Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic–pituitary–ovarian axis development and function in Wistar rats

Accepted Manuscript Title: Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic-pituitary-ovarian axis development and function in Wistar rats Author: Eva Rollerova Jana Jurcovicova Alzbeta Mlynarcikova Irina Sadlonova Dagmar Bilanicova Ladislava Wsolova Alexander Kiss Jevgenij Kovriznych Juraj Kronek Fedor Ciampor Ivo Vavra Sona Scsukova PII: DOI: Reference:

S0890-6238(15)30005-8 http://dx.doi.org/doi:10.1016/j.reprotox.2015.07.072 RTX 7164

To appear in:

Reproductive Toxicology

Received date: Revised date: Accepted date:

19-2-2015 8-7-2015 9-7-2015

Please cite this article as: Rollerova Eva, Jurcovicova Jana, Mlynarcikova Alzbeta, Sadlonova Irina, Bilanicova Dagmar, Wsolova Ladislava, Kiss Alexander, Kovriznych Jevgenij, Kronek Juraj, Ciampor Fedor, Vavra Ivo, Scsukova Sona.Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic-pituitaryovarian axis development and function in Wistar rats.Reproductive Toxicology http://dx.doi.org/10.1016/j.reprotox.2015.07.072 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic-pituitary-ovarian axis development and function in Wistar rats Eva Rollerovaa,*[email protected] Jana Jurcovicova [email protected] Alzbeta Mlynarcikova [email protected] Irina Sadlonova [email protected] Dagmar Bilanicova [email protected] Ladislava Wsolova a [email protected] Alexander Kiss [email protected] Jevgenij Kovriznych a [email protected] Juraj Kronek [email protected] Fedor Ciampor f [email protected] Ivo Vavra [email protected] Sona Scsukova b [email protected] Slovak Medical University, Department of Toxicology, Bratislava, Slovak Republic Institute of Experimental Endocrinology, Slovak Academy of Science, Bratislava Slovak Republic Hameln rds a.s., Department of Toxicology, Modra, Slovak Republic Qi Technologies s.r.l., Pomezia, Italy Institute of Polymers, Slovak Academy of Science, Bratislava, Slovak Republic Institute of Electrical Engineering, Slovak Academy of Sciences, Bratislava, Slovak Republic Institute of Virology, Slovak Academy of Science, Bratislava, Slovak Republic *

Corresponding author at: Slovak Medical University, Department of Toxicology, Limbova 14, 833 01 Bratislava, Slovakia Republic. Tel.:+ +421 2 59370326. Highlights     

Neonatal exposure to polymeric nanoparticle PEG-b-PLA in female Wistar rats. Delayed effects on hypothalamic-pituitary-ovarian axis development and function were studied. Acceleration of the puberty onset and irregular cyclicity were observed. Pituitary hyperemia, vascular dilatation and congestion, altered pituitary LH release and increased progesterone serum levels were detected. Possible neuroendocrine disrupting effect of PEG-b-PLA nanoparticles is proposed.

ABSTRACT We studied delayed effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether (PEG-b-PLA) on the endpoints related to pubertal development and reproductive function in female Wistar rats from postnatal day 4 (PND4) to PND 176. Female pups were injected intraperitoneally, daily, from PND4 to PND7 with PEG-b-PLA (20 or 40 mg/kg b.w.). Both doses of PEG-b-PLA accelerated the onset of vaginal opening compared with the control group. In the low-dose PEG-b-PLA-treated group, a significantly reduced number of regular estrous cycles, increased pituitary weight due to hyperemia, vascular dilatation and congestion, altered course of hypothalamic gonadotropin-

releasing hormone-stimulated luteinizing hormone secretion, and increased progesterone serum levels were observed. The obtained data indicate that neonatal exposure to PEG-bPLA might affect the development and function of hypothalamic-pituitary-ovarian axis (HPO), and thereby alter functions of the reproductive system in adult female rats. Our study indicates a possible neuroendocrine disrupting effect of PEG-b-PLA nanoparticles. Abbreviations: CNS central neural system; DES diethylstilbestrol; DLS dynamic light scattering;; ED endocrine disruptor; EDSPTG Endocrine disruptor screening program test guidelines; ELS electrophoretic light scattering; FDA the Food and Drug Administration; FSH follicle stimulating hormone; GD gestational day; LH luteinizing hormone; LHRH luteinizing hormone releasing hormone; MDR multidrug resistance; MTD maximum tolerated dose; NP nanoparticle; OVX ovariectomized; PCB polychlorinated biphenyls; PEG350 polyethylene glycol, Mn350; PEG-b-PLA20 20 mg/kg b.w. of PEG-b-PLA; PEG-bPLA40 40 mg/kg b.w. of PEG-b-PLA; PND postnatal day; PRL prolactin; TEM transmission electron micrography Keywords: Polymeric nanoparticles; PEG-b-PLA; Endocrine disruptors; Reproductive toxicity; Nanotoxicology; Estrous cycle; Pituitary; Progesterone 1 Introduction Nowadays, the exposure to engineered nanomaterials (ENMs) dramatically increases due to nanoparticles (NPs) actively used in the food industry, cosmetics and medicine. The working environment also leads to not negligible exposure [1]. Last but not least, intentional exposure to ENMs in biomedical applications such as diagnostic and therapeutic tools and devices can increase the possible risks for human health [2,3]. In the context of nanomedicine, polymeric NPs have been developed as delivery systems with excellent drug, protein, and DNA loading and release properties, long shelf life, low toxicity, good biodegradability, and almost no immunogenicity [4,5]. The polymeric NP poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA), an amphiphilic block copolymer (ABC), has been designed as a drug carrier for poorly water-soluble drugs that results in an improved drug pharmacokinetics. Moreover, NPs enhance the effectiveness of the chemotherapeutics in cancer therapy uniquely due to the inhibition of P-glycoprotein (Pgp) function and suppression of Pgp-mediated multidrug resistance (MDR) efflux transporter. In the USA, PEG-b-PLA micelles have entered phase III clinical trials as a substitute for Cremophor EL in the delivery of paclitaxel (PTX) in cancer therapy [6]. They have already been approved in Korea for cancer treatment in formulation Genexol-PM [6]. Polymeric NPs, including PEG-b-PLA, represent one of the most promising approaches also for CNS drug delivery, due to their ability to cross the blood brain barrier (BBB) without infliction of any damage to the BBB [5,7–9] and to exert direct actions on the brain areas. Defective neuroendocrine sex differentiation of the brain and subsequent disrupted hypothalamic control of sexual function after exposure to endocrine disrupting compounds (EDs) during the critical developmental periods might be responsible for a variety of perturbations in adult rats [10]. Moreover, early oocyte development affected during the critical window might demonstrate damaged ovarian cascade [11]. Therefore, a starting point

