Acute and chronic nephrotoxicity of platinum nanoparticles in mice
© Yamagishi et al.; licensee Springer. 2013
Received: 30 May 2013
Accepted: 1 September 2013
Published: 23 September 2013
Platinum nanoparticles are being utilized in various industrial applications, including in catalysis, cosmetics, and dietary supplements. Although reducing the size of the nanoparticles improves the physicochemical properties and provides useful performance characteristics, the safety of the material remains a major concern. The aim of the present study was to evaluate the biological effects of platinum particles less than 1 nm in size (snPt1). In mice administered with a single intravenous dose of snPt1, histological analysis revealed necrosis of tubular epithelial cells and urinary casts in the kidney, without obvious toxic effects in the lung, spleen, and heart. These mice exhibited dose-dependent elevation of blood urea nitrogen, an indicator of kidney damage. Direct application of snPt1 to in vitro cultures of renal cells induced significant cytotoxicity. In mice administered for 4 weeks with twice-weekly intraperitoneal snPt1, histological analysis of the kidney revealed urinary casts, tubular atrophy, and inflammatory cell accumulation. Notably, these toxic effects were not observed in mice injected with 8-nm platinum particles, either by single- or multiple-dose administration. Our findings suggest that exposure to platinum particles of less than 1 nm in size may induce nephrotoxicity and disrupt some kidney functions. However, this toxicity may be reduced by increasing the nanoparticle size.
KeywordsNanosized materials Platinum particles Kidney Nephrotoxicity Safety evaluation
Nanomaterials have been developed and used as innovative materials in a wide range of industrial fields, including electronics, medicine, food, clothing, and cosmetics; these reagents are expected to provide significant benefits to humans. Nanomaterials are defined as substances that have at least one dimension size below 100 nm. The reduced size provides novel physicochemical properties, including increased thermal electrical conductivity, durability, and strength [1–3]. Although these characteristics may yield improved performance and novel functions, several reports have suggested that various types of nanomaterials, such as carbon nanotubes, titanium dioxide, fullerenes, quantum dots, and silica, exhibit harmful biological effects [4–12]. Additionally, some reports have shown that the characteristics of nanoparticles (e.g., size and surface features) can affect their biological and pathological actions [10, 13–16]. Therefore, evaluation of the potential health risks attributable to nanomaterials is indispensable for the safe handling and use of these materials. However, little information is available regarding the safety evaluation of materials less than 1 nm in size.
Platinum nanoparticles have been utilized in a number of manufacturing applications, including catalysis, cosmetics manufacturing, and the processing of dietary supplements. As products using platinum nanoparticles become more familiar in our daily lives, the chances of exposure to platinum nanoparticles are increasing, as are concerns about unanticipated harmful biological effects of these materials [17, 18]. In fact, there are some reports that platinum nanoparticles can induce inflammation in mice or impair the integrity of DNA [19, 20]. On the other hand, platinum nanoparticles have anti-oxidant activity and inhibit pulmonary inflammation (e.g., as caused by exposure to cigarette smoke) [21–23]. These reports indicate that the biological effects of platinum nanoparticles remain poorly defined; the biological safety of sub-nanosized platinum particles (those of less than 1 nm in size; snPt1) remains unknown. Recently, we reported that snPt1 can induce hepatotoxicity . However, the biological effects of snPt1 on other organs remain unclear. In this study, we evaluated the effect of snPt1 on tissues after single- and multi-dose administration in mice. In addition, we investigated the relationship between platinum particle size and biological response by also testing platinum particles of 8 nm in size (snPt8).
Platinum particles with nominal mean diameters of less than 1 nm (snPt1) and 8 nm (snPt8) were purchased from Polytech & Net GmbH (Rostock, Germany). The particle sizes were confirmed using a Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). The particles were stocked as 5 mg/ml aqueous suspensions. The stock solutions were suspended using a vortex mixer before use. Other reagents used in this study were of research grade.
BALB/c and C57BL/6 male mice were obtained from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan) and were housed in an environmentally controlled room at 23°C ± 1.5°C with a 12-h light/12-h dark cycle. Mice had ad libitum access to water and commercial chow (Type MF, Oriental Yeast, Tokyo, Japan). BALB/c mice were injected intravenously with snPt1 or snPt8 at 5 to 20 mg/kg body weight. C57BL/6 mice were injected intraperitoneally with snPt1 or snPt8 at 10 mg/kg body weight, or with an equivalent volume of vehicle (water). At 24 h after the injection of the vehicle or test article, the kidney and liver were collected. For testing the chronic effects of platinum particles, C57BL/6 mice were injected intraperitoneally with snPt1 or snPt8 at 10 mg/kg body weight, or with an equivalent volume of vehicle (water). Intraperitoneal doses were administered as twice-weekly injections for 4 weeks. At 72 h after the last injection of vehicle or test article, the kidney and liver were collected. All experimental protocols conformed to the ethical guidelines of the Graduate School of Pharmaceutical Sciences at Osaka University.
