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Oral Exposure of Silver Nanoparticles or Silver Ions May Aggravate Fatty Liver Disease in Overweight Mice Jianbo Jia, Feifei Li, Hongyu Zhou, Yuhong Bai, Sijin Liu, Yiguo Jiang, Guibin Jiang, and Bing Yan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02752 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 21, 2017
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Environmental Science & Technology
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Oral Exposure to Silver Nanoparticles or Silver Ions May Aggravate
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Fatty Liver Disease in Overweight Mice
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Jianbo Jia,† Feifei Li,† Hongyu Zhou,‡ Yuhong Bai,† Sijin Liu,§
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Yiguo Jiang,ǁ Guibin Jiang,§ and Bing Yan†,*
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†
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250100, P.R. China
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‡
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health and Guangdong Key Laboratory of Environmental Pollution and Health, Jinan
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University, Guangzhou, 510632, P.R. China
School of Environmental Science and Engineering, Shandong University, Jinan,
School of Environment, Guangzhou Key Laboratory of Environmental Exposure and
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§
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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing,
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100085, P.R. China
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ǁ
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Guangzhou Medical University, Guangzhou, 511436, P.R. China
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research
State Key Laboratory of Respiratory Disease, Institute for Chemical Carcinogenesis,
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*
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Professor Bing Yan
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School of Environmental Science and Engineering, Shandong University, Jinan,
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250100, P.R. China
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Tel.: +8613969072308; E-mail:
[email protected] To whom correspondence should be addressed.
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Abstract
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As the applications and environmental release of silver ions and nanoparticles are
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increasing, increasing human exposure to these pollutants has become an emerging
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health concern. The impeding effects of such pollutants on susceptible populations are
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severely under-studied. Here, we demonstrate that silver nanoparticles (Ag NPs), at a
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dose that causes no general toxicity in normal mice, promotes the progression of fatty
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liver disease from steatosis to steatohepatitis only in overweight mice. Exposure to Ag+
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ions induces the same effects in overweight mice. Ag NPs rather than Ag+ ions cause
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this disease progression based on our findings that Ag+ ions are partly reduced to Ag
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NPs in fatty livers, and the toxic effect is correlated with the liver dose of Ag NPs, not
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Ag+ ions. Furthermore, the Ag NP-induced pro-inflammatory activation of Kupffer cells
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in the liver, enhancement of hepatic inflammation, and suppression of fatty acid
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oxidation are identified as key factors in the underlying mechanisms.
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Introduction
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Due to their antibacterial properties, silver nanoparticles (Ag NPs) have been
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extensively used in a wide range of healthcare and consumer products, including
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clothing, consumer electronics and appliances, dietary supplements and medical
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supplies1, 2. The number of Ag NP-based consumer products on the market had reached
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442 (out of 1827 nanomaterial-based products) by May of 20172. The production,
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usage, and disposal of Ag NP-based products have inevitably increased the
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environmental accumulation of and human exposure to Ag NPs3, 4. In addition to Ag
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NPs, Ag+ pollution is also prevalent. Wide applications of silver compounds in medical
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fields include silver nitrate, acetate, and sulfadiazine5. Ag+ pollution also comes from
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the photographic6, electrochemical7 and silver mining industries8. A telling example is
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that in a photoprocessing wastewater sample, Ag+ content is as high as 300 µg/L9.
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Metal ions are readily transformed to nanoparticles under regular environmental
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conditions. Natural waters containing Ag(I) and Au(III) ions generate Ag NPs and Au
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NPs in the presence of natural organic matter upon exposure to either UV or sunlight
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irradiation10-12. Metal oxide/sulfide nanoparticles, such as FeS2, Fe2O3, SiO2, Al2O3, and
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MnO2, can be formed in nature, depending on environmental factors such as
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temperature, pH, oxygen, light, concentration, and natural organic matter13. Metal ions
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can also be converted to nanoparticles in biological bodies such as in plants14,
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microorganisms13, and animal cells15. Therefore, when Ag+ ions are distributed into
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animal organs, it is likely that a proper reductive microenvironment may convert them 3 ACS Paragon Plus Environment
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to Ag NPs. The toxicity of Ag+ ions has long been recognized6, 7. Recently, toxicity of
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Ag NPs has also been extensively reported16, 17. However, in most cases, whether the
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observed toxic effects are from Ag+ ions or from Ag NPs remains elusive. Furthermore,
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the impacts of these pollutants on susceptible populations, such as the overweight
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population, are severely under-studied.
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Overweight and obesity have become a prevalent health threat worldwide in both
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developed18, 19 and developing countries19, 20. According to the WHO, more than 1.9
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billion adults were overweight or obese in 2014, among which 69% (1.3 billion) were
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classified as overweight individuals, while 31% (600 millions) were obese (BMI ≥ 30
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kg/m2)21. This significantly large population is still increasing rapidly due to lifestyle
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changes in many countries and regions. In addition to the risk of heart disease22,
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diabetes23 and cancer24, overweight and obesity also increase the risk of non-alcoholic
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fatty liver disease (NAFLD), which is a spectrum of liver abnormalities such as simple
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steatosis (steatosis without hepatocellular injury), non-alcoholic steatohepatitis
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(steatosis with inflammation and hepatocyte ballooning degeneration), fibrosis, cirrhosis
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and, ultimately, hepatocellular carcinoma25, 26.