of our work hypothesis was: “The critical neonatal exposure to PEG-b-PLA could disrupt the development of HPO axis and evoke permanent damage to reproductive function in adult female rats” [12]. The association between the exposure to ENMs and their adverse effects on reproductive/neuroendocrine development and function is apparent from a number of in vivo and in vitro studies [13–17]. Several NPs can adversely affect female reproductive tract development and function, including cytotoxic effects on ovarian structural cells, impaired oogenesis and follicle maturation, altered normal sex hormone levels, and accelerated the onset of puberty [18–20]. Compared to the peripheral effects, the direct effects of ENMs on central regulation of reproductive processes have not been specifically investigated up to now. There are several studies demonstrating alteration of regions related to dopamine systems in the offspring mice brain after developmental TiO2 exposure [21,22]. The accumulation of gold nanoparticles recorded among other brain parts also in the mice hypothalamus [23] might suggest potential HPO axis dysfunction. Polyethylene glycol (PEG), polylactide acid (PLA), and polylactide co-glycolic acid (PLGA) are approved components of drug carriers by US Food and Drug Administration (FDA), but clinical trials testing their use as drug carriers are still lacking. PEG is not known to be metabolized in humans; it is absorbed minimally and rapidly excreted in feces with no known confirmed toxicity [24]. However, Gajdova et al. [25] reported that Tween 80 with PEG Mn = 350 g/mol as an active ingredient has the potential to behave as a hormone/estrogen active agent. Neonatal exposure of female rats to PEG 350 significantly accelerated their sexual maturation, prolonged the estrous cycle, and induced persistent vaginal estrus and squamous cell metaplasia of the epithelial lining of the uterus. Moreover, ovaries were without corpora lutea and had degenerative follicles. Since nano-forms of the chemicals are frequently more active as conventional formulations of the same material, we decided to select PEG-b-PLA (PEG average Mn = 350 g/mol, PLA average Mn = 1000 g/mol) NPs for our study. More efficient adverse effects demonstrated in a significantly different efficiency of the tested doses of nanoscaled PEG-b-PLA vs. non-nanoscaled PEG have been confirmed in our in vitro studies. Treatment of porcine ovarian granulosa cells (GCs) with PEG-b-PLA (0.2100 μg/ml) and PEG (Mn=300 g/mol; 0.2-100 mg/ml) significantly inhibited (decrease about 25 to 90 %) basal as well as follicle-stimulating hormone (FSH)-induced progesterone secretion by GCs above the concentration 20 μg/ml and 20 mg/ml, respectively (Scsukova et al. unpublished data). Based on aforementioned data on PEG, we have considered potential neuroendocrine disruptive effects of tested NP. Nanoreprotoxicity of polymeric NPs that are intentionally produced with very specific properties has not been investigated until now. Therefore, the aim of the present study was to reveal the in vivo potential endocrine disrupting/nanoreprotoxic effects of neonatally administered PEG-b-PLA on the endpoints related to pubertal and somatic development, and reproductive function, i.e. vaginal and eye opening, and estrous cyclicity in female Wistar rats. The observed effects of PEG-b-PLA were compared with the known action of synthetic estrogen/endocrine disruptor diethylstilbestrol (DES) on the female reproductive system. In addition, we analyzed in vivo luteinizing hormone releasing hormone (LHRH)-induced pituitary LH and follicle-stimulating hormone (FSH) release, levels of peripheral P4, and basic histological pattern of adenohypophysis, as well as immunohistochemical distribution of prolactin in the pituitary of the adult female rats neonatally exposed to PEG-b-PLA. 2 Material and methods

2.1 Preparation of NP suspension and NP characterization Fresh NP micelles of PEG-b-PLA were prepared by modified solvent evaporation method according to Du et al. [26] and Shin et al. [27]. Briefly, copolymer PEG-b-PLA [CH3O(CH2CH2O)x(COCHCH3O)yH, PEG average Mn = 350 g/mol, PLA average Mn = 1000 g/mol, CAS 9004-74-4, Sigma-Aldrich, Steinheim, Germany] (20 mg) was dissolved in 2 ml of tetrahydrofuran (THF; anhydrous, inhibitor free, purity ≥99.9%; Sigma-Aldrich, Steinheim, Germany) and stirred for 2 h at room temperature (RT). Under moderate stirring (100 rpm, MR Hei-Standard Heidolph, Germany), the ultrapurified water (10 ml) (Milipore Mili-Q Synthesis, 18.5 MΩ,) was added dropwise. Two hours later, THF was evaporated under mild vacuum (rotating evaporator LABOROTA 4010–digital, Heidolph, Germany) for 1 h at 48 °C to obtain polymer micelles. After THF evaporation, water was added to the suspension to obtain the final PEG-b-PLA concentration 20 mg/10 ml. Immediately before administration, PEG-b-PLA suspension was vortexed at the highest speed for 1 minute. Suspension of PEG-b-PLA was characterized by transmission electron micrography (TEM), electrophoretic light scattering (ELS), and dynamic light scattering (DLS) methods. Physical particle size, general state of agglomeration/aggregation and morphology were determined by TEM. For this purpose, the specimens were prepared by drying a small drop of the NP suspension (2 mg/ml) negatively stained with uranyl acetate (2%, Sigma-Aldrich, Steinheim, Germany) on the surface of a formvar-coated slot cooper grid. The samples were imaged by transmission electron microscope JEM 1200 (JOEL, Tokyo, Japan) with 120 kV voltage (Fig. 1 Fig. 1A). Zeta potential was measured by Nicomp Submicron Particle Sizer Autodilute Model 380 (Santa Barbara, CA, USA) using the ELS method. Size distribution of PEG-b-PLA was evaluated by DLS with a NICOMPTM 380 ZLS Particle Sizer (Santa Barbara, CA, USA). Size measurement was performed at 25 °C and a scattering angle of 90°. The employed NICOMP software can automatically recognize up to three size distributions of particles concurrently present through a patented software algorithm. 2.2 Background for choice of study design The modified study design according to Endocrine disruptor screening program test guidelines (EDSPTG) OPPTS 890.1450: Pubertal development and thyroid function in intact juvenile/peripubertal female rats [28] and studies by Newbold et al. [29] and Bertolasio et al. [30] were used. The experiments were carried out on one generation neonate rat model described by Newbold‘s group which represents an ontogenic model consistent with the early postnatal period in human infants [29,31]. Two doses of PEG-b-PLA were used (20 and 40 mg/kg b.w.) according to EDSPTG OPPTS 890.1450 [28], which requires a high dose level at or just below the maximum tolerated dose (MTD), and a low dose level at half the high dose level. Because MTD for PEG-b-PLA is not known from the available literature, we derived dose levels of tested NP from the pharmacokinetic study by Shin et al. [32]. In this study, a dose of 50 mg/ml PEG-b-PLA (Mn of PEG = 4200 g/mol and Mn of PLA = 1900 g/mol) was used for preparation of drug-loaded PEG-b-PLA micelles for single i.v. injection of drugs to adult five to six-week-old female FVB albino mice [32]. In our study, 1/25 of this dose was administered to neonatal female

