Abstract
Background
Silver nanoparticles (AgNPs) are widely used in biomedicine due to their strong antimicrobial, antifungal, and antiviral activities. Concerns about their possible negative impacts on human and environmental health directed many researchers towards the assessment of the safety and toxicity of AgNPs in both in vitro and in vivo settings. A growing body of scientific information confirms that the biodistribution of AgNPs and their toxic effects vary depending on the particle size, coating, and dose as well as on the route of administration and duration of exposure. This study aimed to clarify the sex-related differences in the outcomes of oral 28 days repeated dose exposure to AgNPs.
Methods
Wistar rats of both sexes were gavaged daily using low doses (0.1 and 1 mg Ag/kg b.w.) of polyvinylpyrrolidone (PVP)-coated small-sized (10 nm) AgNPs. After exposure, blood and organs of all rats were analysed through biodistribution and accumulation of Ag, whereas the state of the liver and kidneys was evaluated by the levels of reactive oxygen species (ROS) and glutathione (GSH), catalase (CAT) activity, superoxide dismutase (SOD) and glutathione peroxidase (GPx), expression of metallothionein (Mt) genes and levels of Mt proteins.
Results
In all animals, changes in oxidative stress markers and blood parameters were observed indicating the toxicity of AgNPs applied orally even at low doses. Sex-related differences were noticed in all assessed parameters. While female rats eliminated AgNPs from the liver and kidneys more efficiently than males when treated with low doses, the opposite was observed for animals treated with higher doses of AgNPs. Female Wistar rats exposed to 1 mg PVP-coated AgNPs/kg b.w. accumulated two to three times more silver in the blood, liver, kidney and hearth than males, while the accumulation in most organs of digestive tract was more than ten times higher compared to males. Oxidative stress responses in the organs of males, except the liver of males treated with high doses, were less intense than in the organs of females. However, both Mt genes and Mt protein expression were significantly reduced after treatment in the liver and kidneys of males, while they remained unchanged in females.
Conclusions
Observed toxicity effects of AgNPs in Wistar rats revealed sex-related differences in response to an oral 28 days repeated exposure.
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Background
Silver nanoparticles (AgNPs) are one of the most exploited and investigated engineered nanomaterials. They are widely utilized in medicine, cosmetics, textile engineering and electronics due to their strong antimicrobial, antifungal, and antiviral activity [1,2,3]. However, the question about their safety and potential adverse effects on human and environmental health is still prevailing [4,5,86], using the ImageJ 1.50 g software (Windows version of NIH Image, https://imagej.nih.gov/ij/index.html), and includes normalization against reference protein actin.
Statistical analysis
Statistical analysis was performed according to recommendations published previously by Marusteri and Bacera [87]. As data were not normally distributed and more than 2 groups were compared, we performed non-parametric Kruskal–Wallis one-way ANOVA test using the Statistica Software 13.5.0.17 (TIBCO Software Inc., Palo Alto, USA). Data are represented as mean values obtained from four animals including standard deviations (SD). Significant differences (p < 0.05 and p < 0.005) between controls and treated animals, lower and high dose, respectively, are indicated by the asterisks (* and **, respectively). Significant differences (p < 0.05) between animals from the LD and HD groups of the same sex are indicated with hashtags (#), while significant differences (p < 0.05) between males and females are denoted with section signs (§).
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article (and its additional file).
