the ability of DTC

Jo

including MS to induce oxidative stress in neuronal cells (Barlow et al., 2005; Domico et al., 2007; Pruett et al., 2009). There is evidence that they cause the generation of reactive oxygenated and/or nitrated (ROS/RNS) species, as well as significant changes in either antioxidant pools or ROS/RNS-scavenging enzyme system, or both (Banerjee et al., 1999; Halliwell and Gutteridge, 1999; Astiz et al., 2009). In this regard, it is important to emphasize that the brain is particularly vulnerable to oxidative stress (Cobley et al., 2018). Indeed, the

22

oxidative stress was associated to numerous neurobehavioral abnormalities in rodents such as anxiety-like, learning and memory impairment as well as neuro-inflammation and neurodegenerative diseases (Navarro et al., 2002; Rammal et al., 2008; Salim et al., 2010; Patki et al., 2013; Solanki et al., 2017). In addition to this, the endocrine disruption ability of MS, mentioned above, could conceivably lead to several neurobehavioral deficits (Bjørling-Poulsen et al., 2008). In fact, it is well established that neuroendocrine system shapes the vertebrate brain

of

such that sex specific physiology and behaviors emerge. Much of this occurs in discrete

ro

developmental windows that span gestation through the prenatal period (Patisaul and Adewale, 2009). Indeed, animal studies suggested that maternal exposure to endocrine disruptors induced

-p

significant changes in rearing behavior, anxiety-like and depression-like behaviors, learning/memory impairment in offspring, as well as neuronal abnormalities (Masuo and Ishido,

re

2011; Kajta et al., 2017). DTC compounds can also cause thyroid inhibition (Marinovich et al.,

lP

1997; Goldner et al., 2010; Piccoli et al., 2016). A key concern with thyroid inhibitors is that impaired thyroid function may alter hormone-mediated events during development, thereby

ur na

possibly leading to permanent alterations in brain morphology and function (Cooper and Kavlock, 1997; Colborn, 2004). Furthermore, Smallridge and Ladenson (2001) have shown that even mild degrees of hypothyroidism may likely cause functional deficits during brain development. At present, we can only speculate that one of these mechanisms or all of them

Jo

combined could be associated to the different impairments obtained in the present work. In summary, our experiments have provided several main findings to support our hypothesis that the exposure to the pesticide MS in utero results in numerous neurobehavioral deficits. Some of these impairments may be irreversible since they were detected even later at adulthood. Further studies are clearly necessary to point the underlying mechanisms by which MS induced the fore

23

mentioned deficits. The results of these studies may cast new light on the risks of MS exposure on human mothers and their offspring.

of

AUTHOR CONTRIBUTION

ro

Nour-eddine Kaikai, Saadia Ba-M’hamed, Mohamed Bennis, Abderrazzak Ghanima

-p

Nour-eddine KAIKAI, Saàdia BA M’HAMED, Mohamed BENNIS and Abderrazzak GHANIMA designed the experiments, performed the analysis of the data and wrote and edited the

ur na

lP

re

manuscript. Nour-eddine KAIKAI performed the experiments and assembled the figures.

Conflict of Interest:

Jo

The authors declare that they have no conflict of interest.

24

References Ait-Bali, Y., Ba-M’hamed, S., Bennis, M., 2016. Prenatal Paraquat exposure induces neurobehavioral and cognitive changes in mice offspring. Environmental toxicology and pharmacology, 48, pp.53-62. Arakawa, H., Erzurumlu, R.S., 2015. Role of whiskers in sensorimotor development of C57BL/6 mice. Behavioural brain research, 287, pp.146-155. Aruga, J., Minowa, O., Yaginuma, H., Kuno, J., Nagai, T., Noda, T., Mikoshiba, K., 1998.

of

Mouse Zic1 is involved in cerebellar development. Journal of Neuroscience, 18(1), pp.284293.

ro

Astiz, M., de Alaniz, M.J., Marra, C.A., 2009. Effect of pesticides on cell survival in liver and brain rat tissues. Ecotoxicology and environmental safety, 72(7), pp.2025-2032.

