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Insight into the Mechanisms of Combined Toxicity of Single-Walled Carbon Nanotubes and Nickel Ions in Macrophages: Role of P2X7 Receptor Xuejing Cui, Bin Wan, Liang-Hong Guo, Yu Yang, and Xiao-Min Ren Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03842 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016
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Environmental Science & Technology
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Insight into the Mechanisms of Combined Toxicity of
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Single-Walled Carbon Nanotubes and Nickel Ions in
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Macrophages: Role of P2X7 Receptor
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Xuejing Cui,1, 2 Bin Wan,1, 2,* Liang-Hong Guo,1, 2, 3,* Yu Yang,1 Xiaomin Ren,1, 2
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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing
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100085, China
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research
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2
University of Chinese Academy of Sciences, Beijing 100049, China
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3
Institute of Environment and Health, Jianghan University, Wuhan, Hubei 430056,
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China
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*Correspondence to: Dr. Bin Wan, Dr. Liang-Hong Guo, State Key Laboratory of
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Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental
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Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, P.O. Box 2871, Beijing
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100085, China.
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Telephone: (86) 10-62849338. Fax: (86) 10-62849685.
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Email:
[email protected];
[email protected].
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ABSTRACT: Co-existence of nanomaterials and environmental pollutants requires
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in-depth understanding of combined toxicity and underlying mechanism. In this work,
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we found that co-exposure to the mixture of non-cytotoxic level of single-walled
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carbon nanotubes (SWCNTs) (10 µg/mL) and Ni2+ (20 µM) induced significant
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cytotoxicity in macrophages. However, almost equal amount of intracellular Ni2+ was
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detected after Ni2+/SWCNT co-exposure or Ni2+ single exposure, indicating no
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enhanced cellular uptake of Ni2+ occurred. SDS-PAGE analysis revealed 50% more
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SWCNTs retained in Ni2+/SWCNT exposed cells than that with SWCNT exposure
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alone, regardless of the exposure sequence (co-exposure, Ni2+ pre- or post-treatment),
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suggesting inhibited SWCNT exocytosis by Ni2+. The increased cellular dose of
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SWCNTs could quantitatively account for the elevated toxicity of Ni2+/SWCNT
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mixture to cells. It was then found that agonist (ATP) and antagonist (o-ATP) of
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P2X7R could regulate intracellular SWCNT amount and the cytotoxicity accordingly.
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In addition, inhibition of P2X7R by P2X7-targeting siRNA diminished the inhibitory
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effect of Ni2+. It was therefore concluded that Ni2+ impeded SWCNT exocytosis by
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inhibiting P2X7R, leading to higher intracellular retention of SWCNTs and elevated
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cytotoxicity. Our work identified exocytosis inhibition as an important mechanism for
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SWCNT/Ni2+ toxicity, and revealed the crucial role of P2X7R in mediating such
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inhibitory effect.
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INTRODUCTION
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Chemicals released into the environment usually co-exist with other environmental
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pollutants as complex mixtures.1-3 Concurrent or sequential human exposure to these
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chemicals dictates the necessity of risk assessment of chemical mixtures. From both
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the public health and regulatory perspectives, combined toxic effects among mixture
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components are of particular concerns. Recently, the rapid development of
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nanotechnology has brought about large amount of nanomaterials that not only
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improve the quality of life but also raise great concerns on their potential risks on
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environment and human health. It has been demonstrated that co-exposure of some
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nanomaterials and contaminants induce altered toxicity in living organisms, and the
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interactive outcomes generally fall into the following three categories. First of all,
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co-presence of nanomaterials and contaminants results in no increased toxicity.4
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Secondly, nanomaterials alleviate the toxicity of contaminants compared to the
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exposure of individual contaminant.5-7 The lowered toxicity is often interpreted by the
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adsorption
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bioavailability of contaminants in living organisms. Thirdly, of the most importance
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and environmental significance, nanomaterials aggravate the toxicity of contaminants
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in living organisms. This combined toxicity has been observed in binary mixtures
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composed of either fullerene C60 or nano-TiO2 and a variety of chemicals such as
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PAHs,8, 9 PBDEs,10 TBT,11 and heavy metals.12, 13 In most of the studies, the combined
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toxicity is attributed to the so-called ‘Trojan Horse’ effect of nanomaterials, in which
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they facilitate the ingestion of contaminants by the organisms, increase the cellular
of
contaminants
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nanomaterials
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dose and therefore amplify the deleterious effects of these toxicants to organisms. The
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mechanism is also applied to explain the combined effects of carbon nanotubes (CNTs)
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and a variety of contaminants.14-18 In an alternative mechanism, TiO2 nanoparticles
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were shown to accelerate the biotransformation of pentachlorophenol to more toxic
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metabolites, thus enhance the overall toxicity.19 These studies have made great
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progress in understanding the impacts of nanomaterials on the toxicity of
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environmental contaminants. However, the alteration of nanomaterials toxicity by the
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chemical contaminant in the mixtures is mostly ignored, and deserves equal attention.
