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Abstract

The widespread industrial application of nanomaterials (NMs) has dramatically increased the likelihood of environmental and occupational exposure of humans to such xenobiotics. This issue, together with the increasing public health interest in understanding the effects of chemicals on endocrine system, encouraged to investigate the disruptive potential of NMs on the endocrine function. Therefore, the aim of this study was to evaluate the effects of palladium nanoparticles (Pd-NPs) on the female reproductive system of Wistar rats, intravenously exposed to different doses (0.12, 1.2, and 12 µg/kg), through the assessment of possible quantitative changes in the serum concentrations of several sex hormones. Our results demonstrated that the highest exposure doses significantly reduced the estradiol and testosterone concentrations, while increased the luteinizing hormone levels in treated animals compared to controls. Such alterations are indicative for an abnormal reproductive axis function. However, further investigations are needed to clarify the role of the different NP physicochemical properties in determining such effects, and possible underlining molecular mechanisms, as well as their relevance for the development of diseases in the female reproductive system. Overall, this may be helpful to define accurate risk assessment and management strategies to protect the health of the general and occupational populations exposed to Pd-NPs.

Keywords

Palladium nanoparticles endocrine disruptors hypothalamic–pituitary–gonadal axis sex hormones risk assessment and management

Introduction

The World Health Organization defined an endocrine-disrupting chemical (EDC) as “…an exogenous substance or mixture that alters functions of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.”¹ EDCs are able to affect any aspect of hormone action and consequently, altering homeostasis and influencing physiology and development during the whole life span of an individual (from fetal development to adulthood), they have been called into question in the etiopathogenesis of different metabolic, reproductive, and tumor pathologies, in their progression or state of susceptibility.² Moreover, as if that was not enough, it should be emphasized that EDCs are ubiquitous and humans (both general population and workers) are exposed to these chemicals through multiple routes from several sources, mainly via environmental contamination of the food chain or contact with contaminated environmental matrices.³ Indeed, the current evidence suggests that there are over a thousand suspected EDCs and the chemical substances that belong to this category are highly heterogeneous including industrial solvents and lubricants, dioxins, bisphenol A, polychlorinated biphenyls, persistent organic pollutants, plastic compounds, plasticizers, pesticides, phthalates, and heavy metals.^4
–6 However, recently, Wagner⁶ showed that the vast majority of the studies investigating this issue dealt with only a very small number of substances, then highlighting the need for a greater research effort aimed at identifying the several compounds that could interfere with the endocrine system in order to obtain a more comprehensive understanding of the exposome.

Therefore, it is not surprising that in the last years, a growing number of studies, evaluating the potential adverse health effects of nanomaterials (NMs) and nanoparticles (NPs), investigated also the possible role of these xenobiotics as EDCs.⁷ This important and increasing scientific interest in this topic relies on several reasons. First, NMs and/or NPs are manufactured and used in very wide-ranging industrial sectors such as biomedicine, electronic, textile, energy, plastic, chemical, construction, cosmetic, and food industry; and hence, the likelihood of an ever-expanding public and occupational human exposure will inevitably increase in the coming years.⁸ Second, although the findings of nanotoxicology studies proved that these materials may impact different organ systems (i.e. respiratory, nervous, cardiovascular, immune, and endocrine systems) through several molecular mechanisms of action (i.e. reactive oxygen species production, plasma membrane electron transport system disruption, and alteration of signaling pathway), our current understanding of their toxicokinetic and toxicodynamic behavior is quite incomplete and fragmentary.^8,9 Third, as a consequence of their small size and unique physicochemical properties, the toxicological profiles of NMs and NPs may differ considerably from those of larger micron-sized particles composed of the same materials, and thus it cannot be ruled out that chemical substances, currently nonclassified as EDCs, may disrupt the homeostasis and regulatory mechanisms of the endocrine system at nanoscale level.^7,10

Current evidence showed that different types of NMs are capable of altering the normal and physiological activity of the endocrine system.^7,11,12 In detail, several studies have focused on the effects of these materials on the female reproductive system where NMs were able to exert cytotoxic and/or genotoxic effects on ovarian structural cells and to damage oogenesis and follicle maturation.⁷ Interestingly, NMs and NPs caused also significant alterations in normal sex hormone levels. Nevertheless, existing data of potential endocrine interactions and toxicities of NMs are still limited and often conflicting since, other studies, after NM exposure, failed to confirm the aforementioned adverse effects.⁷