-p

Axelstad, M., Boberg, J., Nellemann, C., Kiersgaard, M., Jacobsen, P.R., Christiansen, S., Hougaard, K.S., Hass, U., 2011. Exposure to the widely used fungicide mancozeb causes

re

thyroid hormone disruption in rat dams but no behavioral effects in the offspring. Toxicological sciences, 120(2), pp.439-446.

lP

Baddeley, A., 1992. Working memory. Science, 255(5044), pp.556-559. Baddeley, A.D., 1997. Human memory: Theory and practice. Psychology Press.

ur na

Banerjee, B.D., Seth, V., Bhattacharya, A., Pasha, S.T., Chakraborty, A.K., 1999. Biochemical effects of some pesticides on lipid peroxidation and free-radical scavengers. Toxicology letters, 107(1-3), pp.33-47.

Barlow, B.K., Lee, D.W., Cory-Slechta, D.A., Opanashuk, L.A., 2005. Modulation of antioxidant defense systems by the environmental pesticide maneb in dopaminergic

Jo

cells. Neurotoxicology, 26(1), pp.63-75.

Barlow, B.K., Thiruchelvam, M.J., Bennice, L., Cory‐Slechta, D.A., Ballatori, N., Richfield, E.K., 2003. Increased synaptosomal dopamine content and brain concentration of paraquat produced by selective dithiocarbamates. Journal of neurochemistry, 85(4), pp.1075-1086. Beard, J.D., Umbach, D.M., Hoppin, J.A., Richards, M., Alavanja, M.C., Blair, A., Sandler, D.P., Kamel, F., 2014. Pesticide exposure and depression among male private pesticide

25

applicators in the agricultural health study. Environmental health perspectives, 122(9), pp.984-991. Bernabeu, R., Bevilaqua, L., Ardenghi, P., Bromberg, E., Schmitz, P., Bianchin, M., Izquierdo, I., Medina, J.H., 1997. Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proceedings of the National Academy of Sciences, 94(13), pp.7041-7046. Best, P.J., Orr Jr, J., 1973. Effects of hippocampal lesions on passive avoidance and taste

of

aversion conditioning. Physiology & Behavior, 10(2), pp.193-196. Birks, L., Casas, M., Garcia, A.M., Alexander, J., Barros, H., Bergström, A., Bonde, J.P.,

ro

Burdorf, A., Costet, N., Danileviciute, A., Eggesbø, M., 2016. Occupational exposure to

endocrine-disrupting chemicals and birth weight and length of gestation: A European meta-

-p

analysis. Environmental health perspectives, 124(11), pp.1785-1793.

Bisson, M., Hontela, A., 2002. Cytotoxic and endocrine-disrupting potential of atrazine,

re

diazinon, endosulfan, and mancozeb in adrenocortical steroidogenic cells of rainbow trout exposed in vitro. Toxicology and Applied Pharmacology, 180(2), pp.110-117.

lP

Bjørling-Poulsen, M., Andersen, H.R., Grandjean, P., 2008. Potential developmental neurotoxicity of pesticides used in Europe. Environmental Health, 7(1), p.50. Blakemore, S.J., Tavassoli, T., Calò, S., Thomas, R.M., Catmur, C., Frith, U., Haggard, P., 2006.

ur na

Tactile sensitivity in Asperger syndrome. Brain and cognition, 61(1), pp.5-13. Bowler, R.M., Mergler, D., Huel, G., Cone, J.E., 1994. Psychological, psychosocial, and psychophysiological sequelae in a community affected by a railroad chemical disaster. Journal of traumatic stress, 7(4), pp.601-624.

Jo

Camina, E., Güell, F., 2017. The neuroanatomical, neurophysiological and psychological basis of memory: Current models and their origins. Frontiers in pharmacology, 8, p.438.

Cecconi, S., Paro, R., Rossi, G., Macchiarelli, G., 2007. The effects of the endocrine disruptors dithiocarbamates on the mammalian ovary with particular regard to mancozeb. Current pharmaceutical design, 13(29), pp.2989-3004.