Abbreviations
- Actb:
-
β-Actin
- ADME:
-
Absorption, distribution, metabolism, elimination
- AgNPs:
-
Silver nanoparticles
- ALP:
-
Alkaline phosphatase
- ALT:
-
Alanine transaminase
- AST:
-
Aspartate transaminase
- CAT:
-
Catalase
- CK:
-
Creatine kinase
- Ctl:
-
Control
- DCF:
-
2′,7′-Dichlorofluorescein
- DCFH-DA:
-
2′,7′-Dichlorofluorescin diacetate
- DHE:
-
Dihydroethidium
- DLS:
-
Dynamic light scattering
- DTNB:
-
5,5′-Dithiobis-(2-nitrobenzoic acid)
- EDTA:
-
Ethylenediaminetetraacetic acid
- ELS:
-
Electrophoretic light scattering
- EOH:
-
2-Hydroethidium
- GPx:
-
Glutathione peroxidase
- GRA:
-
Granulocytes
- GSH:
-
Glutathione
- HCT:
-
Hematocrit
- HD:
-
High dose (1 mg Ag/kg b.w.) of AgNPs
- HGB:
-
Hemoglobin
- ICP-MS:
-
Inductively coupled plasma mass spectrometer
- LD:
-
Low dose (0.1 mg Ag/kg b.w.) of AgNPs
- LDH:
-
Lactate dehydrogenase
- LYM:
-
Lymphocyte
- MBCl:
-
Monochlorobimane
- MCH:
-
Mean corpuscular hemoglobin
- MCHC:
-
Mean corpuscular hemoglobin concentration
- MCV:
-
Mean corpuscular volume
- MON:
-
Monocyte
- MPV:
-
Mean platelet volume
- MT:
-
Metallothionein
- PB:
-
Phosphate buffer
- PLT:
-
Platelets
- PVP:
-
Polyvinylpyrrolidone
- RBC:
-
Red blood cells
- RDW:
-
Red cell distribution width
- ROS:
-
Reactive oxygen species
- SOD:
-
Superoxide dismutase
- SD:
-
Standard deviation
- TEM:
-
Transmission electron microscopy
- WBC:
-
White blood cell
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Acknowledgements
We would like to thank Dr Maja Beus for English editing and careful reading.
Funding
We acknowledge the Croatian Science Foundation (Grant No. HRZZ-IP-2016-06-2436) and the EU H2020 project (H2020-NMBP-13-2018 RIA) RiskGONE (Science-based Risk Governance of Nano-Technology; grant agreement n° 814425) for financial support of this work.
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MĆ designed and supervised animal experiments, analysed and interpreted results of the study and was the manuscript’s lead author; RB performed synthesis and characterisation of AgNPs, biochemical analyses, blood characterisation and statistical analyses; SD performed protein and mRNA expression analysis and interpreted results; MLj was involved in animal experiments and designed methodological approach for protein and mRNA expression analysis; WG performed ICP-MS analysis, interpreted results on Ag level in tissues and provided instrumental resources for the study; VM led and designed animal experiments; IŽ and IP contributed to the animal experiments and biochemical analyses and provided instrumental resources for the study; MP contributed to animal experiments and preparation of samples for further analysis; LB analysed data, perfomed statistics and prepared figures and tables; IVV created the concept and methodological approach of the study, supervised the whole study, provided funding and revised the manuscript. All authors revised and approved the final manuscript.
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The study was conducted according to the ethical codex for animal welfare of the Croatian Society for Laboratory Animal Science and with international standards. It was approved by the Ethical Committee of the Institute for Medical Research and Occupational Health (protocol title: "Zdravstveni učinci nanočestica srebra", date of approval: 27th November 2014, protocol class: 01-18/14-02-2/8, protocol number: 100-21/14-2).
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Title of data: Numerical results.Data description: Tables showing data and statistics of the results of determination of biodistribution and bioaccumulation of AgNPs, biochemical analyses of the liver, kidneys and blood parameters, and Mt mRNA and protein analyses.
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Ćurlin, M., Barbir, R., Dabelić, S. et al. Sex affects the response of Wistar rats to polyvinyl pyrrolidone (PVP)-coated silver nanoparticles in an oral 28 days repeated dose toxicity study. Part Fibre Toxicol 18, 38 (2021). https://doi.org/10.1186/s12989-021-00425-y
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DOI: https://doi.org/10.1186/s12989-021-00425-y