Therefore, considering that toxicological investigation of the potential NM adverse effects on endocrine functions is still in a formative and developing phase, it is evident that further investigation is needed to achieve a greater and more thorough knowledge of the possible threats to mammalian reproduction and fertility posed by NMs. In this context, the aim of the present study was to evaluate the effects of palladium NPs (Pd-NPs) on the female reproductive system of Wistar rats exposed to this xenobiotic for 14 days, by assessing possible quantitative changes in the serum concentrations of several sex hormones (estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), and testosterone (T)). This is one of the most important reproductive endpoints that have already been investigated by several in vivo studies using different types of NMs (Table 1).^{13

–20} Indeed, the knowledge gained from the assessment of hormonal changes serves as a foundation for investigations focused on evaluating NMs as potential endocrine-disrupting toxicants.¹²

Table 1.

In vivo nanotoxicological studies investigating the sex hormone levels in female laboratory animals after exposure to NMs and NPs.

Type of NMs	Physicochemical properties	Experimental protocol	Laboratory animals	Results	References
Anatase TiO₂-NPs	Average particle size: 5–6 nm Hydrodynamic diameter: 208–330 nm Specific surface area: 174.8 m²/g Zeta potential: 7.57 and 9.28 mV	Daily intragastric administration of 10 mg/kg of body weight of TiO₂-NPs for 90 consecutive days	One hundred fifty CD-1 (ICR) female mice	Increases of E2 levels Reduction in P, FSH, LH, and T levels	¹³
ZnO-NPs	Morphology: elongated Average particle size: 20–30 nm Purity: 99.983% Specific surface area: 41 m²/g	Single oral administration (oral gavage) of 333.33 mg/kg of body weight of ZnO-NPs	Twelve female Wistar rats	The sexual hormone levels (E2, FSH, and LH) showed no significant difference between control and treatment group	¹⁴
NR-DE	Average particle size: 22–27 nm >Particle composition: organic carbon (69–73%) and elemental carbon (21–37%) Number concentration: 1.83 × 10⁶ particle/cm³	Inhalation exposure for 5 h daily (from day 1 to 19 of gestation) to 148.86 µg/m³ (1.83 × 10⁶ particle/cm³)	Pregnant Fischer rats (344/DuCrlCrli)	No significant difference between control and treatment group in T levels Slight (not statistically significant) increase in E2 levels Statistically significant increase in LH level Slight (not statistically significant) decrease in FSH levels Statistically significant decrease in P levels	¹⁵
Anatase TiO₂-NPs	Morphology: spherules and irregular shape Particle size: 20–60 nm (spherules) and 40–60 nm (irregular shape) Purity: 99% Specific surface area: 45–55 m²/g	Daily (for five consecutive days) oral administration (oral gavage) of 2 and 1 mg/kg of body weight of TiO²-NPs	Forty-two young sexually mature Sprague-Dawley rats	Significant decrease in T levels at 2 mg/kg No significant difference between control and treatment groups in E2 levels	¹⁶
Ni-NPs	Morphology: spherical Average particle size: 90 nm Hydrodynamic diameter: 260–879 nm Purity: 99%	Daily (for 18 weeks) oral administration (oral gavage) of 45, 15, and 5 mg/kg of body weight of Ni-NPs	Hundred female Sprague-Dawley rats	45 and 15 mg/kg of Ni-NPs significantly increased serum FSH concentrations 45, 15, and 5 mg/kg of Ni-NPs significantly increased serum LH concentrations 45, 15, and 5 mg/kg of Ni-NPs significantly decreased serum E2 concentrations	¹⁷
PEG-b-PLA	Morphology: spherical Hydrodynamic diameter: 50 nm Zeta potential value: 28.73 ± 1.44 mV Size of aggregated NPs: 64.9 ± 10.5 nm and 911.4 ± 177.6 nm	Daily intraperitoneal administration (for 4 days from PND 4 to 7) of 20 or 40 mg/kg of body weight of PEG-b-PLA	Fifty-two female SPF Wistar rats	No significant difference between control and treatment groups in FSH and LH levels Statistically significant increase in P serum levels following exposure to 20 mg/kg	¹⁸
Cu-NPs	Particle size: approximately 100 nm	Daily intraperitoneal administration (for 14 days) of 3.12, 6.35, and 12.5 mg/kg of body weight of Cu-NPs	Twenty-four female Sprague-Dawley rats	12.5 mg/kg of Cu-NPs significantly decreased serum FSH, LH, and P concentrations 6.25 and 3.12 mg/kg did not induce significant difference in FSH, LH, and P levels between control and treatment groups No significant difference between control and treatment group in E2 levels	¹⁹
Ag-NPs	Particle size: 50–60 nm	Daily (for 35 days) oral and dermal administration of 0.5, 1, and 5 mg/kg of body weight of Ag-NPs	Twenty-four female SPF Wistar rats	Significant, dose-dependent decrease in E2 levels	²⁰

