Neurodevelopmental effects of perinatal fenarimol exposure on rats

Reproductive Toxicology 23 (2007) 98–105

Neurodevelopmental effects of perinatal fenarimol exposure on rats Vera L.S.S. de Castro a,∗ , Michele A. de Mello b , Carlos Diniz c , Lia Morita c , Tˆania Zucchi b , Paola Poli b,d b

a Embrapa Meio Ambiente, Laborat´ orio de Ecotoxicologia, Rodovia SP 340, km 127.5, 13820-000 Jaguari´una, SP, Brazil Depto de Parasitologia, Instituto de Ciˆencias Biom´edicas, USP, Av. Prof. Lineu Prestes, 1374 Cidade Universit´aria, 05508-900 S˜ao Paulo, SP, Brazil c Universidade Federal de S˜ ao Carlos (UFSCAR), S˜ao Carlos, SP, Brazil d Dipartimento di Genetica, Biologia dei Microrganismi, Antropologia, Evoluzione, Universit` a degli Studi di Parma, Parco Area delle Scienze 11/a, 43100 Parma, Italy

Received 30 August 2005; received in revised form 12 August 2006; accepted 6 September 2006 Available online 12 September 2006

Abstract Knowledge about the potential toxic effects of fenarimol, a widely used fungicide, is still limited. Fenarimol is an aromatase inhibitor and therefore can affect estrogen/androgen levels in vivo in rodents. In view of these facts, the aim of the present work was to study the effects of fenarimol maternal exposure during different critical phases in the development of central nervous system in rat pups, on early physical and neurobehavioral endpoints essential to their development. For that, the effects of the fungicide fenarimol (150 and 300 mg/kg) were examined at three different developmental stages in the rat: during the first 6 days of gestation, prenatal (15–21 days), or first 6 days of lactation. Three categories of the impact of fenarimol on neonatal growth and neurobehavioral development of offspring were assessed: (1) physical, (2) reflex and strength, and (3) motor coordination. Findings on the pups’ physical development did not indicate any significant alterations of the postnatal age at which specific developmental milestones were observed (pinna detachment, development of the fur, eruption of the incisor teeth, opening of the ears and eyes and testes descent). However, there was a reduced rate of weight gain in pups of mothers treated during lactation related to the earlier testing time periods (1–23 days of life). The study of the functional state of the rat pup nervous systems at different stages of postnatal development revealed some neurodevelopmental delays in righting reflex, climbing and grip response and locomotion (20–90 days of life) in the treated groups. Taken together, findings of this study emphasize that, as a result of fenarimol maternal exposure, some neuromuscular and behavioral deficits in nursing pups may occur principally during the last gestational period and lactation. These results could be the basis for further studies on molecular actions of fenarimol in order to predict better the biological consequences of this fungicide. © 2006 Elsevier Inc. All rights reserved. Keywords: Fenarimol; Animal development; Biomarkers; Rats

1. Introduction The development of major regulatory systems underlying behavior and physiology in the neonatal rat is primarily determined by the dam, who serves as the primary source of the nutrition, grooming and warmth required for immediate survival [1], thereby playing a crucial role in the postpartum development of the architecture of the brain. Damage to a particular structure in the circuit or to connecting pathways in the developing nervous system, which is more susceptible than the adult nervous system, can produce structural or functional changes that result

∗

Corresponding author. Fax: +55 19 38678740. E-mail address: [email protected] (V.L.S.S. de Castro).

0890-6238/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2006.09.001

in behavioral changes [2–8]. In addition, the vulnerability of the developing brain to toxic insults is dependent on exposure and on the stage of development of the potential target organ or system. Since the maturity of enzyme systems can influence the effects of exposure to xenobiotic compounds, the developmental effects of such exposure cannot be accurately evaluated unless the developing organism is exposed to the substance at times when the target organ is most likely to be affected [9,10]. Neurobehavioral performance can be a sensitive biomarker of the neurodevelopmental consequences of environmental exposure, as has been reported in cases of pesticide exposure [11]. Fenarimol is an organic fungicide that has both preventive and curative properties. It is effective in controlling apple scab, as well as powdery mildew (of pea, cherry, grape and others), but