rats on postnatal days (PND) 4–7 after birth (in low dose group of PEG-b-PLA = 2 mg/ml). PEG-b-PLA was administered by intraperitoneal route to mimic the most likely route of human exposure (intravenous administration). 2.3 Experimental animals and exposure Nulliparous female (n=10, 220-270 g) and male (n=8, 320-350 g) specific pathogen free (SPF) Wistar rats obtained from Breeding Facility Masaryk University Brno (Czech Republic) were acclimatized to the laboratory conditions for 7 days prior to mating. The animals were placed in plastic cages with wire lids and standard bedding (JRS Lignocel®, Hygienic Animal Bedding, Germany). They were maintained in standard conditions at 22 ± 2 °C and 50 ± 5 % relative humidity with 12 h light:dark schedule (light from 6.00 a.m.). Standard laboratory chow (complete certified laboratory rodent chow M3, BONAGRO, CZ10174, Czech Republic) and tap water in glass bottles were available ad libitum. Healthy female rats were mated with male breeders in an experimental animal house of the Slovak Medical University. Mating was confirmed by the presence of sperm in the vaginal smear. The following 24 h were designated as gestational day 0 (GD 0). Pregnant female rats were individually housed 4 days before spontaneous delivery and checked for births every morning. Offspring was counted on the day of birth (PND 0), and sex was determined. To allow uniform breast-feeding and growth rates, litter sizes were culled to 10 pups per dam with equal or female predominance gender ratio. Male littermates were retained among females and separated on the day of weaning (PND 21). Male pups were sacrificed by anesthesia overdose at the day of weaning. In total, 52 female rats from eight litters (litter size ranged from 11 to 14 pups; litters delivered by GD 21–22) were used. The female pups were weighted, identified individually on the tail, and assigned to the treatment groups 1–4. To avoid significant stress from crossfostering, all female pups from one dam received the same treatment excluding 2 litters which were divided into two groups. The treatment groups were assembled as follows: PEGb-PLA20 n = 13 female pups from 3 litters, PEG-b-PLA40 n = 14 female pups from 3 litters, Control n = 13 from 2 litters, and positive control DES n = 12 from 2 litters. Neonatal female SPF Wistar rats were dosed intraperitoneally (i.p.) daily with 20 mg/kg b.w. (PEG-b-PLA20; n = 14) or 40 mg/kg b.w. (PEG-b-PLA40; n = 13), respectively, vehicle alone (ultrapurified water after evaporation of THF; negative control; n = 13) or 4 μg/kg b.w. of DES (purity 99%, F.W. 268.36; Sigma-Aldrich, Steinheim, Germany) (positive control for estrogenic activity; n = 12) in volume of 10 ml/kg b.w. from PND 4 to 7 between 08.00– 09.00 a.m. Pup body weight during the administration period ranged between 9.14 and 13.91 g (Table 3 Table 3). After weaning the treatment, group-housed female rats were kept under the same conditions. At the end of the study, the animals were sacrificed by decapitation after ketamin/xylasine anesthesia (60/10 mg/kg b.w.) [Ketamin (Narketan) VEtoquinol LTD, Czech Republic; Xylasine (Xylariem) Riemser Arzneimittel AG, Germany] on the day of the first estrus after PND 176. Four female rats in group PEG-b-PLA20 were sacrificed on the day of diestrous because they failed to enter the estrous stage of the estrus cycle. The protocol of the study was approved by the State Veterinary and Food Administration, Slovak Republic. Animal care was in compliance with the Standard Operation Procedures

(GLP) of the Department of Toxicology, Slovak Medical University, Bratislava, and the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123). The study meets the WHO International Ethical Guidelines for Biomedical Research involving experimental animals and is in compliance with the Slovak Statutory order No 377/2012 Z. z. and No 436/2012 Z. z. (Collection of laws). 2.4 Clinical signs, body, and organ weights Each animal was evaluated for clinical signs once a day. The body weight of the female rats was registered at selected time points from PND 4 to the day of sacrifice (the day of the first occurrence of estrus after PND 176). The ovary, uterus, pituitary, kidney, adrenal, and thyroid glands were dissected at the day of necropsy and weighted. 2.5 Vaginal opening and estrous cyclicity All female pups were examined daily for vulvovaginal canilation and eye opening from PND 8 until each animal acquired these developmental landmarks. The age and body weight at which opening occurred were recorded and used for statistical analysis. Estrous cyclicity was evaluated in all female rats at 3 and 5 months of age. Changes in vaginal epithelial cells were monitored: 1) for 23 days from 3 months of age and 2) for 23 days from the end of 5 months of age. Vaginal lavages were obtained daily between 08.00– 09.00 a.m. by pipetting of aqua pro injectione into the vagina. The lavages were spread on slides, fixed with methanol, stained by Giemsa-Romanowski (VAKOS XT ltd., Prague, Czech Republic), and examined under a low-power light microscope. The stage of the estrous cycle was determined based on the presence of leukocytes (metestrous, diestrus), nucleated epithelial cells (proestrus), and cornified epithelial cells (estrus) as described by Cooper and Goldman [33] and Goldman et al. [34]. Constant 4- or 5-day vaginal estrous cycle was regarded as a regular estrous cycle. Extended estrus was defined as exhibiting cornified cells with no leukocytes for 3 or more days, and extended diestrus was defined as the presence of leukocytes for 4 or more days [33]. The number of regular and irregular cycles, percentage of female rats having constant and aberrant cycles, and number of days in stages of the estrus cycle were recorded. 2.6 In vivo LHRH-induced gonadotropin release and hormones measurements On the day of necropsy, blood samples were collected from the jugular vein (0 min) of the adult female rats neonatally exposed to PEG-b-PLA20, PEG-b-PLA40, DES, or vehicle alone under the anesthesia (ketamine/xylazine, 60/10 mg/kg b.w.). Human LHRH (acetate salt, purity ≥98%, Sigma-Aldrich, Steinheim, Germany) (100 ng/kg b.w. in saline solution) was administered into the same vein, and blood samples were taken at 15, 30, and 50 min after LHRH application. The collected blood samples were centrifuged (3000 rpm for 15 min at 4 °C), and serum was harvested and stored at -20 °C until use for hormonal assays. Serum concentrations of FSH, LH, and P4 were measured by rat and human radioimmunoassay (RIA) kits, respectively (Institute of Izotopes, Ltd., Budapest, Hungary). Assay sensitivities were 1.6 ng/ml for rFSH, 0.8 ng/ml for rLH, and 0.14 ng/ml for P4. The respective intra- and inter-assay coefficients of