Ag: silver; Cu: copper; Ni: nickel; E2: estradiol; FSH: follicle stimulating hormone; LH: luteinizing hormone; NM: nanomaterial; NP: nanoparticle; NR-DE: NP-rich diesel exhaust; P: progesterone; PEG-b-PLA: polymeric NP poly(ethylene glycol)-block-polylactide methyl ether; PND: postnatal day; SPF: specific pathogen free; T: testosterone; TiO₂: titanium dioxide; ZnO: zinc oxide.

Methods

Preparation and characterization of uncoated Pd-NP hydrosol

The details of the synthesis and characterization of Pd-NPs have been described by our group in previously published studies.^21

–24 Briefly, Pd(II) stock standard solution (1000 mg/L, Pd(NO₃)₂ in 0.5 mol/L nitric acid (HNO₃); Merck, Darmstadt, Germany) was used as a precursor for Pd-NP hydrosol preparation by chemical reduction. Sodium borohydride (NaBH₄; p.a., purity ≥96%; Merck) served as a reduction agent; 100 mL of aqueous reduction solution was prepared by dissolving 11 mg of NaBH₄ in 10 mL of ultrapure water (obtained from a Milli-Q system, Millipore Corporation of Billerica, Massachusetts, USA; resistivity 18.2 MΩ/cm) and then filling up to volume with ultrapure water; 500 µL of the Pd(II) stock standard solution was added to this reducing solution. The mixture was shaken thoroughly and reduction of Pd(II) to Pd(0) takes place immediately, which can be seen by the coloring of the originally transparent solution to dark gray. The molar excess of reductant (60 times) assures complete reaction and formation of Pd-NPs. The hydrosol was allowed to stand for 12 h in the dark at room temperature in order to achieve equilibrium in NP formation before further experiments were performed.

Zeta potential of the Pd-NPs was found to be −25.9 ± 5.6 mV in this stock solution (pH = 4.7) measured using Zetasizer Nano ZS (Malvern Instruments GmbH, Herrenberg, Germany) indicating a moderate stability of Pd-NPs in dispersion. Poly dispersity index measured by dynamic light scattering was found to be 0.17 in the stock solutions after ultrasonic treatment. Particle size distribution of Pd-NPs in the hydrosol (Figure 1(a)) was investigated by transmission electron microscopy (TEM; Zeiss EM 10, Carl Zeiss Microscopy GmbH, Jena, Germany; operating voltage 80 kV); 500 individual particles were depicted and sizes were evaluated using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). Figure 1(b) shows an exemplary image of the Pd-NPs. Comparison of measurements of size distribution directly after synthesis (10 ± 6 nm) and after 2 weeks of storage in a refrigerator at 4°C (10 ± 2 nm) revealed that the hydrosol is at least stable for this time period.

Figure 1.

Size characterization of Pd-NPs after 2 weeks of storage. (a) Size distribution histogram of Pd-NPs. Mean size was 10 ± 6 nm; (b) exemplary TEM image of Pd-NPs. The mean size was obtained by evaluating and measuring maximum particle length of 500 individual NPs, depicted by TEM images. Pd-NP: palladium nanoparticle; TEM: transmission electron microscopy.

Aliquots of this Pd-NP hydrosol stock were taken after thorough homogenization by vigorous shaking or ultrasonic treatment. Aliquots were diluted in ultrapure water to obtain the final concentrations used in the exposure experiments.

