Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Exposure to Palladium Nanoparticles Affects Serum Levels of Cytokines in Female Wistar Rats

  • Ivo Iavicoli ,

    iavicoli.ivo@rm.unicatt.it

    Affiliation Institute of Public Health, Section of Occupational Medicine, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168, Rome, Italy

  • Luca Fontana,

    Affiliation Institute of Public Health, Section of Occupational Medicine, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168, Rome, Italy

  • Maddalena Corbi,

    Affiliation Institute of General Pathology, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168, Rome, Italy

  • Veruscka Leso,

    Affiliation Institute of Public Health, Section of Occupational Medicine, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168, Rome, Italy

  • Alessandro Marinaccio,

    Affiliation Epidemiology Unit, Occupational Medicine Department, Research Division, Italian Workers’ Compensation Authority (INAIL), Via Alessandria, 220/E, 00198, Rome, Italy

  • Kerstin Leopold,

    Affiliation Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Albert Einstein-Str. 11, 89081, Ulm, Germany

  • Roland Schindl,

    Affiliation Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Albert Einstein-Str. 11, 89081, Ulm, Germany

  • Alessandro Sgambato

    Affiliation Institute of General Pathology, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168, Rome, Italy

Exposure to Palladium Nanoparticles Affects Serum Levels of Cytokines in Female Wistar Rats

  • Ivo Iavicoli, 
  • Luca Fontana, 
  • Maddalena Corbi, 
  • Veruscka Leso, 
  • Alessandro Marinaccio, 
  • Kerstin Leopold, 
  • Roland Schindl, 
  • Alessandro Sgambato
PLOS
x

Abstract

Background

Information currently available on the impact of palladium on the immune system mainly derives from studies assessing the biological effects of palladium salts. However, in the last years, there has been a notable increase in occupational and environmental levels of fine and ultrafine palladium particles released from automobile catalytic converters, which may play a role in palladium sensitization. In this context, the evaluation of the possible effects exerted by palladium nanoparticles (Pd-NPs) on the immune system is essential to comprehensively assess palladium immunotoxic potential.

Aim

Therefore, the aim of this study was to investigate the effects of Pd-NPs on the immune system of female Wistar rats exposed to this xenobiotic for 14 days, by assessing possible quantitative changes in a number of cytokines: IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α.

Methods

Twenty rats were randomly divided into four exposure groups and one of control. Animals were given a single tail vein injection of vehicle (control group) and different concentrations of Pd-NPs (0.012, 0.12, 1.2 and 12 μg/kg). A multiplex biometric enzyme linked immunosorbent assay was used to evaluate cytokine serum levels.

Results

The mean serum concentrations of all cytokines decreased after the administration of 0.012 μg/kg of Pd-NPs, whereas exceeded the control levels at higher exposure doses. The highest concentration of Pd-NPs (12 μg/kg) induced a significant increase of IL-1α, IL-4, IL-6, IL-10, IL-12, GM-CSF and INF-γ compared to controls.

Discussion and Conclusions

These results demonstrated that Pd-NP exposure can affect the immune response of rats inducing a stimulatory action that becomes significant at the highest administered dose. Our findings did not show an imbalance between cytokines produced by CD4+ T helper (Th) cells 1 and 2, thus suggesting a generalized stimulation of the immune system with a simultaneous activation and polarization of the naïve T cells towards Th1 and Th2 phenotype.

Introduction

Palladium (Pd) is a noble metal that belongs to the platinum group elements (PGEs). Over the past few decades, Pd found increasing application as an active catalyst material in modern three-way automobile catalytic converters [1, 2].

The mandatory use of these devices has resulted in a significant reduction in the emission into the atmosphere of hazardous pollutants from lean-burn engines with more than 90% of carbon monoxide, hydrocarbons, and nitrogen oxides (NOx) being converted into less harmful carbon dioxide, water and nitrogen [35]. Unfortunately, although these devices reduce emissions of the aforementioned pollutants, they have become a primary anthropogenic source of Pd, which is released into the environment, both in the fine and ultrafine (<100 nm) airborne particle fraction, due to the physico-chemical [69]. This release has inevitably increased the Pd levels in the general living and occupational environments [1016], therefore enhancing the likelihood of human exposure to Pd particles, also in the nano-metric scale. In this emerging exposure scenario, concerns have been raised regarding the possible adverse effects Pd-NPs may exert on the human health, and particularly on the immune system of exposed subjects.

Recent evidence, in fact, demonstrated the Pd ability to induce allergic reactions in susceptible individuals generally exposed to the metal through jewellery and dental restoration contact [1724], which could be mediated by the release of Pd ions acting as potent sensitizers [25]. Additionally, exposure to Pd-salts was demonstrated to significantly affect the production and release of different cytokines (Table 1). An increase of the interleukin (IL)-6 levels was detected in an in vitro skin equivalent model, consisting of human fibroblasts and keratinocytes [26]. Comparably, an enhanced secretion of IL-6 and IL-8 was observed in a three-dimensional human tissue model based on TR146 cells isolated from a squamous cell carcinoma of the buccal mucosa [27], while an and inhibiting effect on the release of IL-5, interferon (INF)-γ, and tumor necrosis factor (TNF)-α was reported in human peripheral blood mononuclear cells (PBMC) obtained from healthy male volunteers [28]. Similarly, our previous in vivo studies (Table 1) showed that Pd has a significant immuno-modulating effect able to alter the T-helper (Th)1/Th2 cytokine balance in Wistar rats subacutely and subchronically exposed to a Pd salt [29, 30].