26

Chen, Z., Huang, C., Ding, W., 2016. Z-Guggulsterone improves the scopolamine-induced memory impairments through enhancement of the BDNF signal in C57BL/6J mice. Neurochem. Res. 41 (12), 3322–3332. Cobley, J.N., Fiorello, M.L., Bailey, D.M., 2018. 13 reasons why the brain is susceptible to oxidative stress. Redox biology, 15, pp.490-503. Cogan, D.C., Reeves, J.L., 1979. Passive avoidance learning in hippocampectomized rats under different shock and intertrial interval conditions. Physiology & behavior, 22(6), pp.1115-

of

1121. Colborn, T., 2004. Neurodevelopment and endocrine disruption. Environmental health

ro

perspectives, 112(9), pp.944-949.

Cooper, R.L, Kavlock, R.J., 1997. Endocrine disruptors and reproductive development: a weight-

-p

of-evidence overview. Journal of endocrinology, 152(2), pp.159-166.

David, D.J., Samuels, B.A., Rainer, Q., Wang, J.W., Marsteller, D., Mendez, I., Drew, M., Craig,

re

D.A., Guiard, B.P., Guilloux, J.P., Artymyshyn, R.P., 2009. Neurogenesis-dependent andindependent effects of fluoxetine in an animal model of anxiety/depression. Neuron, 62(4),

lP

pp.479-493.

De Saint Blanquat, P., Hok, V., Alvernhe, A., Save, E., Poucet, B., 2010. Tagging items in spatial working memory: A unit-recording study in the rat medial prefrontal

ur na

cortex. Behavioural Brain Research, 209(2), pp.267-273. Dela Pena, I., Yoon, S.Y., Kim, H.J., Park, S., Hong, E.Y., Ryu, J.H., Park, I.H., Cheong, J.H., 2014. Effects of ginseol k-g3, an Rg3-enriched fraction, on scopolamine-induced memory impairment and learning deficit in mice. Journal of ginseng research, 38(1), pp.1-7.

Jo

Dhara, V.R., Dhara, R., 2002. The Union Carbide disaster in Bhopal: a review of health effects. Archives of Environmental Health: An International Journal, 57(5), pp.391-404.

Dobbing, J., Smart, J.L., 1974. Vulnerability of developing brain and behaviour. British Medical Bulletin. Domico, L.M., Cooper, K.R., Bernard, L.P., Zeevalk, G.D., 2007. Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its

27

contribution to neuronal toxicity in mesencephalic cells. Neurotoxicology, 28(6), pp.10791091. Douglas, R.J., 1989. Spontaneous alternation behavior and the brain. In Spontaneous alternation behavior (pp. 73-108). Springer, New York, NY. EPA, 2004. Metam Sodium (Sodium N-Methyldithiocarbamate) risk characterization document. Medical Toxicology Branch. Department of Pesticide Regulation. California Environmental Protection Agency

Revised Toxicology Disciplinary Chapter for the Risk Assessment

of

EPA, 2005. Appendix J – HED Toxicological Chapter for Metam Sodium and MITC. 4th

ro

Fanselow, M.S., Dong, H.W., 2010. Are the dorsal and ventral hippocampus functionally distinct structures?. Neuron, 65(1), pp.7-19.

-p

Fowden, A.L., Forhead, A.J., Coan, P.M. and Burton, G.J., 2008. The placenta and intrauterine programming. Journal of neuroendocrinology, 20(4), pp.439-450.

lP

concepts. Brain Research, 2(1), pp.3-20.

re

Fox, M.W., 1966. Neuro-behavioral ontogeny: A synthesis of ethological and neurophysiological

Fox, W.M., 1965. Reflex-ontogeny and behavioural development of the mouse. Animal behaviour, 13(2-3), pp.234-IN5.

ur na

Frank, N., Christmann, A. and Frei, E., 1995. Comparative studies on the pharmacokinetics of hydrophilic prolinedithiocarbamate, sarcosinedithiocarbamate and the less hydrophilic diethyldithiocarbamate. Toxicology, 95(1-3), pp.113-122. Gallegos, C.E., Bartos, M., Bras, C., Gumilar, F., Antonelli, M.C. and Minetti, A., 2016. Exposure to a glyphosate-based herbicide during pregnancy and lactation induces

Jo

neurobehavioral alterations in rat offspring. Neurotoxicology, 53, pp.20-28.