Continuum source graphite furnace atomic absorption spectrometry (contrAA 600; Analytik Jena, Jena, Germany; used spectral line at 244.791 nm) was used to determine Pd concentration in this hydrosol. For this purpose, aqueous calibration applying adequate dilutions of a Pd stock standard solution (1000 mg/L, Pd(NO₃)₂ in 0.5 mol/L HNO₃, traceable to Standard Reference Materials from the National Institute of Standards and Technology, Merck) in 0.5 mol/L HNO₃ was performed. The Pd concentration in the hydrosol was found to be 4.71 ± 0.05 mg Pd/L. Moreover, the Pd-NP hydrosol was checked for any relevant metal contaminations by total reflection X-ray spectrometry (S2 Picofox; Bruker AXS GmbH, Karlsruhe, Germany); elements found were c(K) = 34 µg/L, Cu = 0.8 µg/L, Zn = 0.3 µg/L, and Fe ≤ 0.1 µg/L.

Animal husbandry

The Experimental Animal Production Plant of the Catholic University of the Sacred Heart of Rome, Italy, supplied 23-month-old female, pathogen-free Wistar rats. Before starting the experiment, the animals were allowed to acclimatize for 2 weeks. The choice to use Wistar rats for our investigation was motivated by the fact that this outbred strain of albino rats is largely employed in all fields of medical and biological research as a general multipurpose model.²⁵ Metabolic pathway similarities to humans, similar anatomical and physiological characteristics, and a large database for comparative purposes support the advantage to employ rats in toxicological research.²⁶ During the entire experiment, the animals were kept in Makrolon cages (model 1291, with overall dimensions of 425 × 266 × 185 mm and floor area of 800 cm²; Tecniplast S.p.A., Buguggiate, Italy) containing a wooden dust-free bedding (model Scobis Uno; Mucedola s.r.l., Settimo Milanese, Italy), at a room temperature of 23.1°C, a relative humidity of 55%, and a 12-h light/dark cycle. The mean weight of animals was 264 ± 18 g and a solid “R” maintenance diet for rats was provided (Altromin Rieper A. S.p.A., Vandoies, Italy). Diet and purified water were provided ad libitum to the animals. During and at the end of the experiments, no significant changes in body weight were evident.

The Ethical Committee “Commissione per la Valutazione Etica di Sperimentazioni Animali e di Correttezza della Gestione della’Animal Care” of the Catholic University of the Sacred Heart of Rome, Italy, approved the animal study and it has been authorized by the Italian Ministry of Health, according to the Legislative Decree 116/92, which implemented in Italy the European Directive 86/609/EEC on laboratory animal protection.

Animal administration and sampling of biological material

The 20 female Wistar rats were randomly divided into three exposure groups and one control group, with five rats per group. Rats were given a single injection of vehicle (control group) and different concentrations of Pd-NPs (0.12, 1.2, and 12 μg/kg) via the tail vein. The intravenous route of application was chosen to reach a worst case scenario of 100% xenobiotic bioavailability. The treatment doses were selected with the aim to resemble possible occupational and/or environmental exposure scenarios. Indeed, considering the Pd airborne levels (highest mean level of 7.70 ± 4.15 μg/m³) reported in the literature for a workplace setting and a human breathing rate of around 20 m³/day (for a man with a mean weight of 70 kg), a potential occupational exposure to Pd via inhalation corresponds to an exposure dose of 2.20 μg/kg, which is in the range of doses used in our experiments.²⁷

On 14th day after exposure, rats were anesthetized with 0.5 mg of medetomidine and 75 mg of ketamine per kilogram of body weight. Subsequently, blood from each animal was drawn by cardiac puncture and collected in a 1.5 mL vial (Eppendorf srl, Milan, Italy). Serum samples were obtained from blood by centrifugation (3500 r/min per 15 min) and stored at −28°C until analysis. After the blood sampling, rats were killed via exsanguination by cutting both the abdominal aorta and vena cava.

Analysis of sex hormones

E2, FSH, LH, P, and T levels were determined in the serum samples collected 14 days after exposure using five different enzyme-linked immunosorbent assay (ELISA) systems. All reagents and samples were brought to room temperature before use, and the subsequent analytical procedures were carried out according to manufacturer instructions. ELISA kits for the analyses of E2, P, and T were provided by Demeditec Diagnostics GmbH (Kiel, Germany), whereas the determination of FSH and LH levels was carried out using ELISA kits of Cloud-Clone Corp (Wuhan, Hubei Province, China). The absorbance was measured at 450 nm with SpectraMax1 Plus 384 spectrophotometer (Molecular Devices Corp., Sunnyvale, California, USA), and sex hormones concentrations were given in nanograms per milliliter after proper calibration.