thumbnail
Table 1. In vitro and in vivo studies investigating cytokine production after exposure to Pd and Pd-NPs.

https://doi.org/10.1371/journal.pone.0143801.t001

Concerning the immunologic effects induced by Pd nanoparticles (Pd-NPs), recent in vitro investigations have proved the ability of such NPs to modulate the expression and release of different cytokines, although with quite different results compared to the Pd-bulk forms (Table 1). Wilkinson et al. [31] showed that the treatment of primary bronchial epithelial cells (PBEC) and lung carcinoma epithelial cell line (A549) with 0.01–10 μg/ml of Pd-NPs resulted in a concentration-dependent reduction in IL-8, in the lower concentration range, and a slight tendency towards increased levels at the highest concentration, in addition to a decrease of pro-inflammatory cytokine TNF-α in human epithelial cells. The influence of Pd-NPs (5–10 nm) on the release and expression of cytokines was also investigated in PBMC of non-atopic women exposed to 10−5 and 10−6 M of this xenobiotic [32]. In this study, Pd-NPs exerted immuno-modulatory effects enhancing the release of IFN-γ and inhibiting the secretion of TNF-α and IL-17. Similar results were also observed in PBMCs of Pd-sensitized women exposed to comparable concentrations of Pd-NPs [33].

From a public and occupational health perspective, the increasing levels of Pd in living and working environments, its well known hyper-sensitivity potential and the preliminary results concerning the ability of its nano-sized form to induce immunological alterations in vitro, seem to call for greater scientific efforts to define the possible immuno-toxic action of Pd-NPs in animal models. This appear an even more urgent issue of research, considering that the peculiar physico-chemical properties of materials at the nano-sized level may change their biological reactivity and potentially their harmful effects on human health [34, 35]. Therefore, the aim of the present study was to evaluate the effects of Pd-NPs on the immune system of female Wistar rats exposed to this xenobiotic for 14 days, by assessing possible quantitative changes in a number of cytokines (IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), INF-γ and TNF-α).

Materials and Methods

Preparation and characterization of uncoated palladium nanoparticle hydrosol

As a first step, 300 μL of a freshly prepared 0.029 molar sodium borohydride solution, obtained by dissolution of 11 mg of sodium borohydride (p.a., Merck, Darmstadt, Germany) in 10 mL of ultrapure water, were diluted in 100 mL of ultrapure water. Then, 500 μL of a Pd stock standard solution (1000 mg/L, Pd(NO3)2 in 0.5 mol/L HNO3, Merck, Darmstadt, Germany) were added and the mixture was shaken thoroughly. The immediate color change from transparent to dark grey indicated the formation of Pd-NPs. The mixture was kept in the dark at room temperature for 12 hours to allow complete reaction.

The Pd-NPs hydrosol obtained was characterized by continuum source—graphite furnace atomic absorption spectrometry (CS-GFAAS; contrAA 600, Analytik Jena, Jena, Germany) and transmission electron microscopy (TEM; Zeiss EM 10, Carl Zeiss Microscopy GmbH, Jena, Germany) operating at 80 kV. The Pd concentration of the stock hydrosol was determined in a 100-fold dilution of the stock hydrosol in ultra pure water by means of CS-GFAAS using the spectral line at 244.791 nm. Calibration was performed in a concentration ranging from 20 to 80 μg Pd/L by 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, Darmstadt, Germany) in 0.5 mol/L HNO3. This resulted in a linear calibration function with a correlation coefficient of 0.986. The stock hydrosol Pd concentration was found to be 4.71 ± 0.05 mg/L. The measurement of 500 individual particles depicted by TEM images using ImageJ software (National Institutes of Health, Bethesda, MD) revealed the size distribution of the particles to be 10 ± 6 nm (Fig 1). The hydrosol served as a stock solution for all experiments and is stable for at least 2 weeks when stored in refrigerators at 4°C. Before use, the stored Pd-NP hydrosol was homogenized by shaking vigorously. Finally, aliquots of the stock solution were diluted in ultrapure water to obtain the final concentrations used in the experiments.

thumbnail
Fig 1. Palladium nanoparticle characterization.

Mean Size distribution histogram of Pd-NPs (A) obtained from evaluation of TEM images (B) taking into account over 500 nanoparticles. The measurement of 500 individual particles depicted by TEM images revealed the size distribution of the particles to be 10 ± 6 nm.

https://doi.org/10.1371/journal.pone.0143801.g001

Animal husbandry

Twenty three-month-old female, pathogen-free Wistar rats were supplied by the Experimental Animal Production Plant of the Catholic University of the Sacred Heart (Rome, Italy) and allowed to acclimatize for two weeks before starting the experiment. Wistar rats are an outbred strain of albino rats employed in all fields of medical and biological research as a general multipurpose model [36]. In fact, the use of rats offers a series of advantages such as metabolic pathway similarities to humans, similar anatomical and physiological characteristics, a large database for comparative purposes [37]. In this regard, currently, the rat is definitely the species of choice for non-clinical immuno-toxicity and in particular outbred Wistar rats are often used due to their acceptable inter-animal variability [38, 39]. The animals were maintained during the entire experiment in Makrolon cages (model 1291, with overall dimensions of 425x266x185 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 animals had a mean weight of 271 ± 16 g and were fed with the solid “R” maintenance diet for rats (Altromin Rieper A. S.p.A., Vandoies, Italy). Diet and purified water were provided ad libitum to the animals. No significant changes in body weight were observed during and at the end of the experiments.