Giménez-Llort, L., Martínez, E., Ferré, S., 1997. Different effects of dopamine antagonists on spontaneous and NMDA-induced motor activity in mice. Pharmacology Biochemistry and Behavior, 56(3), pp.549-553. Goldman, J.M., Stoker, T.E., Cooper, R.L., Mcelroy, W.K., Hein, J.F., 1994. Blockade of ovulation in the rat by the fungicide sodium N-methyldithiocarbamate: relationship between

28

effects on the luteinizing hormone surge and alterations in hypothalamic catecholamines. Neurotoxicology and Teratology, 16(3), pp.257-268. Goldman, J.M., Stoker, T.E., Cooper, R.L., Mcelroy, W.K., Hein, J.F., 1994. Blockade of ovulation in the rat by the fungicide sodium N-methyldithiocarbamate: relationship between effects on the luteinizing hormone surge and alterations in hypothalamic catecholamines. Neurotoxicology and Teratology, 16(3), pp.257-268. Goldman-Rakic, P.S., Muly III, E.C., Williams, G.V., 2000. D1 receptors in prefrontal cells and

of

circuits. Brain Research Reviews. Goldner, W.S., Sandler, D.P., Yu, F., Hoppin, J.A., Kamel, F., LeVan, T.D., 2010. Pesticide use

ro

and thyroid disease among women in the Agricultural Health Study. American journal of epidemiology, 171(4), pp.455-464.

-p

Gonzalez, P.N., Gasperowicz, M., Barbeito-Andrés, J., Klenin, N., Cross, J.C. and Hallgrímsson, B., 2016. Chronic protein restriction in mice impacts placental function and maternal body

re

weight before fetal growth. PloS one, 11(3), p.e0152227.

Gonzalez-Casanova, I., Stein, A.D., Barraza-Villarreal, A., Feregrino, R.G., DiGirolamo, A.,

lP

Hernandez-Cadena, L., Rivera, J.A., Romieu, I. and Ramakrishnan, U., 2018. Prenatal exposure to environmental pollutants and child development trajectories through 7 years. International Journal of Hygiene and Environmental Health, 221(4), pp.616-622.

ur na

Graham, S.L., Davis, K.J., Hansen, W.H., Graham, C.H., 1975. Effects of prolonged ethylene thiourea ingestion on the thyroid of the rat. Food and cosmetics toxicology, 13(5), pp.493499.

Grube, A., Donaldson, D., Kiely, T., Wu, L., 2011. Pesticides industry sales and usage. US EPA,

Jo

Washington, DC.

Haendel, M.A., Tilton, F., Bailey, G.S., Tanguay, R.L., 2004. Developmental toxicity of the dithiocarbamate pesticide sodium metam in zebrafish. Toxicological Sciences, 81(2), pp.390400.

Halliwell, B., Gutteridge, J.M.C., 1999. Free radicals in medicine and biology. Clarendon: Oxford.

29

Himwich, W.A., 1962a. Biochemical and neurophysiological development of the brain in the neonatal period. In International review of neurobiology (Vol. 4, pp. 117-158). Academic Press. Himwich, W.A., 1962b. Neurochemistry of aging. Postgraduate medicine, 31(2), pp.195-200. Hodge, M.C.E., 1993. Metam sodium: Developmental toxicity in the rabbit. Zeneca Central Toxicology Laborato, Cheshire, UK. Ivanova-Chemishanska, L., 1969. Experimental studies on the effect of some dithiocarbamates

of

on the functional state of thyroid gland. Letopisi Na Hci, 9, p.46. Izquierdo, I., Bevilaqua, L.R., Rossato, J.I., Bonini, J.S., Medina, J.H., Cammarota, M., 2006.

consolidation. Trends in neurosciences, 29(9), pp.496-505.

ro

Different molecular cascades in different sites of the brain control memory

-p

Izquierdo, I., Medina, J.H., 1995. Correlation between the pharmacology of long-term potentiation and the pharmacology of memory. Neurobiology of learning and memory, 63(1),

re

pp.19-32.