Statistical methods

Statistical analysis was carried out by IBM SPSS statistics software (IBM Statistical Package for Social Sciences for Windows, version 22.0., Armonk, New York, USA). Serum levels of sex hormones (E2, FSH, LH, P, and T) were measured after the three levels of exposure on day 14. The normal distribution of observed values was checked using the nonparametric Kolmogorov–Smirnov Z test and variance homogeneity was tested using the Levene test. One-way analysis of variance was then performed to test the significance of differences in parameter means in the exposed and control rat groups. The Dunnett post hoc multiple comparison test was used to test the significance (p value Dunnett t test < 0.05) of differences in values for each parameter at different exposure levels against the control group. Box-plot or linear graphs were obtained for all analyzed parameters at different exposure levels.

Results

The serum levels of the various sex hormones (E2, FSH, LH, P, and T) in female Wistar rats after the intravenous administration of 0.12, 1.2, and 12 μg/kg of Pd-NPs are reported in Table 2 and Figure 2. Our findings revealed that exposure to this xenobiotic caused important alterations of sex hormone serum levels in the tested laboratory animals. In fact, each sex hormone investigated showed alterations in serum concentrations compared to the control levels. In detail, the administration of Pd-NPs induced a dose-dependent increase in FSH, LH, and P, while, on the contrary, the mean serum levels of E2 and T progressively decreased with increasing exposure doses.

Table 2.

Mean serum levels (standard error) and statistical significance of E₂, FSH, LH, P, and T in control and Pd-NP exposed (0.12, 1.2, and 12 μg/kg) female Wistar rats.^a

Sex hormones (ng/mL)	Controls	Doses of exposure (μg/kg)			ANOVA F test	p Value ANOVA
Sex hormones (ng/mL)	Controls	0.12	1.2	12	ANOVA F test	p Value ANOVA
E2	0.032 (0.006)	0.016 (0.001)	0.012^b (0.005)	0.012^b (0.002)	5.2	0.01
FSH	13.62 (1.28)	19.19 (1.78)	20.33 (3.04)	21.46 (1.95)	2.7	0.08
LH	16.48 (1.31)	18.31 (2.52)	20.11^b (1.14)	23.09^b (1.07)	5.8	0.007
P	151.5 (10.02)	185.09 (33.07)	194.06 (5.15)	205.37 (18.13)	1.3	0.28
T	0.80 (0.76)	0.62 (0.06)	0.60 (0.03)	0.47^b (0.04)	5.7	0.007

E2: estradiol; FSH: follicle stimulating hormone; LH: luteinizing hormone; P: progesterone; T: testosterone; ANOVA: one-way analysis of variance; Pd-NP: palladium nanoparticle.

^aANOVA test and statistical significance (p value ANOVA).

^b p < 0.05, significance of the difference between mean in each exposed group and mean in the control group.

^c p < 0.05.

Figure 2.

Serum levels of different sex hormones (E2, FSH, LH, P, and T) in control and Pd-NPs exposed (0.12, 1.2, and 12 μg/kg of body weight) rats. (a) Serum estradiol concentrations; (b) serum FSH concentrations; (c) serum LH concentrations; (d) serum P concentrations; (e) serum T concentrations. Outliers (°) measures between 1.5- and 3-fold that of the interquartile range (75° and 25° percentile). Extreme values (*) more than 3-fold that of the interquartile range. E2: estradiol; FSH: follicle stimulating hormone; LH: luteinizing hormone; P: progesterone; T: testosterone; Pd-NP: palladium nanoparticle.

Interestingly, the highest exposure dose (12 μg/kg) determined a statistically significant reduction in E2 and T concentrations and an increase in LH levels. Moreover, statistically significant differences were also observed for E2 and LH after the exposure to the mild dose of 1.2 μg/kg. With regard to the FSH and P serum concentrations, the comparison between the levels observed in the control group and those determined in the exposure groups never reaches the statistical significance. However, it should be noted that for both hormones, the trend of serum concentrations (as a function of the exposure doses used) seems fairly regular, constantly increasing as exposure doses rise. Therefore, this observation could suggest the hypothesis that the lack of statistical significance for FSH and P is probably related to a problem of low sample size.