Ethics statement

The animal study has been approved by the Ethical Committee “Commissione per la Valutazione Etica di Sperimentazioni Animali e di Correttezza della Gestione dell’”Animal Care” of the Catholic University of the Sacred Heart of Rome, Italy, under permit number 20H, and 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. Animals used in this study were housed and treated according to Legislative Decree 116/92 guidelines and all efforts were made to minimize animal suffering.

Animal administration and sampling of biological material

The twenty female Wistar rats were randomly divided into four exposure groups and one control group, with four rats per group. Rats were given a single injection of vehicle (control group) and different concentrations of Pd-NPs (0.012, 0.12, 1.2 and 12 μg/kg) via the tail vein. On 14th day after exposure, rats were anesthetized with 0.5 mg of medetomine and 75 mg of ketamine per kg 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 (3,500 rpm per 15 min) and stored at -28°C until analysis. After the blood sampling, rats were euthanized via exsanguination by cutting both the abdominal aorta and vena cava.

This particular administration route was chosen for the xenobiotic as the intravenous route of application produced a worst case scenario of 100% bioavailability. The doses used to treat animals, were selected in order to resemble possible occupational and/or environmental exposure scenarios. In fact, if we take into consideration the Pd airborne levels (highest mean level of 7.70±4.15 mg/m3) reported in literature for an occupational setting [40] 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 mg/kg, which is in the range of doses used in our experiments. Therefore, the higher exposure doses (1.2 and 12 mg/kg) simulated possible occupational exposure both under normal and accidental conditions during which re-exposure can occur. The lower doses (0.012 and 0.12 mg/kg) were used to investigate potential adverse effects at exposure levels closely resembling those of the general population and to establish a preliminary dose-response curve for defining the toxicological behavior of Pd-NPs [41].

Analysis of serum cytokines

A multiplex biometric enzyme linked immunosorbent assay (ELISA)-based immunoassay, containing dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used, in accordance with the manufacturer’s instructions (Bioplex Rat Cytokine 9-Plex A panel; BioRad Inc., Hercules, CA), for the simultaneous detection and quantitation of IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α. Cytokine levels were determined using a Bio-Plex array reader an automated flow-based microfluidic device that uses a dual-laser fluorescent detector with real-time digital signal processing for quantitation (Bioplex, Biorad).

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). Levels of cytokines IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α were measured after the four levels of exposure on day 14. The normal distribution of observed values was checked using the non-parametric Kolmogorov–Smirnov Z test and variance homogeneity was tested using the Levene test. One-way analysis of variance (ANOVA) 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

Table 2, Figs 2 and 3 and S1 Fig show serum levels of the various cytokines (IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α) in rats after the intravenous administration of 0.012, 0.12, 1.2 and 12 μg/Kg of Pd-NPs. The results obtained demonstrated that exposure to Pd-NPs was able to affect immune response in female Wistar rats. Indeed, each cytokine investigated showed alterations in serum concentrations compared to the control levels. The mean serum concentrations of all cytokines appeared to decrease after the administration of 0.012 μg/kg Pd-NPs, whereas their values exceeded the control levels at higher doses of exposure (0.12, 1.2 and 12 μg/kg).

thumbnail
Fig 2. Mean serum levels of cytokines in Wistar rats exposed to Pd-NPs compared to control rats.

Compared to control values, a rather particular trend was observed in all cytokine serum levels in the treated rats, with a slight decrease at the lowest exposure dose and an increase thereafter with increasing exposure doses. Indeed, the mean serum concentrations of all cytokines appeared to decrease after the administration of 0.012 μg/kg Pd-NPs, whereas their values exceeded the control levels at higher doses of exposure (0.12, 1.2 and 12 μg/kg).

https://doi.org/10.1371/journal.pone.0143801.g002

thumbnail
Fig 3. Serum levels of different cytokines in control and palladium nanoparticle exposed rats.

In the exposure range from 0.12 to 1.2 μg/kg it was possible to observe a general, but not statistically significant (with the exception of IL-2, IL-4 and IL-10 at 0.12 μg/kg), increase in all cytokine serum levels, while at 12 μg/kg 7 out of 9 of the cytokines examined showed remarkable (and statistically significant) increases in serum concentrations. *Group mean significantly different from controls (p value < 0.05).

https://doi.org/10.1371/journal.pone.0143801.g003

thumbnail
Table 2. Mean serum levels (standard error) and statistical significance of IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α in control and palladium nanoparticles-exposed (0.012, 0.12, 1.2 and 12 μg/kg) female Wistar rats.

https://doi.org/10.1371/journal.pone.0143801.t002

The highest concentration of Pd-NPs (12 μg/kg) induced a statistically significant increase of IL-1α, IL-4, IL-6, IL-10, IL-12, GM-CSF and INF-γ compared to controls, while at the same dose of exposure the values of other cytokines, although higher than in untreated rats, did not show significant differences. A noticeable increase in IL-2, IL-4 and IL-10 serum concentrations was also observed also at 0.12 μg/kg. These results showed that the exposure to 12 μg/kg of Pd-NPs caused an important stimulatory effect on the immune system of female Wistar rats.