variation were 4.2 % and < 11.5 % for rFSH, 6.5 % and < 10.9 % for rLH, and < 10.2 % and < 11.8 % for P4. 2.7 Histopathological examination Pituitary samples were processed for light microscopic examination using a standard paraffin technique and stained with hematoxylin and eosin. Histological preparations were examined under light microscope Axiophot (OPTON) at the magnification 100, 200, and 400 times. 2.8 Statistical analysis All data were expressed as mean ± SEM, and the value of p < 0.05 was considered statistically significant. For the vaginal and eye opening parameters, one-way analysis of variance (ANOVA) was used. When the treatment differences were indicated as significant, the Scheffe multiple comparison test was further used to compare each treatment group with the controls. In the case of heterogeneity of variance among control, DES, and PEG-b-PLA groups, the non-parametric Kruscal-Wallis test was carried out. The differences in the number of rats with regular or irregular cycles were assessed by contingency tables with Fisher exact test. The one-way ANOVA followed by the least significant difference (LSD) test were used for determination of the differences in a number of individual stages of estrous cycle between the individual treatment and control groups. To compare the body weight and weight of the selected organs, one-way ANOVA (for normally divided weights) followed by Bonferroni's correction for multiple comparisons (in case of the equal variance) or Tahman test (in case of the statistically significant differences in variance) were used. To consider the effect of body weight on the time of maturation, the analysis of covariance (ANCOVA) was used. The body weight at PND 4 measured before the treatment was used as an independent covariate. Statistical software SPSS 19.0 was used for analyses. 3 Results 3.1 Characterization of PEG-b-PLA NPs TEM demonstrated spheric shape of NPs and the average primary particle size (PPS) suspended in deionized water about 50 nm (Fig. 1A). Zeta potential value, measured in triplicate at pH 7.0 was 28.73 ± 1.44 mV. Micelle dispersion resulted in size distribution with two main peaks of secondary particle sizes (SPS), averaged as 64.9 ± 10.5 nm and 911.4 ± 177.6 nm (Fig. 1C), which indicates that the PEG-b-PLA micelles were aggregated in solution (Fig. 1B). Similar to the findings in Shin et al. [32], the PEG-b-PLA micelles were stable for 24 h at ambient temperature as monitored by TEM (Fig. 1B). The value of the sample surface area was not determined for non-rigid polymeric PEG-b-PLA NPs under investigation. A dose metric unit has been more practical to derive as well as to compare the dose used in our study with those used in the recent studies [6,27,32]. 3.2 Clinical signs and body weight No visible signs of toxicity were observed in any of the treated animals in our study. One DES-treated female rat had to be sacrificed on PND 128 because of gingivitis and problem with food intake. The final body weights are summarized in Table 3. No treatment-related statistical differences in growth and terminal body weight were found in the female rats in

any experimental group, although the PEG-b-PLA20-exposed animals reached the highest terminal body weight. 3.3 Effects of DES treatment DES, the positive control, significantly accelerated the age of vaginal opening in comparison with vehicle control (negative control) (p < 0.000), the onset of vaginal opening was observed by 22 days earlier compared to the negative control (PND 10.92 vs. PND 33, respectively). Significant decrease of body weight at time of vaginal opening was observed (p < 0.000) against negative control (Table 1 Table 1). As expected, a significant disturbance of estrous cyclicity in female rats neonatally exposed to DES was observed in 100% of animals in comparison with the negative control (first monitoring: the number of regular cycles χ2 = 30.537, p < 0.000 and irregular cycles χ2=20.502, p < 0.000; second monitoring: the number of regular cycles χ2=33.762, p < 0.000 and irregular cycles χ2=27.393, p < 0.000) (Table 2 Table 2). Interestingly, the positive control exerted extended periods of diestrous observed from vaginal opening to appearance of the first estrous followed by several irregular cycles until beginning of the persistent estrous (data not shown). In spite of irregular cycles in 100% animals, no changes were seen in the duration of each stage of the cycle at 3 months after birth. During the second examination (at 5 months of age), animals continued to display irregular estrous cyclicity reflected by significant changes in the stages metestrous/diestrous (F=74.395, p < 0.000) and estrous (F=55.057, p < 0.000). The induction of persistent vaginal estrous was accompanied by a significant reduction of metestrous/diestrous (Table 2). The significant ovarian atrophy (63.5% of negative control, p < 0.001), adrenal gland (124.8% negative control, p < 0.01), and uterine (131.2% of negative control, p < 0.001) hypertrophy were observed in the positive control compared to the negative control (Table 3). No significant changes in the pituitary, thyroid gland, or kidney weights were found in the positive control (Table 3). 3.4 The effects of PEG-b-PLA on the age of complete vaginal and eye opening in prepubertal rats The age of vaginal and eye opening as well as the body weight at coincident days are shown in Table 1. Both treatments decreased the age of vaginal opening (p < 0.000, F = 159.427 and p = 0.002, F = 5.528, respectively) and the body weight at these days (p < 0.000, F = 104.012 and p = 0.041, F = 2.975, respectively). The neonatal exposure to PEG-b-PLA significantly advanced the onset of puberty in both treated groups (PEG-b-PLA20, p = 0.045 and PEG-b-PLA40, p < 0.000) in a dose-dependent manner by 3 and almost 5 days, respectively, when compared to the negative control (Table 1). The acceleration of puberty onset by the effect of PEG-b-PLA was not so marked as in the positive control group (DES vs. PEG-b-PLA groups, p < 0.000). There was no effect of short-time exposure to PEG-b-PLA on the age of eye opening when compared to the negative control (Table 1). A concomitant significant decrease of body weight at the time of vaginal opening was observed in both PEG-b-PLA groups (PEG-b-PLA20, p = 0.028; PEG-b-PLA40, p < 0.000)