Overall, our results demonstrate the ability of Pd-NPs to significantly alter the normal sex hormone levels, thus suggesting the possibility that this xenobiotic may play an important role in disrupting the physiological functions of female reproductive system of Wistar rats.

Discussion

The rapid development and widespread application of NMs, together with the enhancing interest in the public health effects of EDCs, has given stimulus to understand possible alterations induced by NMs on such apparatus, considering also its relevance in maintaining the homeostasis and controlling proper growth and development of the organisms.^7,28

In this scenario, the female reproductive system and its regulating hypothalamic–pituitary–gonadal (HPG) axis have received most attention. The hypothalamus, in fact, is able to secrete into the hypophyseal portal circulation the gonadotropin-releasing hormones which act on the pituitary gland to regulate the synthesis and secretion of the gonadotropins, LH, and FSH. Once in the gonads, these hormones can stimulate the folliculogenesis and the E2 steroid hormone secretion from the ovaries as well as the production of P and E2 by the corpus luteum after ovulation has occurred. In turn, these ovarian hormones can regulate the hypothalamic and pituitary activities through a strictly controlled inhibitory feedback mechanism.^17,29,30

Exogenous chemicals can interfere with the normal functioning of the HPG axis.³¹ In particular, nanosized materials were reported to affect in vivo the female reproductive systems, causing significant alterations in sex hormone levels, cytotoxic, and/or genotoxic effects on ovarian structural cells and damaged oogenesis and follicle maturation.⁷

To the best of our knowledge, this is the first work aimed to assess the effects of Pd-NPs on aspects of serum sex hormone levels (i.e. FSH, LH, E2, P, or T) in female rats. Our findings demonstrate that subacute exposure to Pd-NPs significantly reduced the E2 and T concentrations while increased the LH levels in treated animals. The detected alterations in such hormonal levels are indicative for an abnormal reproductive axis function. These findings are in line with some previous reported data obtained with other kinds of NMs, that is, Ni-,¹⁷ TiO₂-,¹⁶ Ag-,²⁰ and NP-rich diesel exhaust,¹⁵ while are in conflict with other investigations which demonstrated opposite hormonal response trend^13,19 or failed to detect significant changes in sex hormone levels after NM exposure.^14,18

These different results may be due to the features of NMs used in the studies. In fact, NM physicochemical characteristics, that is, particle size, shape, chemical properties, surface area and functionalization, charge, solubility, oxidant generation potential, and degree of agglomeration, may all influence NM interactions with biological systems, therefore determining different toxicological responses.³² It is important to note also that biomolecular interactions once NMs are adsorbed and distributed into the organism may contribute to confer them peculiar properties potentially influencing their effects on the endocrine system.

Additionally, the diverse range of doses and modes of administration employed prevent an accurate comparison between the obtained results. Importantly, most of the studies used unrealistically high NM doses to treat animals, which makes difficult the extrapolation of relevant conclusions for the human population, which may be potentially exposed to lower levels of such chemicals. Our study adopted doses of treatment based on the environmental levels of Pd detected in real workplace settings,²⁷ in order to resemble ordinary and accidental conditions of exposure potentially experienced in occupational and general living environments. This seems relevant from a public health perspective, considering the increasing interest in understanding the effects of EDCs at low-dose conditions of exposure. In this preliminary phase of knowledge, the large range of concentrations explored was also chosen to extrapolate a suitable dose–response relationship which may be helpful in acquiring information to better understand the toxicity of nanosized chemicals on the endocrine system.

In line with this latter issue, the persistence of the detected alterations in the long term, as well as their relevance for the development of diseases in the female reproductive system, including irregularities in menstruation cycle, precocious puberty, polycystic ovary syndrome, or premature ovarian failure; the manifestation of unwanted side effects like increase growth of endometrium or higher risk of breast cancer; or alterations in fertility outcomes, that is, mating success rate and pregnancy rate, should be deeply clarified.^33,34 Therefore, future investigations testing the endocrine disruptive properties of Pd-NPs should include lifelong follow-up to assess not only the persistence of such effects, but also to point out possible latent alterations. In this perspective, further research seems necessary to define if the adverse consequences of Pd-NPs on the reproductive endocrine system may affect the subsequent generations even though they were not directly exposed to the endocrine disruptors. Live birth rate, birth survival and feeding survival rate, and the occurrence of gross malformations in pups should be studied and eventually put in relation with possible endocrine hazardous exposures. Overall, the complexity of the endocrine system and the great variability in NM toxicological behavior require investigation strategies specifically focused on the endocrine disrupting outcomes, which may be quite different compared to those designed to detect acute toxicity.