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Previous studies have shown that the liver is a major organ for deposition of
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nanoparticles after absorption27-31. A single oral dose of Ag NPs induced acute
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inflammation of the liver in healthy male BALB/c mice, as evidenced by lymphocyte
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infiltration and increased expression of genes related to inflammation32. Hepatic
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inflammation, an increased level of alkaline phosphatase, and infiltration of 4 ACS Paragon Plus Environment
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inflammatory cells in the liver were also observed in healthy Sprague-Dawley rats after
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a 28-day oral exposure to Ag NPs29. These findings indicate that Ag NPs may perturb
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immune functions in animals, while inflammation is strongly indicated in the
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development of steatohepatitis33-35. Therefore, a possible role of Ag NPs in causing
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steatohepatitis needs to be examined.
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In this work, we first established a mouse model mimicking the sub-obese yet
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significant overweight population by feeding C57BL/6J mice a high-fat diet (HFD) for
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eight weeks. Oral exposure to a non-toxic dose of Ag NPs or AgNO3 to normal and
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overweight mice for two weeks caused no general toxicity in normal mice. However, an
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accelerated progression from steatosis to steatohepatitis was observed only in
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overweight mice, with Ag NPs as well as Ag+ ions. We found that after absorption and
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liver deposition, Ag+ ions were partially reduced to Ag NPs only in the livers of
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overweight mice. The Ag NPs that were either deposited or formed in situ in the livers
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of overweight mice induced activation of Kupffer cells, led to liver inflammation,
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suppressed fatty acid oxidation, and caused the transition from steatosis to
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steatohepatitis.
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Materials and methods
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Polyvinylpyrrolidone (PVP)-coated silver nanoparticles. PVP-coated Ag NPs
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were purchased from Xuzhou Jiechuang New Material Technology Co., Ltd.
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(Guangzhou, China). The purity of the Ag NPs provided by the manufacturer was ≥ 5 ACS Paragon Plus Environment
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99.99%. The morphological characteristics of PVP-coated Ag NPs were analyzed by
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transmission electron microscope (TEM, JEM-1011, Jeol, Japan). The hydrodynamic
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size and the zeta potential of Ag NPs were measured by a particle size analyzer
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(Malvern Nano ZS90, Malvern, UK). The Ag NPs were dispersed in DI water and
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sonicated for 30 min before administration.
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Animals and diets. Male C57BL/6J mice (5 weeks old) were obtained from the
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institute of Laboratory Animal Science, Chinese Academy of Medical Sciences
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(CAMS) & Peking Union Medical College (PUMC), Beijing, China. All animal
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experiments were approved by the Animal Care and Use Committee of Shandong
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University and were in accordance with the NIH guidelines in ‘Guide for the care and
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use of laboratory animals’. After acclimation for 1 week, 130 mice were randomly
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divided into 2 groups and fed a normal diet (ND) or a high-fat diet (HFD, containing
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54.8% ND, 18.9% lard, 1.3% cholesterin, 0.3% cholate, 11.2% saccharose, 8.7% casein,
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1.8% premix and 3% maltodextrin, acquired from Shanghai Laboratory Animal Center
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(SLAC), Shanghai, China). Body weight was recorded every week. After 8 weeks, mice
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in each group were then randomly divided into 5 groups, with one of these 5 groups
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sacrificed to confirm NAFLD. The other 4 groups of both ND- and HFD-fed mice were
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administered sterile water, Ag NPs ([Ag]=100 or 300 mg/kg) or AgNO3 ([Ag]=18
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mg/kg) for another 2 weeks. Body weight was recorded every day during Ag NP
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administration.
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In vivo biodistribution studies. Tissue samples (the heart, liver, spleen, lung, 6 ACS Paragon Plus Environment
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kidney, small intestine and fat around the small intestine) were added to 10 mL 70%
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nitric acid and digested in microwave ovens. After dilution with DI water, the Ag
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content in the digested solutions was analyzed using an inductively coupled
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plasma-mass spectrometer (ICP-MS, Agilent 7700, Santa Clara CA, USA).
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TEM and EDX analysis. The liver samples were dissected into 1-2 mm3 portions
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in 2.5% glutaraldehyde immediately after removal from the abdominal cavity and fixed
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in 2.5% glutaraldehyde for no less than 24 h. Ultrathin sections were then cut and
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analyzed with TEM (JEM-2100, Jeol, Japan). The elemental composition of the
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observed particles was analyzed in situ with an INCA energy dispersive X-ray
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spectrometer (Oxford Instruments, Oxfordshire, UK).
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Histopathological examination. The liver samples were fixed in 10% buffered
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formalin for 24 h and then embedded in paraffin. Paraffin-embedded sections were
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stained with hematoxylin and eosin staining (H&E staining). NAFLD activity was
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scored by an experienced pathologist blinded to the experimental procedures according
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to the criteria outlined in previous studies36, 37. Frozen sections were stained with Oil
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Red O staining for further confirmation of the steatosis.
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Biochemical analysis. Liver lipids were extracted with a mixture of
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chloroform/methanol (2:1 v/v)38. Liver cholesterol was analyzed using a commercial kit
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according to the manufacturer's protocol (Zhejiang Dongou Diagnostics Co., Ltd.,
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Zhejiang, China). Serum CHOL, TG and LDL levels were measured using an automatic
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biochemical analyzer (P800, Roche, USA). 7 ACS Paragon Plus Environment
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Statistical analysis. SigmaPlot 12.0 was used for statistical analyses. One-way
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ANOVA, followed by least-significant difference or Tukey’s tests, was performed to
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determine significance. Numerical data are presented as the means±s.d. Differences
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were considered significant when P