against the negative control. The significantly decreased body weight at the day of eye opening was also recorded in the high-dose group of PEG-b-PLA40 (p = 0.047) compared to the negative controls (Table 1). To find out the effect of body weight on the time of maturation, the body weight at PND 4 (Table 3), measured before the first treatment, was considered as an independent covariate. There was no significant effect of body weight on the average day of vaginal (p = 0.107, F = 2.705) or eye opening (p = 0.120, F = 2.534). Thus, the effect of treatment and no effect of body weight was confirmed for the advanced vaginal opening (p < 0.000, F = 165.244). 3.5 The effects of PEG-b-PLA on estrous cyclicity in intact adult rats The estrous cycles in the negative control group were completely normal in the sense of cycle length and regularity in the degree of cornification of epithelial vaginal cells in both periods of evaluation (3 and 5 months of age during 23 days), as shown in Table 2. The cornified cells predominated on the last day in a 4-day cycle. In some cases, 2 consecutive days of vaginal cornification were observed in a 5-day cycle (consistently with Goldman et al. [34]). During the first observation at 3 months of age, a significantly reduced number of regular, constant 4- or 5-day vaginal estrous cycles was detected in both PEG-b-PLA-treated groups (PEG-b-PLA20 vs. negative control, p = 0.018 and PEG-b-PLA40 vs. negative control, p = 0.018) with a coincident significantly increased number of irregular cycles in the low group PEG-b-PLA20 vs. negative control (p = 0.045) (Table 2). The significant alteration of estrous cyclicity represented by the decrease of regular estrous cycles persisted in the PEG-b-PLA20 group (vs. negative control p < 0.000) also during the second examination period at 5 months after birth (Table 2). The situation in PEG-b-PLA40 returned to cyclicity similar to the negative control (Table 2). The incidence of abnormal (prolonged and irregular) estrous cycles observed in 38.46% of PEG-b-PLA20-treated female rats at 3 months of age increased up to 53.85% at 5 months of age (Table 2). In the PEG-b-PLA40 group, 28.57% and 21.43% of rats failed to enter a normal estrous cycle at 3 and 5 months of age, respectively (Table 2). During the first monitoring, a significant increase in the proportion of the metestrous/diestrous stage with concomitant decrease in the number of days in proestrous was recorded in both PEG-b-PLA groups (PEG-b-PLA20, p = 0.043 and PEG-b-PLA40, p = 0.029). The proestrous stage decrease reached a significant difference in the PEG-b-PLA20 group when compared with negative controls (p = 0.044) (Table 2). PEG-b-PLA groups were characterized by a predominance of leucocytes in the vaginal smears. However, the proportion of metestrous/diestrous stages significantly increased in the first observation in both PEG-b-PLA groups was not statistically different in comparison with negative controls during the second examination (Table 2). 3.6 The effects of PEG-b-PLA on organ weights Both doses of PEG-b-PLA increased the pituitary weight; in the low-dose group, a significant increase was observed (140.9% of negative control in PEG-b-PLA20, p =0.002 and 121.2% of negative control in PEG-b-PLA40) (Table 3). The tendency to increase the adrenal and thyroid glands and uterine weight was observed in both PEG-b-PLA-treated groups. In the

case of the thyroid gland, the weight was significantly different from the negative control in the PEG-b-PLA40 group (Table 3). The ovary weight was not affected in any PEG-b-PLAtreated group (Table 3). 3.7 Histopathological examination of the pituitary Significant increase of pituitary weight, as a possible indicator of the altered anatomical pattern of this organ, led us to conduct a histopathological examination. The microscopic picture of the anterior pituitary histology in the examined area corresponded well to the morphology seen under the normal physiological conditions. There were 2–3 basic cell types notable in the histological preparations stained with haematoxylin and eosin. Differences between both PEG-b-PLA20 and PEG-b-PLA 40 groups and both control groups were found. A middle hyperemia of blood vessels and capillaries concomitantly with vascular dilatation and congestion was obvious in PEG-b-PLA20-treated rats (Fig. 2 Fig. 2), while only a slight degree was evident in PEG-b-PLA40-treated rats. The histological preparations in negative controls displayed only sporadic findings similar to positive controls demonstrating almost no hyperemia. 3.8 The effects of PEG-b-PLA on in vivo LHRH-induced gonadotropin release There were no differences in basal serum LH levels on the day of necropsy in any treated group (Fig. 3 Fig. 3). The LH concentrations (mean ± SEM) were 1.30 ± 0.24 ng/ml, 1.41 ± 0.39 ng/ml, 1.27 ± 0.28 ng/ml, and 1.75 ± 0.49 ng/ml for negative controls, low and high PEG-b-PLA doses, and positive control, respectively. Stimulation of the pituitary with LHRH significantly increased serum LH levels in all experimental groups in comparison with their basal levels measured 15, 30, and 50 min after the injection (p < 0.000, F = 132.411; p < 0.000, F = 77.535; and p < 0.000, F = 55.471, respectively) (Fig. 3). No significant differences in serum LH between the groups treated with both doses of PEG-b-PLA, positive, and negative controls were found after LHRH stimulation at 15 and 50 min. However, a t-test showed significant differences in LH concentrations in both PEG-b-PLA groups versus the negative control (p <0.05) at 30 min (Fig. 3). No differences were found in the LHRHinduced FSH release in comparison with their basal levels in any of the groups studied (data not shown). 3.9 The effects of PEG-b-PLA on progesterone (P4) serum levels The examined groups of rats showed statistically significant differences in serum P4 levels (p = 0.004) using the Kruskal-Wallis test. The highest values were found in the PEG-b-PLA20treated group and the lowest values in the positive control group. The levels of serum P4 (mean ± SEM) were 23.74 ± 5.97 ng/ml for negative control, 40.20 ± 9.65 ng/ml for PEG-bPLA20, 22.60 ± 3.30 ng/ml for PEG-b-PLA40, and 10.65 ± 1.95 ng/ml for positive control (Fig. 4 Fig. 4). When the Mann-Whitney test was used for analysis, the increase of the P4 levels reached statistical significance in PEG-b-PLA20 in comparison with the negative (p = 0.032) and positive (p = 0.001) controls. 4 Discussion The nanoreprotoxicity should/might be considered one of the major emerging issues related to the hazard of nanomaterials used in every day products and pharmaceutics. Therefore, in