Additionally, nanosized endocrine disruptors may have different effects according to the age of exposure, that is, it cannot be ruled out that the hormone alterations we detected in sexually mature female rats may not be evident or be different in immature animals. In this context, further research should also explore the existence of specific periods of life more sensitive than others in the exposure-related endocrine effect manifestations. From a general and occupational health perspective, this topic requires scientific efforts aimed to identify the vulnerability and risk factors for endocrine disrupting actions, in order to predict effects on single individuals or whole populations, and particularly on those people occupationally exposed to NMs in workplace settings involved in NM synthesis, production, and application. Overall, in a precautionary manner, this may be important also to guide the adoption/implementation of adequate preventive and protective measures to manage risks derived from nanosized EDCs, specifically tailored on physiological, that is, women of childbearing age, or pathological conditions of susceptibility. Furthermore, to understand the environmental fate of dispersed NMs, which largely depends on their persistent nature and stability into the environment, as well as their toxicokinetic and toxicodynamic behavior once adsorbed into the organisms may be helpful in defining the occasions of contact with the substances, the exposure length, potential endocrine target organs, and latency periods for the development of the effects. To date, this seems a quite challenging issue since exposure assessments still lack standardization, and dosimetric parameters responsible for the biological effects of NMs have not been fully elucidated. Moreover, the possible involvement of other environmental or occupational pollutants in affecting the endocrine disrupting activity of NMs should be carefully evaluated since effects of different classes of EDCs may be additive or synergic.

As an ulterior point of discussion, at present, it is quite hard to predict the mechanisms by which Pd-NPs may exert their effects on the reproductive system. They may imitate or partly imitate naturally occurring hormones in the body, potentially producing overstimulation. On the other side, they may behave as antagonists, acting directly on hormone receptors, therefore preventing the normal activation of the signal cascades, as well as on any number of proteins that control the delivery of a hormone to its normal target cell or tissues. However, the possible direct damaging effect of NMs on endocrine tissues and cells, their ability to induce inflammatory and oxidative stress responses in target tissues, as well as possible epigenetic mechanisms induced by NMs should be investigated to define possible interesting modes of action.

Overall, further research may allow to confirm the potential toxicity of Pd-NPs on the endocrine system and provide useful information to be considered in even more accurate risk assessment processes, to help suitable risk communication and comprehensive risk management strategies, and to protect the health of the general populations and, more specifically, that of subjects exposed to Pd-NPs in occupational settings.

In conclusion, this study represents the first attempt to investigate the endocrine disruptive potential of Pd-NPs in an animal model of sexually mature female Wistar rats. An abnormal function of the HPG axis was detected as demonstrated by the reduced serum concentrations of E2 and T and the increased level of LH in treated animals compared to controls.

Although preliminary, these results appear relevant considering the large application of NMs in technological and industrial fields, their increased dispersion in general living environments and in workplaces, as well as the consequent enhanced likelihood of exposure for both the general and occupational involved populations. Importantly, these findings raise challenging questions concerning the possible implications that these hormonal changes may have on the reproductive health of exposed females, particularly in contexts of low-dose long-term exposure, and the potential molecular mechanisms responsible for such effects. Therefore, the intrigued interplay between the complex organization of the reproductive endocrine hormonal pathways and the variable toxicological behavior of differently characterized NPs require further, specifically focused, nanotoxicological investigations aimed to define the endocrine disrupting potential of Pd-NPs. Overall, this seems important to reach an accurate risk assessment process, to plan risk communication strategies, and to define specific preventive and protective measures to manage “endocrine risks” derived from NP exposure for the general and occupational exposed populations, considering also physiological or pathological conditions of hypersusceptibility.

Footnotes

Authors’ contribution

AS and II are co-senior authors. VL and LF have equally contributed to this work.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Palladium nanoparticle effects on endocrine reproductive system of female rats

Abstract

Keywords

Introduction

Methods

Preparation and characterization of uncoated Pd-NP hydrosol

Animal husbandry

Animal administration and sampling of biological material

Analysis of sex hormones

Statistical methods

Results

Discussion

Footnotes

Authors’ contribution

Declaration of Conflicting Interests

Funding

References