V.L.S.S. de Castro et al. / Reproductive Toxicology 23 (2007) 98–105

can be cytotoxic [12]. In rat offspring of exposed dams, 150 and 300 mg/kg of fenarimol acts as a genotoxin, primarily related to direct exposure of the litter via maternal milk, since the DNA damage increases during the time of exposure (2 h to 6 days after the birth) [13]. Fenarimol possesses estrogenic properties [14] and acts both as an estrogen agonist and as an androgen antagonist [15]. In addition, fenarimol affects rat aromatase activity in vivo, inhibiting estrogen biosynthesis in rat microsomes [16] and in human tissues [17]. This compound also affects other enzymes of the cytochrome P450 gene family that are involved in the metabolism of steroids [18]. Studies of reproductive toxicity in rats have shown that fenarimol reduces male fertility, both in exposed adult males and in the male offspring of exposed females. This effect is manifested as an absence of male sexual behavior that might result from altered perinatal development of male patterns of sexual behavior [16,19]. Exposing pregnant rats to fenarimol affects perinatal development of male sexual behavior by placental transfer, minimal in the early gestational stages but more pronounced in later stages, as well as by transfer (in markedly high way) via lactation [19]. Pharmacokinetic data [19] on pregnant dams also suggest the potential for direct access to the brain tissue of fetuses (placental transfer) and neonates (transfer via lactation). The aim of the present work was to study the effects that maternal fenarimol exposure has on early physical and neurobehavioral outcomes. To that end, we exposed groups of pregnant rats to fenarimol during different phases, including the prenatal phase (days 15–21 of gestation) and the first 6 days of lactation, periods of expected high levels fungicide transfer [19], both of which have been shown to be particularly important for offspring brain maturation and behavioral development [1,2,4]. For the purpose of comparison with a period of expected low fungicide transfer, an additional group of pregnant rats was exposed during early gestation (days 1–6). 2. Materials and methods 2.1. Chemicals Fenarimol [␣-(2-chlorophenyl)-␣-(4-chlorophenyl)-5-pyrimidinemethanol; CAS 60168-88-9; PM: 331.20; Rubigan® , containing 120 mg/ml of the active principle] was obtained from Dow AgroSciences (Indianapolis, IN, USA).

2.2. Animals Male and nulliparous female Wistar rats, aged 90 days and weighing 230 ± 15 g, from our breeding colony (Embrapa Meio Ambiente, S˜ao Paulo, Brazil) were housed individually (except during mating) in polycarbonate cages hardwood chip with bedding and given ad libitum access to food (Purina Lab Chow) and tap water. National and institutional guidelines for housing and treatment were followed. Animals were maintained in a temperature-controlled environment (22 ± 2 ◦ C) at 70% humidity and on a 12-hour light/dark cycle. The P-generation breeders in our colony were obtained from the University of Campinas facilities of Cemib (Campinas, SP, Brazil), which is recognized by the International Council for Laboratory Animal Science. The general health status data for our colony are controlled by standard operating procedures (ISO 9001:2000). In order to monitor general health status and neurodevelopmental milestones of these rats, the data obtained are compared to those found in the literature, as well as to

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historical data of our laboratory where similar experiments are repeatedly performed.

2.3. Experimental design Vaginal smears were obtained and examined daily for 12 consecutive days preceding mating and pesticide exposure. The smears were prepared to assess the frequency and duration of the estrous cycle. Smears representing the estrous cycle phase of each female were then examined and compared in order to exclude those with estrous cycle abnormalities. Males, previously determined to be fertile, were housed overnight with females. The day on which a vaginal plug was found or spermatozoa was found in the vaginal smear was considered gestational day 0. Sperm-positive females were randomized by weight to receive subcutaneous injections of fenarimol (suspended in 0.9% saline solution supplemented with two drops of Tween 80). The females were injected subcutaneously with a total dosage of 150 or 300 mg/kg fenarimol (delivered in six daily doses of 0, 25.0 or 50.0 mg/kg d) to animals assigned to each of the three different exposure periods. Those groups were further subdivided based on the phase in which they would be exposed: Pri (gestational days 1–6); Prf (gestational days 15–21); and Lac (the first 6 days of lactation). Controls were injected with saline solution alone. After mating and throughout gestation, all pregnant rats were weighed and examined for signs of toxicity daily. The prenatal and lactation periods were included since offspring sensitivity to fenarimol has been reported to be higher during those phases [13,19]. Analyzing the effect of early gestational exposure allows comparisons to be made between periods of high and low fungicide transfer to the fetus or offspring. From gestational day 21 until delivery, each presumably pregnant female was checked twice daily (at 9:00 and 17:00 h) for completion of or difficulties in parturition. The day of parturition was defined as postnatal day (PND) 1. For litters born after 17:00 h, the following day was considered PND 1. The pups were counted, examined for gross malformations and weighed individually. From PNDs 1 to 23, pup body weights were recorded daily during nursing. Within each litter, the mean body weight was considered the litter weight, and the litter was considered the experimental unit. The pups were also weighed on days 30, 60 and 90 prior to evaluation of open-field locomotion. On PND 23, pups were sorted by sex based on anogenital distance and housed in same-exposure, single-sex pairs or triplets. Reflex responses and locomotor skills were tested, as described below, in males only. After parturition, the neonates were observed for mortality and signs of toxicity. The offspring, tested in an alternating sequence of experimental and control pups, were evaluated for survival, growth, development and behavior. When parturition was complete, the numbers of stillborn and live pups in each litter were recorded. Each litter was examined daily for any change in appearance, behavior or survival, and all deaths were recorded. The litter size was not adjusted, and daily recorded in order to avoid interfering with maternal-litter unit behavior since it is possible that litters with fewer pups are the most affected by fenarimol. Nevertheless, the number of viable pups at birth was not affected by treatment [13].