the present experiment, we studied the effects of short-time neonatal exposure to polymeric NP PEG-b-PLA on pubertal and somatic development and on functions of the reproductive system in female rats, investigating peripheral effects as well as specific neuroendocrine endpoints. Neonatal exposure to PEG-b-PLA accelerated the onset of puberty and consequently led to a premature loss of the regular estrous cyclicity being typical for early reproductive senescence in adult rats [35]. Moreover, significantly increased pituitary weight and altered LH response to LHRH stimulation were observed in adult female rats after restricted 4-day neonatal exposure to PEG-b-PLA. In the group of rats exposed to 20 mg PEG-b-PLA /kg b.w./day, a significant increase in the serum P4 along with the prevalence of the diestrous stage of the estrous cycle were recorded. The present data support our hypothesis that neonatal exposure to PEG-b-PLA might disrupt development of the HPO axis, being demonstrated as delayed reproductive dysfunction. To evaluate a possible impact of the neonatal exposure to PEG-b-PLA on the onset of puberty in the female rats, we carried out vaginal opening (VO) assay that allows a broad overall assessment of endocrine system function. The present study reports that both doses of PEG-b-PLA (20 and 40 mg/kg b.w./day) significantly and in a dose-dependent manner accelerated the onset of puberty by 3 and 4.92 days, respectively, when compared with the negative control group. The most striking acceleration of vaginal patency was also observed in the positive controls (4 μg/kg b.w./day). To the best of our knowledge, there is only one study investigating the effect of prenatal exposure to carbon black NP (Printex 90) on the onset of puberty by analyzing the time of vaginal opening. A low dose of NP (intratracheally, 11 μg/animal) elicited significantly earlier onset of puberty in female offspring compared to controls, while this effect was not observed in higher dose groups (54 and 268 μg/animal) [20]. Our results are consistent with the results of recent studies demonstrating that critical perinatal exposure to EDs accelerates the pubertal onset in the female rodents and that timing of the exposure, doses, and response stages of specific tissues are critical [10,11,20,35–40]. Based on the results of the VO test, we can conclude that the age of exposure falls in the critical developmental window [41], and the VO test might be a good indicator of endocrine disturbance elicited by NP exposure. This part of our study demonstrating significant alteration of pubertal timing suggests interference of NP PEG-b-PLA with the neuroendocrine system as it is consistent with recent records of ED effects [39,42]. Vaginal cytology data were collected to evaluate whether exposure to PEG-b-PLA could affect the time at which female rats began to show aberrant cycles prior to reproductive senescence. Neonatal exposure of rats to PEG-b-PLA significantly reduced the number of regular cycles in both dose groups monitored at 3 months of age, and this adverse effect also persisted in the low dose group (20 mg/kg b.w./day) at 5 months of age. Studies monitoring alteration of estrous cyclicity after NP exposure are to our knowledge not available. In our study, the most frequently observed stage of irregular estrous cycles in both PEG-b-PLA-injected groups was diestrous, showing prolonged presence of leukocytes in the vaginal smears in accordance with the observations of many studies investigating EDs [38,43–46]. In the positive control, consistently with the effect of lower dosages of the

estradiol, the onset of persistent cornification was delayed until a later age, referred to as a delayed anovulatory syndrome [34]. Although the persistent estrous, as one of the irreversible abnormalities after exposure to various EDs, was well described, the other characteristics and mechanisms remain to be determined [47]. Thus, persistent diestrous and pseudopregnancy described in line with the effect of low doses of estradiol have been attributed to elevated prolactin (PRL) secretion [48–50]. It has been demonstrated that estradiol-induced hyperprolactinemia could be a consequence of an increased number of lactotrophs, increased production of hypothalamic or pituitary PRL-regulating factors, or decreased dopamine biosynthesis and density of dopamine D2 receptors on the lactotrophs [49,51,52]. However, in our study, the prevalence of diestrous-type smears observed in the PEG-b-PLA20 group was not accompanied with a significantly changed PRL immunoreaction in the pituitaries (data not shown), and serum PRL levels were not measured with respect to the impropriety of the anesthesia used [53,54]. In addition, histological samples of the anterior pituitary did not exert histopathological changes in the sense of basal tissue structure. The accelerated failure of the regular cycles enables to identify the alteration in neuroendocrine and ovarian functions that can be mediated through the estrogenic as well as nonestrogenic mechanisms [50]. It has been shown that PRL secretion stimulated by estrogens or EDs can rescue and maintain functional corpora lutea, suppress cyclicity, and the persistence of leucocytes in the vaginal smears can be detectable. As a consequence, elevated P4 levels can be detected during persistent diestrous or pseudopregnancy, before ensuing luteolysis [34]. Alternatively, a prolonged diestrous phase was found to be accompanied with increased LH release and enhancement of P4 serum levels in female obese rats. In this study, the authors suggested that dysregulation of female reproductive function in central nervous system as well as ovaries was induced by alteration of insulin signaling [55]. Significantly increased serum P4 levels were observed in adult female rats neonatally treated with PEG-b-PLA20 compared to negative controls, although interindividual susceptibility to NP exposure was observed. The elevated P4 serum concentrations may correlate with a significantly increased number of irregular estrous cycles and prevalence of diestrous observed in this group in 5 months of life. Moreover, a significant difference found between PEG-b-PLA20 and positive control (with typical permanent cornification of vaginal smears within the scope of permanent estrus) may correlate with a physiological difference of P4 levels in the diestrous and estrous stages [40] with a relevant level of intrinsic luteolytic mechanism of corpora lutea [56,57]. Based on the results of estrous cyclicity monitoring, we can conclude that the effect of PEGb-PLA on cycle regularity is probably not mediated through the same mechanism responsible for the effects found following DES exposure. In our study, estrous cyclicity was shown to be a good indicator for delayed toxic effects of PEG-b-PLA NPs on the reproductive system, demonstrating age-dependent but not dose-dependent effects. We hypothesized that neonatal exposure to PEG-b-PLA could result in alteration of the central regulation, including neuroendocrine control of LH secretion. It has been described that transient exposure to EDs (estradiol, o,ṕ-DDT) in early postnatal life followed by early maturation of pulsatile GnRH secretion and subsequent early developmental reduction of LH response to GnRH are possible mechanisms of the sexual precocity [31]. In our study, the effect of PEG-b-PLA on the level of central regulation by means of in vivo LHRH-induced