Discussion

In the last few years there has been a significant increase in the Pd content of catalytic converters since this metal is cheaper and has a very high catalytic activity [2]. This enlarged Pd employment resulted in serious contamination of a number of environmental matrices with a consequent increasing likelihood of general living and occupational exposure to the metal, both in the fine and ultrafine airborne particle fractions [1016]. Therefore, the definition of the potential health effects induced by Pd-NP exposure has become an issue of public health relevance.

Currently, most available information concerning the impact of Pd on the immune system is the result of in vitro and in vivo studies that have assessed the biological effects induced by different Pd salts [21, 2630]. Nevertheless, the findings of these studies may not be sufficient to explain the immunotoxic potential of Pd in the nano-sized scale as potentially released from automobile catalyst abrasion and deterioration [3, 7, 9, 4244]. The unique set of NP physico-chemical characteristics, in fact, may affect their toxico-kynetic and dynamic behavior, therefore directly or indirectly influencing the possible interactions with the immune system, in a potentially different manner compared to their bulk counterpart. This important issue prevents us to extrapolate data from Pd salts to a context of nano-sized Pd exposure [34, 35, 45, 46] and applies for a deep research on the Pd-NP toxicological profile to obtain, in turn, a more comprehensive assessment of the immunological toxicity of the metal.

Therefore, the present study aimed at evaluating the possible adverse effects of Pd-NPs on the immune system of female Wistar rats intravenously exposed to these xenobiotics through the determination of the serum levels of a series of different cytokines.

An important stimulatory action on the immune system, which becomes significant at the highest dose of treatment has been demonstrated. In fact, an overall up-ward trend, although not significant, was observed for all cytokine serum levels in the 0.12–1.2 μg/kg dose range (with the exception of significant increases detected for IL-2, IL-4 and IL-10 at 0.12 μg/kg), while at 12 μg/kg, 7 out of 9 of the cytokines examined showed significantly remarkable increases in serum concentrations (Table 2). This systemic cytokine activation supports a clear pro-inflammatory action of Pd-NPs when administered in vivo, which, not surprisingly, has a rather different profile compared to the immune alterations detected in previous studies exploring a variety of Pd salts. In fact, while we observed a stimulatory response on the production of IFN-γ and a slight increase (though not significant) in TNF-α, the exposure of PBMC to various Pd salts induced inhibitory effects on these cytokine secretion [28]. Moreover, the enhanced IFN-γ and IL-4 levels reported herein were not detected in our previous in vivo studies (sub-acute and sub-chronic exposure of Wistar rats to potassium hexachloropalladate, respectively), even if in each of these experiments some results (increased IL-4 and IL-2 production after sub-acute administration and increased IFN-γ levels following sub-chronic exposure to Pd salt) were somewhat similar to those induced by Pd-NPs [29, 30]. These quite conflicting results, further underlines the need to specifically investigate the Pd-NP interaction with the immune system which seems different from that of Pd salts, probably depending on the diverse biological reactivity determined by the peculiar chemical, optical, magnetic and structural NP properties, as previously mentioned.

Additionally, comparing our results with those obtained with Pd-NPs in vitro, a certain variability concerning the activated Th cell subsets and the induced cytokine profiles emerged [32, 33]. This seems an interesting topic of research, since understanding how the immune system adapts to the insults of specific xenobiotics, maybe through an excessive Th1 or Th2 responses, with different tissue damages or hypersensitivity reactions, respectively, gives the possibility to deeply understand the toxicological behavior of such NPs, therefore identifying early and specific biological alterations [4749]. In this perspective, a clear NP induced imbalance towards a Th1 mediate immune response was recently reported in in vitro studies [32, 33], while our findings demonstrated a Pd-NP induced up-ward trend among all the investigated cytokines, therefore supporting a more complex and generalized inflammatory activation of the immune system in exposed animals. Overall, this suggests that the systemic cytokine activation induced by Pd-NPs in vivo was not related to a specific Th pattern since no imbalance was evident between commonly studied Th1 and Th2 cytokine subsets.

This may reflect the complexity of the Pd-NP interaction with in vivo biological systems which cannot be thoroughly resembled by in vitro results [50]. In fact, several in vivo factors such as exposure mode, penetration of physiological barriers, solubility in biological media as well as the protein corona formation, as the result of a dynamic nano-bio interaction, can dramatically change the effects of challenging the immune system with a given concentration of a specific xenobiotic [51, 52].