2.4. Developmental milestones In order to provide warmth before and after testing and to avoid excessive stress during tests due to manipulation, each pup was removed from its nest in a paper towel and placed on a heating pad. Physical developmental parameters such as pinna detachment (unfolding of the external ear), fur development, incisor eruption, complete opening of both ears and complete opening of both eyes, as well as testes descent (into the scrotum), were expressed as the number of days required for the appearance of these milestones. Oral cavities were examined daily to determine the first day of eruption of the upper and lower incisors, determined as the appearance of the tip (crown) of the incisor. The PNDs on which these milestones appeared were recorded until all pups in a given litter were positive for all developmental parameters. Consequently, the frequency of animals showing each of the above parameters was recorded for each day of the observational period. For that, each litter was considered an experimental unit. The order of testing the litters was determined randomly. All data regarding the timing of the appearance of these markers of physical development were compared with those for normal development of the strain (Wistar) [20].

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2.5. Reflex development Throughout the lactation period, the pups were evaluated in terms of reflex development and neuromuscular maturation. On various PNDs [21,22], the following parameters were tested: 1. Righting reflex (PNDs 2–5). This test measures motor function and coordination. The newborn pup was held on its back on a padded flat surface and then released. The righting reflex was defined as the number of seconds required for a newborn lying on its back to right itself on all four limbs and was considered fully formed if the rat pup did so within 30 s. 2. Climbing response (PNDs 7–10). This test reflects development in grasping reflex and muscular strength. The time required to climb to the top of an inclined (45◦ ) wire mesh screen was recorded. Normality was defined as a climbing time of no longer than 120 s. 3. Grip response (PNDs 3–7). In this test, the pup was encouraged to hang by its forepaws on a horizontal rod 30 cm above a 2.5-cm thick bed of wood shavings. Gripping ability was considered developed when the pup was able to hang from the rod. 4. Neuromotor development evaluation. Other randomly selected and marked pups were separated for neuromotor development evaluation. Only males were tested since sex can influence locomotion [24,25]. Locomotion behavior was evaluated for three pups per litter on PNDs 21, 30, 60 and 90 in an open-field arena (each experimental group: n = 10 litters). Each animal was placed in the center of the arena and assessed for 6 min, divided into two equal periods. After each test, the arena was washed with a solution of 5% alcohol in water. Hand-operated counters and stopwatches were used to score ambulatory frequency (number of units entered). The observers were blinded as to the treatment groups. Control and study pups were examined alternately so that the time of day did not bias the results. Three (tail tattoo) identified male pups from each dam (10 litters/experimental point) were used on each test day as described previously [23]. Since the litter factor is a significant source of variation in results obtained from behavioral measurements, the mean of the three scores was considered the litter performance, and each litter was considered an experimental unit.

Righting reflex, climbing response and grip strength were analyzed using one-way ANOVA followed by a simple effects test (Dunnett’s C test), when appropriate, using the general linear model procedure. The Kruskal–Wallis test was used to measure the statistical significance of pup locomotion score (openfield data). The level of statistical significance was set at p < 0.05. The personnel monitoring the tests were blinded as to the group to which the pup they observed belonged.