LH release assay was investigated. Low basal concentrations of serum LH determined in all experimental groups were consistent with normal physiological hormone status in PND176 female rats, considering the age-dependent decrease of serum LH levels [31,42,55,58]. No differences in basal serum LH were found in any of the experimental groups. Significantly increased LH secretion after 15 min of LHRH stimulation was observed in all experimental groups without any effect of the treatment (DES, PEG-b-PLA). However, 30 min after LHRH injection, significant elevation of LH levels was found in both PEG-bPLA-treated groups when comparing with the negative and positive control groups. Fernandez et al. [42] did not find any changes in the basal LH secretion after neonatal exposure to well-known ED - bisphenol A (BPA) (PND 1-10) in the adult female rats, but they observed a significant decrease of serum LH levels in the BPA high-dose group in comparison with the controls measured 15 min after LHRH stimulation. This inhibitory effect of BPA on the stimulated LH release is consistent with a phenomenon of neonatal estrogenization [31,59]. Our findings, i.e. increased LH release 30 min after LHRH injection concomitantly with enhancement of P4 serum levels along with prolonged diestrous phase of estrous cycle, might coincide with the observation by Akamine et al. [55], indicating possible involvement of insulin signaling. It worth to mention that significantly increased levels of LH and FSH and lowered E2 serum levels after nickel NPs exposure (two doses: 260-725 nm; 5 μg/ml and 400879 nm; 12.5 μg/ml administered orally) were observed by Kong et al. [60] in a onegeneration reproductive toxicity study. Along with the NP-induced alteration of pituitary function, hyperemia, vascular dilatation, and congestion in the pituitary were evident in animals exposed neonatally to both PEG-bPLA doses, with a significant extent of changes in the PEG-b-PLA20 group compared to the negative and positive controls. The same pathological finding was recently described in ovaries as an outcome of the exposure to nickel NPs in a one-generation reproductive toxicity study [60]. Taken together, the alteration of pituitary LH response to LHRH stimulation, higher production of P4 by the corpora lutea, and prevalence of the diestrus stage of the estrus cycle might suggest possible disturbance of central control by a negative feedback mechanism, and the pituitary as one of the possible target sites of PEG-b-PLA action. In our study, a low dose of PEG-b-PLA (20 mg/kg b.w.) has been recorded to be a more effective dose in comparison to the high dose-level of NP tested (40 mg/kg b.w. of PEG-bPLA). Significantly higher numbers of irregular estrous cycles during both observation periods along with increasing percentages of the animals showing aberrant cycles, and pathological finding in the pituitary along with altered pituitary and ovarian functions, suggest that we could consider a non-monotonous dose-response relationship. Many studies have demonstrated that BPA could alter physiological parameters in a non-monotonous dose-response relationship (Ushaped dose-response curve) [38,51,61]. A stimulation of amplification systems through signaling by receptor systems, receptor down regulation, homeostatic feedback responses, or cross talks between signaling systems have been suggested to be responsible for this kind of response [35,62]. Toxicity of ENMs is influenced by their size, shape, surface and biological components or ions adsorbed on their surface, their environment, among others. Furthermore, it is evident that the degree of ENMs agglomeration has an effect on the distribution and biological effects in the body as well as individual cells [63]. In the our study, the agglomerate

formation might lead to lower bioavailability of high NP dose and actually affect the degree of NP adverse potential being more pronounced in low dose group of PEG-b-PLA in accordance with remarks by Bruinink et al. [64]. We suggest that a non-monotonous dose-response effect of PEG-b-PLA might coincide with a different level of accumulation/aggregation of used NP suspension. However, the mechanism of polymeric NP PEG-b-PLA action remains unclear and needs further investigation. 5 Conclusions The present study demonstrates the adverse effects of short-time neonatal exposure to polymeric NP PEG-b-PLA on the somatic and pubertal benchmarks and some endpoints of reproductive functions in female rats. There are also indications that NP PEG-b-PLA might interfere with the activation and function of the hypothalamic-pituitary-gonadal axis, and that hormonal effects might play an important role in nanoreprotoxicity of PEG-b-PLA NPs at both central neuroendocrine and gonadal levels. Further, the results of our study suggest that the effects observed following PEG-b-PLA exposure are not likely mediated through the same mechanism as the well-known effects of DES. We believe that this new piece of knowledge might be helpful for the selection of future nanotechnologies and their direct applications to avoid negative effects of nanotherapy. Moreover, our study could focus the attention to requirements of safety assessments of NPs from the point of endocrine disruption. Conflict of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. Declaration of Interest This work was supported by the Grant Agency for Support of Science and Research (APVV) No APVV 0404-11 (acronym NANOREPRO), VEGA grant No 2/0172/14, and the Intramural Grant of Slovak Medical University Bratislava No 16. The work was also supported by the realization of the project “Center of excellence of environmental health“, ITMS No. 26240120033, based on supporting operational Research and development program financed from the European Regional Development Fund and the project of EC FC7, [INFRA-2010-1.131], Contract No. 262,163. Acknowledgements The authors would like to thank Mrs. L. Derkova and Mr. D. Klamo (Department of Toxicology, Slovak Medical University; SMU) for their excellent technical assistance, Mag. M. Stelzer (Department of Slow Viruses, SMU) for excellent processing of the histological samples, and Dr. A. Kocan (Head of Department of Organic Pollutants, SMU) and Dr. E. Pieckova (Head of Laboratory of Mycology, SMU) for the provision of a rotating evaporator. We are indebted to Dr. J.-Z. Du (Department of Polymer Science and Engineering, and CAS Key Laboratory of Soft Matter Chemistry, University of Science and Technology of China, Hefei), and Dr. S. Florian and Dr. I. Lacik (Institute of Polymers, Slovak Academy of Sciences, Bratislava) for their valuable comments regarding the preparation of the nanoparticles tested, and to Dr. M. Novotova (Institute of Molecular Physiology and

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Fig. 1. Representative TEM microphotograph of PEG-b-PLA micelles (transmission electron microscope JEM 1200, JOEL, Tokyo, Japan, with 120 kV voltage) immediately after preparation (A) and after 24 h (B), and typical size distribution of PEG-b-PLA micelles determined by DLS (C).

Fig. 2. Representative examples of the hyperemia of blood vessels and capillaries and vascular dilatation and congestion in the pituitary gland of the adult female rats neonatally exposed to a low dose of PEG-b-PLA (20 mg/kg b.w.). Light-microscopic examination of the control vehicle group (A) and PEG-b-PLA20 group (20 mg/kg b.w) (B).

Fig. 3. Serum luteinizing hormone (LH) concentrations in adult female rats neonatally treated with diethylstilbestrol (DES: 4 μg/kg/day) and two various doses of PEG-b-PLA (20 mg/kg b.w. and 40 mg/kg b.w.) on postnatal days 4–7, before (0 h) and 15, 30, and 50 min after luteinizing hormone releasing hormone (LHRH, 100 ng/kg b.w. in saline solution) administration. Values are expressed as mean ± SEM, 11-14 animals per group. *Statistically significant differences when compared to the vehicle control group, p ≤ 0.05.