Izquierdo, I., Medina, J.H., 1997. Memory formation: the sequence of biochemical events in the

lP

hippocampus and its connection to activity in other brain structures. Neurobiology of learning and memory, 68(3), pp.285-316.

ur na

Jacobsen, P.R., Christiansen, S., Boberg, J., Nellemann, C., Hass, U., 2010. Combined exposure to endocrine disrupting pesticides impairs parturition, causes pup mortality and affects sexual differentiation in rats. International journal of andrology, 33(2), pp.434-442. Joo, Y., Roh, S., 2016. Risk factors associated with depression and suicidal ideation in a rural population. Environmental health and toxicology, 31.

Jo

Kajta, M., Wnuk, A., Rzemieniec, J., Litwa, E., Lason, W., Zelek-Molik, A., Nalepa, I., Rogóż, Z., Grochowalski, A., Wojtowicz, A.K., 2017. Depressive-like effect of prenatal exposure to DDT involves global DNA hypomethylation and impairment of GPER1/ESR1 protein levels but not ESR2 and AHR/ARNT signaling. The Journal of steroid biochemistry and molecular biology, 171, pp.94-109.

30

Koh, S.B., Kim, T.H., Min, S., Lee, K., Kang, D.R., Choi, J.R., 2017. Exposure to pesticide as a risk factor for depression: A population-based longitudinal study in Korea. Neurotoxicology, 62, pp.181-185. Kreider, J.C., Blumberg, M.S., 2005. Geotaxis and beyond: Commentary on Motz and Alberts (2005). Neurotoxicology and teratology, 27(4), pp.535-538. Li, B., He, X., Sun, Y. and Li, B., 2016. Developmental exposure to paraquat and maneb can impair cognition, learning and memory in Sprague-Dawley rats. Molecular

of

BioSystems, 12(10), pp.3088-3097. Lima, C.S., Ribeiro-Carvalho, A., Filgueiras, C.C., Manhães, A.C., Meyer, A., Abreu-Villaça,

ro

Y., 2009. Exposure to methamidophos at adulthood elicits depressive-like behavior in mice. Neurotoxicology, 30(3), pp.471-478.

-p

Lorenzini, C.A., Baldi, E., Bucherelli, C., Sacchetti, B., Tassoni, G., 1996. Role of dorsal hippocampus in acquisition, consolidation and retrieval of rat's passive avoidance response: a

re

tetrodotoxin functional inactivation study. Brain research, 730(1-2), pp.32-39. Łukawski, K., Kamiński, R.M., Czuczwar, S.J., 2008. Effects of selective inhibition of N-

lP

acetylated-α-linked-acidic dipeptidase (NAALADase) on mice in learning and memory tasks. European journal of pharmacology, 579(1-3), pp.202-207. Manservisi, F., Gopalakrishnan, K., Tibaldi, E., Hysi, A., Iezzi, M., Lambertini, L., Teitelbaum,

ur na

S., Chen, J. and Belpoggi, F., 2015. Effect of maternal exposure to endocrine disrupting chemicals on reproduction and mammary gland development in female Sprague-Dawley rats. Reproductive Toxicology, 54, pp.110-119. Marinovich, M., Guizzetti, M., Ghilardi, F., Viviani, B., Corsini, E., Galli, C.L., 1997. Thyroid

Jo

peroxidase as toxicity target for dithiocarbamates. Archives of toxicology, 71(8), pp.508-512. Masuo, Y., Ishido, M., 2011. Neurotoxicity of endocrine disruptors: possible involvement in brain development and neurodegeneration. Journal of Toxicology and Environmental Health, Part B, 14(5-7), pp.346-369.

Michalak, A., Biala, G., 2017. Calcium homeostasis and protein kinase/phosphatase balance participate in nicotine-induced memory improvement in passive avoidance task in mice. Behavioural brain research, 317, pp.27-36.