When analyzing the dose-response relationship obtained in our study, a rather particular trend was observed in all cytokine serum levels in the treated rats (Fig 2) with a slight decrease at the lowest exposure dose and an increase thereafter with increasing exposure doses. Comparably, Wilkinson et al. [31] observed a similar dose-response trend, with a decrease in the IL-8 release from PBEC and A549 cells at the lower concentration range and a slight tendency towards increased levels at the highest concentration. These dose–response relationships would seem to suggest the presence of a hormetic phenomenon since in some cases the hormetic effects are typically graphed as a J-shaped dose-response curve [53]. In fact, the term “hormesis” is used to describe dose-response curves where the response is reversed between low and high doses of a stressor (Fig 4) representing an index of biological plasticity at multiple levels of biological organization [54]. In this regard, it is possible to hypothesize that the decrease in cytokine levels determined at the lowest dose of exposure may be an adaptive compensatory process following an initial disruption in homeostasis induced by the NP chemical stress, which ultimately may induce increasing alterations in the cytokine concentrations at the higher treatment doses. Greater attention is being given to hormesis in the fields of aging and biogerontology, toxicology, pharmacology, public health and occupational medicine research, and recently this dose-response model has been shown to occur quite frequently also after exposure to different types of NPs [55]. To the best of our knowledge, this is the first time that a similar biphasic dose-response has been reported as a consequence of Pd-NP exposure. Obviously, this result should be considered with caution and further studies are needed. However, the possible presence, at low exposure levels, of effects that may be adaptive, non-adverse or even beneficial is an intriguing issue that deserves further attention particularly on account of the complex regulatory mechanisms of the immune system that favor a balance between pathogenic and protective Th cells and the crucial role that different Th subsets play in immunopathology [56].

thumbnail
Fig 4. Mean serum levels of different cytokines expressed as percentage variation from control values (100%).

The particular trend of the dose–response relationship observed for all cytokines (with a slight decrease at the lowest exposure dose and an increase thereafter with increasing exposure doses) would seem to suggest the presence of a hormetic phenomenon since in some cases the hormetic effects are typically graphed as a J-shaped dose response curve.

https://doi.org/10.1371/journal.pone.0143801.g004

Nevertheless, considering that this is the first in vivo attempt to assess the effects of Pd-NPs on the immune system and that our understanding of their immunotoxicity is still in an initial phase, the biological implications of the aforementioned alterations in serum cytokine concentrations are uncertain. However, it should be noticed that the T helper cells, producing different subsets of cytokines, are critical for a proper immune cell homeostasis and host defence, but may be also major contributors to pathology of autoimmune and inflammatory diseases [48]. Therefore, it is not possible to exclude that prolonged or repeated exposure to these xenobiotics may ultimately result in inflammatory related tissue damages, hypersensitivity or autoimmune responses triggered by the immuno-toxic alterations exerted by Pd-NPs, also in relation to possible inherent or acquired individual susceptibility factors as well as to other environmental or occupational co-exposed substances. Interestingly, extrapolated to a public health and occupational medicine perspective, the detected pro-inflammatory alterations, may act as possible early indicators of biochemical alterations induced by Pd-NPs before un-reversible organ damages or systemic diseases become manifested. These changes should be deeply verified and eventually validated as possible biomarkers to be employed in biological monitoring programs in order to assure adequate risk management strategies.

Concerning the potential mechanisms underlining Pd-NP immune effects, it is worth noting that also other types of NPs have yielded similar findings in in vitro and in vivo experiments. For example in ICR mice the administration of magnetite iron oxide (Fe3O4)-NPs induced an increase in Th1 and Th2 serum cytokine concentrations [57, 58] and titanium dioxide (TiO2)-NPs caused a transient release of both types of cytokines in A549 cells [59]. These results may suggest that different types of NPs may share a common molecular mechanism of action, maybe through oxidative stress reactions, that is able to exert a generalized stimulatory effect on the immune system. Oxidative stress and inflammation, in fact, are interrelated by amplification loops [60]. Pro-inflammatory cytokines, in fact, may induce a massive production of free oxygen radicals which in turn may modulate the release of inflammatory mediators by activating different transcription factors [61]. This amplification between oxidative stress and inflammation may be involved in the adverse effects caused by NPs, some of which may cause DNA damage and cell death by apoptosis [62]. However, given the limited information available on this issue, no definite conclusions can be deduced at this stage of research.

Clearly, further in vitro and in vivo studies are needed to more deeply understand the immune potential of Pd-NPs. In vitro experiments, in fact, may represent a valid instrument to investigate Pd-NP induced cellular changes at bio-molecular levels and to determine their underlying mechanistic processes. In vivo investigations, on the other side, seem necessary to define the toxico-kinetic and dynamic behavior of NPs, as well as to confirm our preliminary results also under conditions of long-term exposure resembling those experienced by the general and occupational exposed populations.

Conclusions

Intravenous administration of Pd-NPs revealed the ability of this xenobiotic to significantly affect the immune system of Wistar rats by enhancing the serum levels of several cytokines secreted by different Th subsets. This generalized stimulatory effect was also observed in other in vitro and in vivo studies that investigated the immune potential of various NPs but differed substantially from the results of previous in vitro studies that evaluated the impact of Pd-NPs on the cytokine expression and release from PBMC cells. In view of the scant quantity of information currently available on the immunotoxicity of Pd-NPs, these conflicting results warrant further studies to evaluate and clarify the potentially toxic effects of Pd-NPs on the immune system and to reach a definitive understanding of this issue. This assessment appears even more urgent if we consider the increase in human exposure to Pd-UFPs and the fact that Pd salts and Pd-NPs exert different effects. Our findings differ considerably from the immunotoxic effects induced by several Pd salts in cell lines or laboratory animals, thus confirming that the unique physico–chemical properties of NPs give them a specific toxicological profile. Lastly, an evaluation of cytokine levels could be an interesting and promising biomarker for providing a more adequate assessment and management of risk with regard to nanomaterial exposure and effects [63].