For animals dosed intravenously with snPt1 or snPt8, the kidney, spleen, lung, heart, and liver were removed at 24 h post-injection and fixed with 4% paraformaldehyde. For animals dosed intraperitoneally with snPt1 or snPt8, the kidney and liver were removed at 24 h (for single administration) or 72 h (for multiple administration) post-injection and fixed with 4% paraformaldehyde. Thin tissue sections were stained with hematoxylin and eosin for histological observation.
Serum blood urea nitrogen (BUN) was measured using a commercially available colorimetric assay kit (Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s protocol. In brief, collected serum (10 μl) was combined with 1 ml color A reagent (including urease) and incubated at 37°C for 15 min. Following the addition of 1 ml Color B reagent, the samples were incubated at 37°C for 10 min. Absorbance of samples was measured at a wavelength of 570 nm.
Data are presented as mean ± SEM. Statistical analysis was performed by Student’s t test. P < 0.05 was considered significant.
Results and discussion
Following exposure, nanoparticles are transported into the blood and reach the systemic circulation, from which the nanoparticles distribute and accumulate in several organs such as the lung, liver, spleen, kidneys, brain, and heart [27–30]. Because the kidney is able to remove molecules from the circulation, renal excretion is an expected route for elimination of nanoparticles. In fact, functionalized single-wall carbon nanotubes (SWCNT), following injection into mice, are rapidly excreted by the kidney . The hepatobiliary system also is an important route for the elimination of foreign substances and particles . Because these organs play pivotal roles in eliminating foreign substances, various nanomaterials are accumulated there and lead to tissue injury. As one example, our previous work showed that snPt1-treated mice exhibited acute hepatotoxicity . In the present study, we investigated the biological effects of snPt1 after intravenous or intraperitoneal administration in mice and demonstrated that snPt1 induced nephrotoxicity and impaired renal function, as evidenced by BUN levels. In contrast, we could not find apparent toxic effects on the heart, lung, or spleen after the single intravenous administration of snPt1, although the disposition of these nanoparticles will need to be assessed further.
The underlying mechanism of snPt1-induced tissue injury still remains unclear. Cisplatin, which is a platinating agent used as part of the anti-cancer regimen for various types of cancers [33, 34], exerts its antitumor activity by binding preferentially to the nucleophilic positions on guanine and adenine of DNA, resulting in the formation of intra- and inter-strand crosslinks. Eventually, the crosslinks lead to DNA-strand breaks and killing of cancer cells . However, cisplatin usage is limited due to nephrotoxicity, leading to lesions in the epithelial tubules [36, 37]. Cisplatin also causes toxicity in the liver and blood . These observations suggest that the toxic effects of cisplatin resemble those of snPt1. A previous study reported that platinum nanoparticles entered human lung fibroblasts (cell line IMR-90) and human glioblastoma cells (U251) and induced cytotoxicity thorough intracellular reactive oxygen species (ROS) production and DNA damage following p53 activation and upregulation of p21, which leads to growth arrest and apoptosis . Our observation of snPt1-induced cytotoxicity in cell culture suggests that snPt1 may be internalized by renal cells, with concomitant induction of ROS production or DNA damage. However, alternative toxic effects (such as cytotoxicity of inflammatory cytokines on renal cells by accumulation of inflammatory cells in the kidney) might emerge during chronic exposure to snPt1.
At equivalent dose levels, platinum particles of 8 nm in size did not induce apparent toxic effects in renal tissues by acute or chronic administration. This result suggests that selection of specific size ranges for the platinum particles might overcome the undesirable side effects. Current studies have shown that organic cation transporter 2 (OCT2) is highly expressed in kidney and plays an important role in the nephrotoxicity of cisplatin [40, 41]. Identification of the snPt1 transporter may help to clarify the mechanism of snPt1-induced nephrotoxicity.
In the present study, we investigated the biological safety of platinum nanoparticles in mice and found that platinum particles of less than 1 nm induced kidney injury, although the injurious effects were reduced by increasing the nanoparticle size. For future nanoparticle applications, it will be critical to further understand the bioactivity and kinetics of materials less than 1 nm in size. Accumulation of toxicity profiles will aid in the creation of the safe and efficacious nanomaterials and contribute to the advancement of the field.
platinum particles less than 1 nm in size
platinum particles of 8 nm in size
blood urea nitrogen
Madin-Darby canine kidney
reactive oxygen species.
The authors thank all members of our laboratory for useful comments. This study was partly supported by a grant from the Ministry of Health, Labour, and Welfare of Japan.
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