31

Navarro, A., Sánchez Del Pino, M.J., Gómez, C., Peralta, J.L, Boveris, A., 2002. Behavioral dysfunction, brain oxidative stress, and impaired mitochondrial electron transfer in aging mice. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 282(4), pp.R985-R992. Negga, R., Stuart, J.A., Machen, M.L., Salva, J., Lizek, A.J., Richardson, S.J., Osborne, A.S., Mirallas, O., McVey, K.A., Fitsanakis, V.A., 2012. Exposure to Glyphosate-and/or Mn/ZnEthylene-bis-Dithiocarbamate-Containing Pesticides Leads to Degeneration of γAminobutyric Acid and Dopamine Neurons in Caenorhabditiselegans. Neurotoxicity

of

research, 21(3), pp.281-290.

ro

O'Malley, M., Barry, T., Verder‐Carlos, M., Rubin, A., 2004. Modeling of methyl isothiocyanate air concentrations associated with community illnesses following a metam‐sodium sprinkler

-p

application. American journal of industrial medicine, 46(1), pp.1-15.

Palfai, T., Walsh, T.J., 1980. Comparisons of the effects of guanethidine, 6-hydroxydopamine

and Behavior, 13(6), pp.805-809.

re

and diethyldithiocarbamate on retention of passive avoidance. Pharmacology Biochemistry

lP

Patisaul, H.B., Adewale, H.B., 2009. Long-term effects of environmental endocrine disruptors on reproductive physiology and behavior. Frontiers in behavioral neuroscience, 3, p.10. Patki, G., Solanki, N., Atrooz, F., Allam, F., Salim, S., 2013. Depression, anxiety-like behavior

ur na

and memory impairment are associated with increased oxidative stress and inflammation in a rat model of social stress. Brain research, 1539, pp.73-86. Perera, F.P., Tang, D., Rauh, V., Lester, K., Tsai, W.Y., Tu, Y.H., Weiss, L., Hoepner, L., King, J., Del Priore, G. and Lederman, S.A., 2005. Relationships among polycyclic aromatic hydrocarbon–DNA adducts, proximity to the World Trade Center, and effects on fetal

Jo

growth. Environmental health perspectives, 113(8), pp.1062-1067.

Piccoli, C., Cremonese, C., Koifman, R.J., Koifman, S., Freire, C., 2016. Pesticide exposure and thyroid function in an agricultural population in Brazil. Environmental research, 151, pp.389398. Pruett, S.B., Cheng, B., Fan, R., Tan, W., Sebastian, T., 2009. Oxidative stress and sodium methyldithiocarbamate–induced modulation of the macrophage response to lipopolysaccharide in vivo. Toxicological sciences, 109(2), pp.237-246.

32

Pruett, S.B., Cheng, B., Fan, R., Tan, W., Sebastian, T., 2009. Oxidative stress and sodium methyldithiocarbamate–induced modulation of the macrophage response to lipopolysaccharide in vivo. Toxicological sciences, 109(2), pp.237-246. Pruett, S.B., Myers, L.P., Keil, D.E., 2001. Toxicology of metam sodium. Journal of toxicology and environmental health part B: critical reviews, 4(2), pp.207-222. Rammal, H., Bouayed, J., Younos, C., Soulimani, R., 2008. Evidence that oxidative stress is linked to anxiety-related behaviour in mice. Brain, behavior, and immunity, 22(8), pp.1156-

of

1159. Rath, N.C., Rasaputra, K.S., Liyanage, R., Huff, G.R. and Huff, W.E., 2011. Dithiocarbamate

ro

toxicity-an appraisal. In Pesticides in the modern world-effects of pesticides exposure. InTechOpen.

-p

Rauch, S.A., Braun, J.M., Barr, D.B., Calafat, A.M., Khoury, J., Montesano, M.A., Yolton, K., Lanphear, B.P., 2012. Associations of prenatal exposure to organophosphate pesticide

re

metabolites with gestational age and birth weight. Environmental health perspectives, 120(7), pp.1055-1060.

lP

Ribas-Fito, N., Cardo, E., Sala, M., Eulalia De Muga, M., Mazon, C., Verdu, A., Kogevinas, M., Grimalt, J.O., Sunyer, J., 2003. Breastfeeding, exposure to organochlorine compounds, and neurodevelopment in infants. Pediatrics 111, 580–585.

ur na

Rice, D., Barone, J. S.,2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environmental health perspectives, 108(suppl 3), 511-533.