Supporting Information

S1 Fig. Detail of cytokines serum levels observed in control rats and in four groups of female Wistar rats exposed to different levels of Pd-NPs.

https://doi.org/10.1371/journal.pone.0143801.s001

(DOCX)

Author Contributions

Conceived and designed the experiments: II AS. Performed the experiments: LF VL. Analyzed the data: AM. Contributed reagents/materials/analysis tools: MC KL RS. Wrote the paper: II LF MC VL AM KL RS AS.

References

  1. 1. Mitzukami F, Malda K, Watanabe M, Masuda K, Sano T, Kuno K. Preparation of thermostable high-surface area aluminas and properties of the alumina supported Pt catalysts. In: Crucq A, editor. Catalysis and Automotive Pollution Control II. New York: Elsevier Science Publishers BV; 1991. pp. 557–568.
  2. 2. Choi B, Jeong J, Son G, Jung M. Conversion characteristics of double-layer washcoat tri-metal three-way catalyst using high cell density substrate. JSME Int J B. 2005; 48:874–881.
  3. 3. Merget R, Rosner G. Evaluation of the health risk of platinum group metals emitted from automotive catalytic converters. Sci Total Environ. 2001; 270:165–173. pmid:11327390
  4. 4. Palacios MA, Gómez MM, Moldovan M, Morrison G, Rauch S, Mcleod C et al. Platinum-group elements: quantification in collected exhaust fumes and studies of catalyst surfaces. Sci Total Environ. 2000; 257:1–15. pmid:10943898
  5. 5. Ravindra K, Bencs L, Van Grieken R. Platinum group elements in the environment and their health risk. Sci Total Environ. 2004; 318:1–43. pmid:14654273
  6. 6. Bencs L, Ravindra K, Van Grieken R. Methods for the determination of platinum group elements originating from the abrasion of automotive catalytic converters. Spectrochim Acta B. 2003; 58: 1723–1755.
  7. 7. Artelt S, Kock H, König HP, Levsen K, Rosner G. Engine dynamometer experiments: platinum emissions from differently aged three-way catalytic converters. Atmos Environ. 1999; 33: 3559–3567.
  8. 8. Kalavrouziotis IK, Koukoulakis PH. The environmental impact of the platinum group elements (Pt, Pd, Rh) emitted by the automobile catalyst converters. Water Air Soil Pollut. 2009; 196: 393–402.
  9. 9. Rauch S, Morrison GM, Moldovan M. Scanning laser ablation-ICP-MS tracking of platinum group elements in urban particles. Sci Total Environ. 2002; 286: 243–251. pmid:11886096
  10. 10. Dubiella-Jackowska A, Polkowska Z, Namieńnik J. Platinum group elements in the environment: emissions and exposure. Rev Environ Contam Toxicol. 2009; 199: 111–135. pmid:19110940
  11. 11. Iavicoli I, Bocca B, Carelli G, Caroli S, Caimi S, Alimonti A et al. Biomonitoring of tram drivers exposed to airborne platinum, rhodium and palladium. Int Arch Occup Environ Health. 2007; 81: 109–114. pmid:17492463
  12. 12. Iavicoli I, Bocca B, Caroli S, Caimi S, Alimonti A, Carelli G et al. Exposure of rome city tram drivers to airborne platinum, rhodium, and palladium. J Occup Environ Med. 2008; 50: 1158–1166. pmid:18849761
  13. 13. Kamala CT, Balaram V, Satyanarayanan M, Kiran Kumar A, Subramanyam KS. Biomonitoring of airborne platinum group elements in urban traffic police officers. Arch Environ Contam Toxicol. 2015; 68: 421–431. pmid:25542147
  14. 14. Mathur R, Balaram V, Satyanarayanan M, Sawant SS, Ramesh SL. Anthropogenic platinum, palladium and rhodium concentrations in road dusts from Hyderabad city, India. Environ Earth Sci. 2011; 62: 1085–1099.
  15. 15. Sobrova P, Zehnalek J, Adam V, Beklova M, Kizek R. The effects on soil/water/plant/animal systems by platinum group elements. Cent Eur J Chem. 2012; 10: 1369–1382.
  16. 16. Zereini F, Wiseman C, Püttmann W. Changes in palladium, platinum, rhodium concentrations, and their spatial distribution in soils along a major highway in Germany from 1994 to 2004. Environ Sci Technol. 2007; 41: 451–456. pmid:17310706
  17. 17. Iavicoli I, Bocca B, Fontana L, Caimi S, Petrucci F, Bergamaschi A et al. Distribution and elimination of palladium in male wistar rats following 14-day oral exposure in drinking water. J Toxicol Environ Health A. 2009; 72: 88–93. pmid:19034798
  18. 18. Iavicoli I, Bocca B, Fontana L, Caimi S, Bergamaschi A, Alimonti A. Distribution and elimination of palladium in rats after 90-day oral administration. Toxicol Ind Health. 2010; 26: 183–189. pmid:20176776