3. Results 3.1. Milestones—physical development All sperm-positive females became pregnant. The neonates did not show any physical signs of toxicity. Fenarimol exposure did not affect maternal weight gain, gestation duration, the numTable 1 Mean weight at different postnatal days of pups of dams exposed to fenarimol during different periods Group

Days 1

30

60

90

Pri Control Fen 150 Fen 300

5.9 ± 0.1 6.9 ± 1.2 6.4 ± 0.6

92.4 ± 6.6 83.5 ± 3.7 88.7 ± 7.6

214.1 ± 23.1 203.9 ± 44.8 209.6 ± 16.9

298.1 ± 20.9 297.0 ± 27.3 299.9 ± 15.0

Prf Control Fen 150 Fen 300

6.2 ± 0.5 5.6 ± 0.6 5.5 ± 0.9

88.9 ± 8.3 77.6 ± 7.7 81.1 ± 6.4

171.2 ± 41.5 212.6 ± 26.1 222.0 ± 25.2

288.4 ± 20.5 302.6 ± 18.7 293.6 ± 20.0

2.6. Statistical analysis

Lac Control Fen 150 Fen 300

The data were analyzed using the statistical and graphical functions of SPSS 11 (SPSS Inc., Chicago, IL, USA). The litter was considered as the unit of analysis. Parametric and nonparametric statistical tests were used depending on the specific type of data. Physical milestones were analyzed using one-way ANOVA with repeated measurements.

6.0 ± 0.5 5.9 ± 1.0 6.2 ± 0.3

75.4 ± 14.8 78.8 ± 6.4 74.7 ± 8.3

186.0 ± 33.9 213.2 ± 1 9.2 211.0 ± 8.2

277.9 ± 42.1 303.3 ± 12.4 295.7 ± 6.3

Control: saline solution; Fen 150 and Fen 300: 150 and 300 mg/kg fenarimol, respectively. Pri: first 6 days of gestation; Prf: prenatal (15–21 days); Lac: first 6 days of lactation. Data were expressed as mean ± S.D. (g) The litter was considered as the experimental unit (10 litters/treatment).

Fig. 1. Weight increase (g) of offspring (mean of 10 litters/treatment, PND 1–23) of dams exposed to fenarimol (Fen 150 and Fen 300) or control solution at the beginning of pregnancy (1st–6th day; Pri), final period of pregnancy (15th–21st day; Prf), or first 6 days of lactation (Lac).

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Table 2 Physical developmental milestones in pups of dams exposed to fenarimol during different periods (mean ± S.D.a ) Group

Postnatal day Pinna detachment

Fur development

Incisor eruption

Ear opening

Eye opening

Testes descent

Pri Control Fen 150 Fen 300

3.7 ± 0.4 2.9 ± 0.4 3.1 ± 0.3

5.3 ± 0.8 5.4 ± 0.5 5.2 ± 0.4

4.3 ± 1.2 5.3 ± 0.8 4.7 ± 0.5

11.7 ± 0.8 10.6 ± 1.9 12.9 ± 0.8

15.1 ± 0.7 14.3 ± 1.4 13.9 ± 1.8

17.6 ± 1.3 16.6 ± 1.3 17.6 ± 0.9

Prf Control Fen 150 Fen 300

3.6 ± 0.5 3.6 ± 0.5 3.6 ± 0.5

4.2 ± 0.4 4.8 ± 0.4 5.0 ± 0.0

5.3 ± 0.5 5.4 ± 0.5 5.3 ± 0.5

13.1 ± 1.5 14.1 ± 0.6 13.8 ± 0.9

14.6 ± 0.5 15.3 ± 0.9 14.9 ± 0.6

17.6 ± 1.0 18.4 ± 0.5 17.9 ± 0.7

Lac Control Fen 150 Fen 300

3.3 ± 0.5 3.5 ± 0.8 3.4 ± 0.5

5.1 ± 0.3 5.1 ± 0.3 5.0 ± 0.0

5.7 ± 0.5 4.7 ± 0.7 5.0 ± 0.9

13.6 ± 0.7 12.9 ± 1.7 14.6 ± 0.9

14.7 ± 0.7 14.3 ± 1.2 15.0 ± 1.0

16.7 ± 1.3 16.2 ± 2.1 16.8 ± 1.6

Control: saline solution; Fen 150 and Fen 300: 150 and 300 mg/kg fenarimol, respectively. Pri: first 6 days of gestation; Prf: prenatal (15–21 days); Lac: first 6 days of lactation. a The litter was considered as the experimental unit. The values correspond to the days of life required for the entire litter to show each milestone (mean ± S.D., n = 10 whole litter/treatment).