Salim, S., Asghar, M., Chugh, G., Taneja, M., Xia, Z., Saha, K., 2010. Oxidative stress: a

Jo

potential recipe for anxiety, hypertension and insulin resistance. Brain research, 1359, pp.178185.

Santillán, M.E., Vincenti, L.M., Martini, A.C., de Cuneo, M.F., Ruiz, R.D., Mangeaud, A., Stutz, G., 2010. Developmental and neurobehavioral effects of perinatal exposure to diets with different ω-6: ω-3 ratios in mice. Nutrition, 26(4), pp.423-431. Sapey‐Triomphe, L.A., Lamberton, F., Sonié, S., Mattout, J. and Schmitz, C., 2019. Tactile hypersensitivity and GABA concentration in the sensorimotor cortex of adults with autism. Autism Research.

33

Secher, T., Novitskaia, V., Berezin, V., Bock, E., Glenthøj, B., Klementiev, B., 2006. A neural cell adhesion molecule–derived fibroblast growth factor receptor agonist, the FGL-peptide, promotes early postnatal sensorimotor development and enhances social memory retention. Neuroscience, 141(3), pp.1289-1299. Short Jr, R.D., Russel, J.Q., Minor, J.L., Lee, C.C., 1976. Developmental toxicity of ferric dimethyldithiocarbamate and bis (dimethylthiocarbamoyl) disulfide in rats and mice. Toxicology and applied pharmacology, 35(1), pp.83-94.

of

Smallridge, R.C. and Ladenson, P.W., 2001. Hypothyroidism in pregnancy: consequences to neonatal health. The Journal of Clinical Endocrinology & Metabolism, 86(6), pp.2349-2353.

ro

Solanki, N., Salvi, A., Patki, G., Salim, S., 2017. Modulating oxidative stress relieves stressinduced behavioral and cognitive impairments in rats. International Journal of

-p

Neuropsychopharmacology, 20(7), pp.550-561.

Soleo, L., Defazio, G., Scarselli, R., Zefferino, R., Livrea, P., Foà, V., 1996. Toxicity of

re

fungicides containing ethylene-bis-dithiocarbamate in serumless dissociated mesencephalicstriatal primary coculture. Archives of toxicology, 70(10), pp.678-682.

lP

Stevens, M., Freeman, J., 2018. Efficacy of dimethyl disulfide and metam sodium combinations for the control of nutsedge species. Crop protection, 110, pp.131-134. Sullivan, F.M., 1993. Impact of the environment on reproduction from conception to

ur na

parturition. Environmental health perspectives, 101(suppl 2), pp.13-18. Tamaki, Y., Hidaka, H., 1976. Behavioral depression in Sidman avoidance learning induced by dopamine β-hydroxylase inhibitors. Psychological reports, 38(2), pp.437-438. Tilton, F., La Du, J.K., Vue, M., Alzarban, N. and Tanguay, R.L., 2006. Dithiocarbamates have a

Jo

common toxic effect on zebrafish body axis formation. Toxicology and applied pharmacology, 216(1), pp.55-68.

Tinston, D.J., 1993. Metam sodium developmental toxicity study in the rat. Zeneca Central Toxicology Laboratory, Cheshire, UK. Vanz, F., Bicca, M.A., Linartevichi, V.F., Giachero, M., Bertoglio, L.J., de Lima, T.C.M., 2018. Role of dorsal hippocampus κ opioid receptors in contextual aversive memory consolidation in rats. Neuropharmacology, 135, pp.253-267.

34

Worthing, C.R., Walker, S.B., 1983. The Pesticide Manual, British Crop Protection Council. British Crop Protection Council, Croydon, p.89. Yoshida, S., Numachi, Y., Matsuoka, H., Sato, M., 1998. Impairment of cliff avoidance reaction induced by subchronic methamphetamine administration and restraint stress: comparison between two inbred strains of rats. Progress in neuro-psychopharmacology & biological

Jo

ur na

lP

re

-p

ro

of

psychiatry, 22(6), pp.1023-1032.