  19. 19. Brasch J, Geier J. Patch test results in schoolchildren. Results from the Information Network of Departments of Dermatology (IVDK) and the German Contact Dermatitis Research Group (DKG). Contact Dermatitis. 1997; 37: 286–293. pmid:9455632
  20. 20. Durosaro O, el-Azhary RA. A 10-year retrospective study on palladium sensitivity. Dermatitis. 2009; 20: 208–213. pmid:19804697
  21. 21. Larese Filon F, Uderzo D, Bagnato E. Sensitization to palladium chloride: a 10-year evaluation. Am J Contact Dermat. 2003; 14: 78–81. pmid:14749025.
  22. 22. Wohrl S, Hemmer W, Focke M, Gotz M, Jarisch R. Patch testing in children, adults, and the elderly: influence of age and sex on sensitization patterns. Pediatr Dermatol. 2003; 20: 119–123. pmid:12657006
  23. 23. Faurschou A, Menné T, Johansen JD, Thyssen JP. Metal allergen of the 21st century—a review on exposure, epidemiology and clinical manifestations of palladium allergy. Contact Dermatitis. 2011; 64: 185–195. pmid:21392026
  24. 24. Muris J, Goossens A, Gonçalo M, Bircher AJ, Giménez-Arnau A, Foti C et al. Sensitization to palladium in Europe. Contact Dermatitis. 2015; 72: 11–19. pmid:25348727
  25. 25. World Health Organization (WHO). Environmental Health Criteria 226: palladium. International Programme on Chemical Safety. Geneva: World Health Organization, 2002.
  26. 26. Schmalz G, Schuster U, Schweikl H. Influence of metals on IL-6 release in vitro. Biomaterials. 1998; 19: 1689–1694. pmid:9840004
  27. 27. Schmalz G, Schweikl H, Hiller KA. Release of prostaglandin E2, IL-6 and IL-8 from human oral epithelial culture models after exposure to compounds of dental materials. Eur J Oral Sci. 2000; 108: 442–448. pmid:11037761
  28. 28. Boscolo P, Di Giampaolo L, Reale M, Castellani ML, Ritavolpe A, Carmignani M et al. Different effects of platinum, palladium, and rhodium salts on lymphocyte proliferation and cytokine release. Ann Clin Lab Sci. 2004; 34: 299–306. pmid:15487704.
  29. 29. Iavicoli I, Carelli G, Magrini A, Marinaccio A, Fontana L, Boscolo P et al. The effects of sub-acute exposure to palladium on cytokines in male Wistar rats. Int J Immunopathol Pharmacol. 2006; 19 Suppl 4: 21–24. pmid:17291402.
  30. 30. Iavicoli I, Carelli G, Marinaccio A, Fontana L, Calabrese E. Effects of sub-chronic exposure to palladium (as potassium hexachloro-palladate) on cytokines in male Wistar rats. Hum Exp Toxicol. 2008; 27: 493–497. pmid:18784202
  31. 31. Wilkinson KE, Palmberg L, Witasp E, Kupczyk M, Feliu N, Gerde P et al. Solution-engineered palladium nanoparticles: model for health effect studies of automotive particulate pollution. ACS Nano. 2011; 5: 5312–5324. pmid:21650217
  32. 32. Boscolo P, Bellante V, Leopold K, Maier M, Di Giampaolo L, Antonucci A et al. Effects of palladium nanoparticles on the cytokine release from peripheral blood mononuclear cells of non-atopic women. J Biol Regul Homeost Agents. 2010; 24: 207–214. pmid:20487634.
  33. 33. Reale M, Vianale G, Lotti LV, Mariani-Costantini R, Perconti S, Cristaudo A et al. Effects of palladium nanoparticles on the cytokine release from peripheral blood mononuclear cells of palladium-sensitized women. J Occup Environ Med. 2011; 53: 1054–1060. pmid:21866053
  34. 34. Borm PJ, Robbins D, Haubold S, Kuhlbusch T, Fissan H, Donaldson K et al. The potential risks of nanomaterials: a review carried out for ECETOC. Part Fibre Toxicol. 2006; 3: 11. pmid:16907977
  35. 35. Nel A, Xia T, Madler L, Li N. Toxic potential of materials at the nanolevel. Science. 2006; 311: 622–627. pmid:16456071
  36. 36. Clause BT. The Wistar Rat as a right choice: establishing mammalian standards and the ideal of a standardized mammal. J Hist Biol. 1993; 26: 329–349. pmid:11623164
  37. 37. Kacew S, Festing MFW. Role of Rat Strain in the Differential Sensitivity to Pharmaceutical Agents and Naturally Occurring Substances. CEJOEM. 1999; 5: 201–231.
  38. 38. Descotes J. An introduction to Immunotoxicology. Taylor & Francis Ltd, 1 Gunpowder Square, London, 2003.
  39. 39. Richter-Reichhelm HB, Dasenbrock CA, Descotes G, Emmendörffer AC, Ernst HU, Harleman JH et al. Validation of a modified 28-day rat study to evidence effects of test compounds on the immune system. Regul Toxicol Pharmacol. 1995; 22: 54–56. pmid:7494903