ber of newborns or pup mortality. Pups of dams exposed to the higher dose of fenarimol during the lactation period (Fen Lac 300) presented lower body weights from PND 1 through PND 23 than did those in the control and Fen Lac 150 groups (Fig. 1). However, after PND 30, body weights were comparable among all groups (Table 1). All litters were observed daily to evaluate the possible effects of maternal fenarimol exposure. The results were analyzed (ANOVA) to determine whether there was significant delay or hastening of the appearance of some physical milestones (pinna detachment, fur development, eruption

of incisor teeth, opening of ears, opening of eyes and testes descent). Maternal exposure to fenarimol did not appear to affect these parameters significantly under the observational conditions employed in the present study. The time required, measured in days of life, to reach each one of the milestones evaluated did not differ in relation to dose or phase during which the dam was exposed (Table 2). Nevertheless, a few developmental markers appeared later in the Prf Fen 150 group than in the Pri Fen 150 and Lac groups (ear opening and testes descent, respectively), and those differences were statistically significant.

Fig. 2. Pup’s climbing response of dams treated with saline solution (control) or 150 and 300 mg/kg fenarimol (Fen 150 and Fen 300) at the beginning of pregnancy (1st–6th day; Pri), final period of pregnancy (15th–21st day; Prf), or first 6 days of lactation (Lac). Data were expressed as the time (mean ± S.E. of 10 litters/treatment; the mean of the values determined on three pups was considered the litter performance) necessary to show the considered behavior at different postnatal days (age).

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Fig. 3. Pup’s righting reflex (B) of dams treated with saline solution (control) or 150 and 300 mg/kg fenarimol (Fen 150 and Fen 300) at the beginning of pregnancy (1st–6th day; Pri), final period of pregnancy (15th–21st day; Prf), or first 6 days of lactation (Lac). Data were expressed as the time (mean ± S.E. of 10 litters/treatment; the mean of the values determined on three pups was considered the litter performance).

3.2. Milestones—reflex development Reflex development and neuromuscular maturation were evaluated for three pups from each dam. The parameters considered were righting reflex and climbing response (both expressed in seconds), as well as grip response (expressed as the mean number of days required for the pups to present the response). The study of the functional state of the rat pup nervous systems at different stages of postnatal development revealed differences in terms of when the reflex was expressed. In general, fenarimol exposure delayed the appearance of the climbing response in pups (Fig. 2), with a peculiar modulation of the effect depending on the period of maternal exposure. For the Pri and Prf treatments, taking age-related variability (five different PNDs) and age–dose interactions into consideration, the mean climbing time in fenarimol groups differed significantly from that measured in their corresponding controls (Dunnett’s C, p < 0.05). However, there was no clear dose-dependent increase in the marginal means (Pri control: 98.4 s; Pri Fen 150: 169.7 s; Pri Fen 300: 152.1 s, F2,136 = 19.249, p < 0.001; Prf control: 63.4 s; Prf Fen 150: 158.5 s; Prf Fen 300: 156.4 s, F2,136 = 46.794, p < 0.001). For the Lac treatment, a clear and significant relationship was found between mean climbing time and fenarimol dose (Dunnett’s C test, p < 0.05; Lac control: 58.6 s; Lac Fen 150: 127.7 s; Lac Fen 300: 223.3 s, F2,136 = 120.775, p < 0.001). Furthermore, at the end of the observational period (PND 10), all fenarimol groups presented climbing times that were approximately double those recorded for the respective control groups. In short, fenarimol exposure increased the climbing time independently of the exposure period, although the greatest difference was recorded when the dams were exposed during the lactation phase.