35

re

-p

ro

of

Figure 1: Experimental design of treatment administration and behavioral testing.

lP

Figure 2: Evaluation of sensorimotor development. A: Latency of surface righting reflex test at P5, 7 and 9. B: Cliff avoidance reflex at P6. C: Percentage of tactile response in body stimulation test. D: Performance in negative geotaxis test at P10 and 12. Results are presented as £££

ur na

mean ± SEM. *P<0.05; **P<0.01; ***P<0.001; #P<0.05; ###P<0.001; £P<0.05; ££P<0.01; P<0.001. ‘*’ treated groups (50, 100 or 150 mg/kg) vs control group (0 mg/kg), ‘#’ treated

Jo

group with 150 or 100 vs group of 50 mg/kg, ‘£’ group of 150 vs group of 100 mg/kg.

-p

ro

of

36

Jo

ur na

lP

the open field. B: The moving velocity.

re

Figure 3: Effect of prenatal exposure to MS on locomotor activity. A: Total distance traveled in

Figure 4: Effect of prenatal exposure to MS on depression-like behavior. A: Time of immobility recorded during tail suspension test. B: Grooming time measured during splash test. Results are

37

presented as mean ± SEM. * P<0.05; ** P<0.01; ***P<0.001; #P<0.05; ##P<0.01. ‘*’ treated groups (50, 100 or 150 mg/kg) vs control group (0 mg/kg), ‘#’ group of 150 or 100 vs group of

lP

re

-p

ro

of

50 mg/kg.

Figure 5: Effect of MS exposure on memory retention in step-through passive avoidance test 2h and 24h following foot shock administration. Results are presented as mean ± SEM. **P<0.01;

Jo

ur na

***P<0.001. ‘*’ treated groups (50, 100 or 150 mg/kg) vs control group (0 mg/kg).

Jo

ur na

lP

re

-p

ro

of

38

Figure 6: Effect of MS exposure on working memory in Y-maze test. A: Percentage of spontaneous alternation. B: Total number of arm entries.

of

ro

-p

re

lP

ur na

Jo

39

40

Table 1: Effect of MS on fertility and reproduction parameters as well as body weight gain in dams and offspring. Results are presented as mean ± SEM. *P<0.05; **P<0.01; #P<0.05; ##

P<0.01; £P<0.05 and ££P<0.01. ‘*’ treated groups (50, 100 or 150 mg/kg) vs control group (0

mg/kg), ‘#’ group treated with 150 or 100 mg/kg vs group treated with 50 mg/kg of MS, ‘£’ 150 vs 100 mg/kg treated groups.

MS exposure (mg/kg/day)

Parameters

50

100

Number of females with vaginal plug

10

9

10

Gestation index (%)

50

66.66

70

87.5

Percentage of aborted females (%)

0

0

0

28.57

Mortality number during gestation

0

0

Gestation period (days)

19±0.63

18.42±0.29

Deliverance index (%)

100

Live-birth index (%)

-p

ro

Fertility parameters

150

of

Control

8

0

1

18.71±0.18

20±0.32 #

100

100

57.14

92.67±4.52

83.33±16.67

97.22±1.82

89.90±6.15

Viability index (%)

100±0

61.79±16.98 *

98.57±1.43 #

87.12±8.05

Lactation index (%)

100±0

68.49±5.92 **

94.32±4.00 ##

61.21±9.82 **££

Number of pups/litter

9±1.26

7.28±1.44

8.71±0.68

8±2.04

Body weight gain at GD7

0.05±0.37

0.06±0.19

-1.08±0.90

0.18±0.26

Body weight gain at GD15

9.50±2.15

11.15±1.74

12.73±2.56

9.34±1.42

Body weight gain at GD18

14.45±1.38

15.15±1.89

18.20±2.70

14.34±1.70

Body weight at P0

1.60±0.01

1.37±0.03

1.24±0.01

1.38±0.01

Body weight at P5

3.19±0.04

2.82±0.23

2.61±0.06

2.61±0.04

Body weight at P12

5.36±0.14

5.28±0.67

4.91±0.07

5.43±0.06

Body weight at P24

8.93±0.67

8.73±0.40

8.73±0.18

8.67±0.11

% of males

40

50

37.25

52.38

ur na

lP

re

Reproduction parameters

Maternal body weight gain (g)

Jo

Offspring body weight (g)