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In recent years, single-walled carbon nanotubes (SWCNTs), due to their unique
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physical, chemical and electronic properties, have been widely used in the fields of
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biomedicine, biotechnology, and electronics.20,
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applications and production rates, SWCNTs will inevitably enter the environment
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during their life circle. Lines of evidences revealed that SWCNTs could enter
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organisms via the respiratory or intestinal tract and accumulate in living organisms for
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as long as three months.22-25 In vivo studies showed that CNTs through inhalation
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(intratracheal instillation and pharyngeal aspiration) could induce acute inflammation,
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progressive granulomas and fibrosis in animals.25, 26 In vitro, CNTs were shown to
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cause lower cell viability and depressed cell proliferation in human lung-tumor and
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keratinocyte cells27,
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regulation in embryo kidney cells.29 The toxicities have also been detected in other
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cell types, namely mesenchymal stem cells,30 and epithelial cells.31 Most recently, our
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studies and others showed that SWCNTs could readily enter macrophages and
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With ever increasing range of
and decrease in cell-adhesion ability and altered cell-cycle
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interfere with the biogenesis and gene expression of lysosome, proteasome and
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mitochondrial respiration chains, inducing inflammatory response,32 resulting in
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suppressed engulfing function of primary monocytes/macrophages.33, 34
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Nickel is ubiquitously present in the environment, and the exposure to low doses
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of Ni compounds is unavoidable. In the field of nanotechnology, nickel is often used
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as a catalyst for the synthesis of commercial SWCNTs, or as a coating material on the
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surface of nanoparticles, which lead to the co-existence of nanoparticles and nickel.
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The presence of nickel in these products might be toxic as it can form reactive oxygen
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species (ROS) that is detrimental to cells. As a Group I carcinogen to humans,35
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environmental nickel has already raised health concern stemming from the knowledge
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of its toxicity and pathogenic properties derived from occupational epidemiology and
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animal studies including carcinogenicity, genotoxicity, and neurotoxicity.36-38
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Especially, nickel ions (II) were shown to inhibit the activity of an important
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purinergic receptor, P2X7R,39,
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inflammasomes.41,
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secretion of lysosomes,43,
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SWCNTs are localized after cellular internalization.45-47 Normally, at the cellular level,
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the toxic properties and extent of SWCNTs depend on the amount of SWCNTs inside
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cells, which are determined by a variety of cell processes including exocytosis. It is
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possible that SWCNTs and other contaminants such as nickel ions interact with each
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other, resulting in altered toxicological profile. Thus, we hypothesized that nickel ions
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might interfere with toxicokinetics and thus the toxicity of SWCNTs.
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which mediates the production and release of
Most importantly, this receptor was reported to mediate the 44
where most carbonaceous nanomaterials including
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To test the hypothesis, monocytic/macrophage cells were employed based on two
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considerations. Macrophages are abundant in P2X7R expression.48 They are also
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pivotal players in both innate and adaptive immunity. As a part of the first line of
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defense against invading foreign substances including nanoparticles, macrophages are
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the primary target for nanoparticle toxicity.32, 34 Therefore, we compared the toxicity
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of SWCNTs and nickel ions both alone and in combination on macrophage survival,
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and investigated the molecular mechanisms underlying the combined effects.
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MATERIALS AND METHODS
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Materials. The murine macrophage cell line Raw264.7 cells (ATCC: TIB-71)
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and human monocytic cell line THP-1 cells (ATCC: TIB-202) were acquired from
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American Type Culture Collection. All ingredients for the culture media were
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purchased from Gibco, Invitrogen (UK). SWCNTs (CNTs purity >95%, SWCNT
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purity >90%, ash