Maternal fenarimol exposure also seemed to delay development of the righting reflex in newborns (Fig. 3). For Prf and Lac exposure, a significant difference (Dunnett’s C test, p < 0.05) was detected between the control and fenarimol groups in terms of mean righting time, taking age-related variability (four different PNDs) and age-dose interactions into consideration (Prf control: 1 s; Prf Fen 150: 3.1 s; Prf Fen 300: 2.8 s, F2,109 = 8.557, p < 0.001; Lac control: 1.8 s; Lac Fen 150: 3.1 s; Lac Fen 300: 3.3 s, F2,109 = 4.102, p < 0.01). No significant differences were found for the Pri group, although the mean righting time was Table 3 Grip response in pups of dams exposed to fenarimol during different periods (mean ± S.D.a ) Group

Postnatal day

Pri Control Fen 150 Fen 300

3.2 ± 0.4 3.3 ± 0.6 3.3 ± 0.7

Prf Control Fen 150 Fen 300

3.3 ± 0.5 3.6 ± 0.8 3.5 ± 0.8

Lac Control Fen 150 Fen 300

3.0 ± 0.2 3.8 ± 1.0* 3.7 ± 0.9*

Control: saline solution; Fen 150 and Fen 300: 150 and 300 mg/kg fenarimol, respectively. Pri: first 6 days of gestation; Prf: prenatal (15–21 days); Lac: first 6 days of lactation. a The values correspond to the days of life necessary to the litter shows the grip response (mean ± S.D., n = 10 litters/treatment; the mean of the values determined on three pups was considered the litter performance). * Significant differences in relation to the control (Dunnett’s C, p < 0.05).

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Fig. 4. Open-field performance of offspring of dams treated with saline solution (control) or 150 and 300 mg/kg fenarimol (Fen 150 and Fen 300) at the beginning of pregnancy (1st–6th day; Pri), final period of pregnancy (15th–21st day; Prf), or first 6 days of lactation (Lac). The parameter recorded was locomotion (number of unit entered during 6 min) at different postnatal days (age). Data were expressed as mean ± S.E. of 10 litters/treatment (the mean of the values determined on three pups was considered the litter performance).

shorter in the control group (Pri control: 1.6 s; Pri Fen 150: 2.5 s; Pri Fen 300: 2.8 s, F2,109 = 2.527, p = 0.081). Nevertheless, pups in all groups achieved complete maturation of this reflex within the period of observation. All newborns tested developed the grip response (Table 3), with significant differences (Dunnett’s C test, p < 0.05) between the control and fenarimol groups depending on the phase during which the dam was exposed (Lac but not Pri and Prf), although without a dose-dependent difference (Pri: F2,27 = 0.239, p = 0.788; Prf: F2,27 = 2.028, p = 0.138; Lac: F2,27 = 8.385, p < 0.001). Offspring locomotion behavior at different ages (PND 21, 30, 60 and 90) is presented in Fig. 4. Open-field testing

detected significant alterations in mean locomotion scores on PND 21 (Table 4, Kruskal–Wallis, p < 0.05), mainly dependent on the period of exposure as clearly shown by the number of unit entered for each treatment (Prf Fen 300: 22.3 ± 14.2; Prf Fen 150: 33.4 ± 26.1; Prf control: 50.6 ± 18.4; Lac Fen 300: 15.4 ± 14.8; Lac Fen 150: 31.9 ± 22.8; Lac control: 48.8 ± 21.8. This alteration was maintained up until PND 90 in the Lac group (Table 4, Kruskal–Wallis, p < 0.05). 4. Discussion Fenarimol given to pregnant rats did not cause any overt sign of maternal toxicity nor did it affect maternal weight. However,

Table 4 Kruskal–Wallis test data for offspring locomotion behavior at different ages Postnatal day

Group

N

Mean rank

Chi-square

Degrees of freedom

21

Prf Control Prf Fen 150 Prf Fen 300

10 10 10

22.80 14.85 8.85

12.645

2

Total

30

Lac Control Lac Fen 150 Lac Fen 300

10 10 10

23.50 14.50 8.50

10.539

2

Total

30

Lac Control Lac Fen 150 Lac Fen 300

10 10 10

20.50 17.70 8.30

14.716

2

Total

30

21

90

Prf group, PND21; Lac group, PND21 and PND90. The litter was considered as the experimental unit (10 litters/treatment).