Fig. 4. Effect of neonatal exposure to PEG-b-PLA on serum progesterone levels (ng/ml) in adult female rats. DES - diethylstilbestrol, PEG-b-PLA 20 - low dose 20 mg/kg b.w., PEG-bPLA 40 - high dose 40 mg/kg b.w. Values are expressed as median ± Min-Max, 11–14 animals per group. *Statistically significant differences when compared to the vehicle control group, p ≤ 0.05.

Table 1 Effects of intraperitoneal injection of PEG-b-PLA at PND 4–7 on day of vaginal and eye opening. Treatment groups

Negative control

DES

PEG-b-PLA 20

PEG-b-PLA 40

Dose (mg/kg BW/day)

-

0.004

20

40

No. of animals

12

11

13

14

Age of eye opening (days)

15.31 ± 0.13

15.75 ± 0.18

15.00 ± 0.00

15.36 ± 0.48

Body weight (g)

30.21 ± 0.56

30.83 ± 1.24

31.63 ± 0.33

32.15 ± 0.64**

Age of vaginal opening (days)

32.85 ± 0.31

10.92 ± 0.54***

29.85 ± 0.45**

27.93 ± 1.22***

Body weight (g)

101.46 ± 10.83

21.64 ± 1.63***

87.44 ± 11.18**

78.50 ± 4.71***

at eye opening

at vaginal opening BW - body weight. Data are presented as mean ± SEM. **p<0.01 significantly different from Control analyzed by Scheffe multiple comparison test. ***p<0.001 significantly different from Control analyzed by Scheffe multiple comparison test.

Table 2 Effects of intraperitoneal injection of PEG-b-PLA at PND 4–7 on estrous cyclicity in adult rats. Treatment groups

Negative control DES

PEG-b-PLA 20

PEG-b-PLA 40

Dose (mg/kg BW/day)

–

0.004

20

40

No. of rats

13

12

13

14

12* (100%)

5 (38.46%)

4 (28.57%)

1st monitoring at 3 months of age for 23 days No. of rats with 0 (0%) irregular estrous cycles No. of cycles regular

4.00 ± 0.21

0.50 ± 0.15***

2.62 ± 0.35*

2.71 ± 0.32*

irregular

0.25 ± 0.13

1.67 ± 0.23***

0.92 ± 0.18

0.64 ± 0.13

Proestrus

3.33 ± 0.26

4.33 ± 0.63

1.92 ± 0.40#

2.07 ± 0.39

Estrus

5.67 ± 0.41

8.67 ± 2.08

5.31 ± 0.47

5.50 ± 0.47

Diestrus

14.00 ± 0.33

10.00 ± 1.97

15.77 ± 0.50#

15.43 ± 0.33#

12* (100%)

7 (53.85%)

3 (21.43%)

Stage of cycle (days)

2nd monitoring at 5 months of age for 23 days No. of rats with 0 (0%) irregular estrous cycles No. of cycles regular

3.80 ± 0.24

0.25 ± 0.18***

1.62 ± 0.33***

3.70 ± 0.35

irregular

0.42 ± 0.19

2.67 ± 0.31***

1.15 ± 0.22

0.50 ± 0.17

Proestrus

2.67 ± 0.33

1.92 ± 0.38

2.62 ± 0.50

1.93 ± 0.37

Estrus

6.50 ± 0.45

16.92 ± 1.00###

5.23 ± 0.94

5.64 ± 0.41

Diestrus

13.83 ± 0.47

4.08 ± 0.80###

15.15 ± 0.75

15.43 ± 0.37

Stage of cycle (days)

BW - body weight. Data are presented as mean ± SEM.

*p<0.05 significantly different from Control analyzed by Fisher exact test. ***p<0.001 significantly different from Control analyzed by Fisher exact test. #

p<0.05 significantly different from Control analyzed by one-way ANOVA and LSD test.

###

p<0.001 significantly different from Control analyzed by one-way ANOVA and LSD test.

Table 3 Body weights on the days of treatment (PND4 and PND7), body and organ weights on the day of necropsy. Treatment groups

Negative

DES

PEG-b-PLA 20

PEG-b-PLA 40

control Dose (mg/kg BW/day)

–

0.004

20

40

No. of animals

12

11

13

14

PND4

8.91 ± 0.68

8.90 ± 0.46

9.16 ± 0.48

9.54 ± 0.58

PND7

13.84 ± 0.92

13.73 ± 0.92

13.94 ± 0.68

14.09 ± 0.76

300.31 ± 6.80

324.84 ± 5.23

309.09 ± 5.45

Body weight (g)

Day of necropsy 301.92 ± 5.33 Pituitary weight (mg)

13.20 ± 1.04

13.51 ± 0.70

18.59 ± 0.90**

15.98 ± 0.76

(mg%)a

4.91 ± 0.29

4.45 ± 0.28

5.71 ± 0.28

5.13 ± 0.21

(mg)

90.33 ± 7.14

57.32 ± 4.47***

89.01 ± 4.39

90.54 ± 5.22

(mg%)a

32.90 ± 2.65

19.06 ± 1.39***

27.44 ± 1.32

29.35 ± 1.67

(mg)

564.90 ± 26.01

741.39 ± 28.58***

623.78 ± 26.24

659.99 ± 16.32

(mg%)a

205.87 ± 10.25

247.55 ± 9.22**

193.06 ± 9.31

214.09 ±5.51

(mg)

14.47 ± 1.08

15.95 ± 1.31

16.42 ± 1.08

19.62 ± 0.98**

(mg%)a

5.29 ± 0.40

5.31 ± 0.44

5.07 ± 0.34

6.36 ± 0.32

(mg)

52.63 ± 2.57

65.69 ± 2.33**

61.38 ± 2.24

60.06 ± 1.49

(mg%)a

19.09 ± 0.88

21.99 ± 0.92

18.96 ± 0.77

19.47 ± 0.48

Ovary weight

Uterus weight

Thyroid weight

Adrenal weight

Treatment groups

Negative

DES

PEG-b-PLA 20

PEG-b-PLA 40

control Kidney weight (g)

1.89 ± 0.04

2.08±0.04

2.06 ± 0.05

2.08 ± 0.08

(g%)b

0.69 ± 0.02

0.69 ± 0.01

0.63 ± 0.01

0.67 ± 0.02

BW - body weight. Data are presented as mean ± SEM. **p<0.01 significantly different from Control analyzed by ANOVA followed by Bonferroni‘s correction for multiple comparisons. ***p<0.001 significantly different from Control analyzed by ANOVA followed by Bonferroni‘s correction for multiple comparisons. a

Organ weight (mg)/body weight (g) x 100.

b

Organ weight (g)/body weight (g) x 100.