  40. 40. Cristaudo A, Picardo M, Petrucci F, Forte G, Violante N, Senofonte O et al. Clinical and allergological biomonitoring of occupational hypersensitivity to platinum group elements. Anal Lett. 2007; 40: 3343–3359.
  41. 41. Fontana L, Leso V, Marinaccio A, Cenacchi G, Papa V, Leopold K et al. The effects of palladium nanoparticles on the renal function of female Wistar rats. Nanotoxicology. 2015; 9: 843–851. pmid:25405262
  42. 42. Morawska L, Ristovski Z, Jayaratne ER, Keogh DU, Ling X. Ambient nano and ultrafine particles from motor vehicle emissions: characteristics, ambient processing and implications on human exposure. Atmos Environ. 2008; 42: 8113–8138.
  43. 43. Shi JP, Khan AA, Harrison RM. Measurements of ultrafine particle concentration and size distribution in the urban atmosphere. Sci Total Environ. 1999; 235: 51–64.
  44. 44. Dongarrá G, Varrica D, Sabatino G. Occurrence of platinum, palladium and gold in pine needles of Pinus pinea L. from the city of Palermo (Italy). Appl Geochem. 2003; 18:109–116.
  45. 45. Tsuji JS, Maynard AD, Howard PC, James JT, Lam CW, Warheit DB et al. Research strategies for safety evaluation of nanomaterials, part IV: risk assessment of nanoparticles. Toxicol Sci. 2006; 89: 42–50. pmid:16177233
  46. 46. Chang C. The immune effects of naturally occurring and synthetic nanoparticles. J Autoimmun. 2010; 34: J234–246. pmid:19995678
  47. 47. Cosmi L, Maggi L, Santarlasci V, Liotta F, Annunziato F. T helper cells plasticity in inflammation. Cytometry A. 2014; 85: 36–42. pmid:24009159
  48. 48. Raphael I, Nalawade S, Eagar TN, Forsthuber TG. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 2015; 74: 5–17. pmid:25458968
  49. 49. Schmitt N, Ueno H. Regulation of human helper T cell subset differentiation by cytokines. Curr Opin Immunol. 2015; 34: 130–136. pmid:25879814
  50. 50. Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I et al. Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis. 2005; 16: 157–202. pmid:16536334
  51. 51. Iavicoli I, Fontana L, Leso V, Bergamaschi A. The effects of nanomaterials as endocrine disruptors. Int J Mol Sci. 2013; 14: 16732–16801. pmid:23949635
  52. 52. Walkey CD, Chan WC. Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chem Soc Rev. 2012; 41: 2780–2799. pmid:22086677
  53. 53. Calabrese EJ. Getting the dose-response wrong: why hormesis became marginalized and the threshold model accepted. Arch Toxicol. 2009; 83: 227–247. pmid:19234688
  54. 54. Calabrese EJ. Hormesis is central to toxicology, pharmacology and risk assessment. Hum Exp Toxicol. 2010; 29: 249–261. pmid:20332169
  55. 55. Iavicoli I, Fontana L, Leso V, Calabrese EJ. Hormetic dose-responses in nanotechnology studies. Sci Total Environ. 2014; 487: 361–374. pmid:24793332
  56. 56. Berger A. Th1 and Th2 responses: what are they? BMJ. 2000; 321: 424. pmid:10938051
  57. 57. Chen BA, Jin N, Wang J, Ding J, Gao C, Cheng J et al. The effect of magnetic nanoparticles of Fe(3)O(4) on immune function in normal ICR mice. Int J Nanomedicine. 2010; 5: 593–599. pmid:20856834
  58. 58. Park EJ, Kim H, Kim Y, Yi J, Choi K, Park K. Inflammatory responses may be induced by a single intratracheal instillation of iron nanoparticles in mice. Toxicology. 2010; 275: 65–71. pmid:20540983
  59. 59. Medina-Reyes EI, Déciga-Alcaraz A, Freyre-Fonseca V, Delgado-Buenrostro NL, Flores-Flores JO, Gutiérrez-López GF et al. Titanium dioxide nanoparticles induce an adaptive inflammatory response and invasion and proliferation of lung epithelial cells in chorioallantoic membrane. Environ Res. 2015; 136: 424–434. pmid:25460664
  60. 60. Gaillet S, Rouanet JM. Silver nanoparticles: their potential toxic effects after oral exposure and underlying mechanisms—a review. Food Chem Toxicol. 2015; 77: 58–63. pmid:25556118
  61. 61. Acker H. The oxygen sensing signal cascade under the influence of reactive oxygen species. Philos Trans R Soc Lond B Biol Sci. 2005; 360: 2201–2210. pmid:16321790
  62. 62. Franco R, Sánchez-Olea R, Reyes-Reyes EM, Panayiotidis MI. Environmental toxicity, oxidative stress and apoptosis: ménage à Trois. Mutat Res. 2009; 674: 3–22. pmid:19114126
  63. 63. Iavicoli I, Leso V, Manno M, Schulte PA. Biomarkers of nanomaterial exposure and effect: current status. J Nanopart Res. 2014; 16: 2302.