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we found some effects on the pups, probably due to maternal transfer to the newborns during lactation (Fig. 1). Although, in the absence of evident signs of maternal toxicity, the offspring presented some growth retardation, it is unclear whether fenarimol had a direct effect on the fetus. In addition, our experimental design did not allow the effects in offspring to be related to potential maternal damage, be it physical or behavioral, which warrants further studies. The postnatal effects of maternal fenarimol exposure on pup development were studied. However, the data obtained in the present study show that fenarimol exposure does not affect the maturation of physical developmental milestones in a dosedependent manner. The effect on weight gain in offspring of mothers treated during lactation (Fig. 1) could partially explain some of the functional deficits in neuromotor maturation. In addition, some physical anomalies were observed in the Pfr Fen 150 group. Although we were unable to detect a consistent effect of increasing doses of fenarimol when given either at pregnancy or at lactation, nonlinear dose responses are not uncommon and actually seem reasonable in view of the complexity of the systems they target. The absence of a dose–effect relationship might be attributable to the reactions of a complex biological system to a toxicant since not all brain regions develop within the same time frame [4]. Further studies are needed in order to better elucidate the mechanisms of milestone parameter deficits following exposure to fenarimol. The results of the open-field locomotion test revealed changes related to fenarimol exposure during the perinatal and lactation periods, with a consequent decrease in locomotion, mainly during lactation. These findings could indicate long-term neurotoxicity [8,11,28], although they do not shed light on the question of whether the lower number of units recorded is related to a motor effect or to other factors such as alterations to the physiological systems that control anxiety [26], exploratory activity and emotional reactivity. The last two (exploratory activity and emotional reactivity) represent independent dimensions, rather than the two extremes of a unitary variable, and can interfere with each other [27,28]. Locomotor activity can serve as a tool to evaluate the effects of stressors that alter the functioning of the hypothalamic-pituitary-gonadal (HPG) axis [29]. In addition, the medial hypothalamus could play an important role in the modulation of defense responses measured by rat exploratory behavior in open-field and elevated plus-maze tests [30]. Based on these observations, fenarimol can act as a stressor that affects the HPG axis. It has been demonstrated that, in the neonatal hypothalamus, fenarimol concentrations peak and then gradually decline, probably due to a high-affinity binding site as demonstrated by studies with 14 C labeled fenarimol [19]. Fenarimol has also been shown to alter aromatase activity within the central nervous system [16]. The data on the functional state of the rat pup nervous systems (development of the righting reflex, grip response and climbing response) at different stages of postnatal development also revealed differences in terms of when the reflex was expressed, especially when the dams were exposed during the lactation period. The present data, in agreement with those from a pre-

vious study conducted by our group [13] and with those of Hirsch et al. [19], indicate the importance of the timing of maternal exposure to fenarimol: exposure during the late gestational period and the first days of lactation delays pup reflex development and neuromuscular maturation. The delayed appearance of neurodevelopmental milestones makes it clear that fenarimol is neurotoxic and is capable of damaging the physiological systems controlling spontaneous motor activity and motor coordination. Similarly, perinatal exposure to xenobiotics such as 2,3,7,8tetrachlorodibenzo-p-dioxin has been associated with delayed development of the righting reflex and impaired rotarod performance in rats, behaviors that normally depend on proper cerebellar development and function [31]. Since fenarimol can produce some neurobehavioral abnormalities associated with both cognitive and locomotor systems, it is important to determine whether developing cerebellar granule neuroblasts are potential targets for its toxicity. The reduced activity, as well as the other effects on neuromuscular function, could be at least partially the result of lower body weight. Further detailed studies should evaluate these hypotheses. Beyond these findings, such experiments raise some general issues related to strategies of developmental testing. The dosing schedules used in the experiments resulted in certain neuromotor and behavioral defects but not in significant dose-dependent physical immaturities. A possible explanation for this is that the times of treatment did not permit the chemical to affect physical landmark development. Indeed, some of the differences in developmental milestones are apparent in control groups. Another reason that may account for not seeing physical landmark changes probably is related to exposure periods that were not as long as necessary to show delays in physical milestones and early neuromotor development. Also, their experimental design could be detailed to better measure the effects promoted by fenarimol, since these parameters were considered suited to the study of the potential for other pesticide-related fetal effects including delayed development [6,22,32]. However, the results of our behavioral tests suggest a delay in achieving milestones of the neurodevelopment in young pups, as evidenced by functional biological changes together with overt signs of neurotoxicity. Our results then serve to highlight the potential for inducing perinatal disruption after maternal exposure. Since fenarimol is one of the most frequently used pesticides, it is necessary to study its molecular effects in order to predict the biological consequences of the use of this fungicide. Acknowledgement This work was supported by grants from the Brazilian Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP, Foundation for the Support of Research in the State of S˜ao Paulo). References [1] Huot R, Gonzalez M, Ladd C, Thrivikraman K, Plotsky P. Foster litters prevent hypothalamic-pituitary-adrenal axis sensitization mediated by neonatal maternal separation. Psychoneuroendocrinology 2004;29:279–89.

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