Scholarly article on topic 'Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease'

Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease Academic research paper on "Veterinary science"

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Abstract of research paper on Veterinary science, author of scientific article — Sarah S. Poulsen, Anne T. Saber, Alicja Mortensen, Józef Szarek, Dongmei Wu, et al.

Abstract Adverse lung effects following pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) are well documented in rodents. However, systemic effects are less understood. Epidemiological studies have shown increased cardiovascular disease risk after pulmonary exposure to airborne particles, which has led to concerns that inhalation exposure to MWCNTs might pose similar risks. We analyzed parameters related to cardiovascular disease, including plasma acute phase response (APR) proteins and plasma lipids, in female C57BL/6 mice exposed to a single intratracheal instillation of 0, 18, 54 or 162μg/mouse of small, entangled (CNTSmall, 0.8±0.1μm long) or large, thick MWCNTs (CNTLarge, 4±0.4μm long). Liver tissues and plasma were harvested 1, 3 and 28days post-exposure. In addition, global hepatic gene expression, hepatic cholesterol content and liver histology were used to assess hepatic effects. The two MWCNTs induced similar systemic responses despite their different physicochemical properties. APR proteins SAA3 and haptoglobin, plasma total cholesterol and low-density/very low-density lipoprotein were significantly increased following exposure to either MWCNTs. Plasma SAA3 levels correlated strongly with pulmonary Saa3 levels. Analysis of global gene expression revealed perturbation of the same biological processes and pathways in liver, including the HMG-CoA reductase pathway. Both MWCNTs induced similar histological hepatic changes, with a tendency towards greater response following CNTLarge exposure. Overall, we show that pulmonary exposure to two different MWCNTs induces similar systemic and hepatic responses, including changes in plasma APR, lipid composition, hepatic gene expression and liver morphology. The results link pulmonary exposure to MWCNTs with risk of cardiovascular disease.

Academic research paper on topic "Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease"

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YTAAP-13288; No of Pages 13

Toxicology and Applied Pharmacology xxx (2015) xxx-xxx

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Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/ytaap

qi Changes in cholesterol homeostasis and acute phase response link

2 pulmonary exposure to multi-walled carbon nanotubes to risk of

3 cardiovascular disease

Q2 Sarah S. Poulsen a,b'*, Anne T. Saber a, Alicja Mortensenc, Jozef Szarek d, Dongmei Wu e, Andrew Williams e,

5 Ole Andersen b, Nicklas R. Jacobsen a, Carole L. Yauk e, Hakan Wallin a,f, Sabina Halappanavar e, Ulla Vogela,g

6 a National Research Centre for the Working Environment, Copenhagen DK-2100, Denmark

7 b Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde, Denmark

8 c National Food Institute, Technical University of Denmark, Soborg, Denmark

9 d Faculty of Veterinary Medicine, University ofWarmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland

10 e Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada

11 f Department of Public Health, University of Copenhagen, DK-1014 Copenhagen K, Denmark

12 g Department of Micro- and Nanotechnology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

article info

abstract

14 Article history:

15 Received 24 October 2014

16 Revised 7 January 2015

17 Accepted 12 January 2015

18 Available online xxxx

44 413 47

Keywords:

Nanotoxicology

Atherosclerosis

Toxicogenomics

Acute phase response Histology

Adverse lung effects following pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) are well Q3

documented in rodents. However, systemic effects are less understood. Epidemiological studies have shown 27

increased cardiovascular disease risk after pulmonary exposure to airborne particles, which has led to concerns 28

that inhalation exposure to MWCNTs might pose similar risks. 29

We analyzed parameters related to cardiovascular disease, including plasma acute phase response (APR) 30

proteins and plasma lipids, in female C57BL/6 mice exposed to a single intratracheal instillation of 0,18, 54 or 31

162 ng/mouse of small, entangled (CNTSmall, 0.8 ± 0.1 ^m long) or large, thick MWCNTs (CNTLarge, 4 ± 0.4 ^m 32

long). Liver tissues and plasma were harvested 1,3 and 28 days post-exposure. In addition, global hepatic gene 33

expression, hepatic cholesterol content and liver histology were used to assess hepatic effects. 34

The two MWCNTs induced similar systemic responses despite their different physicochemical properties. APR 35

proteins SAA3 and haptoglobin, plasma total cholesterol and low-density/very low-density lipoprotein were 36

significantly increased following exposure to either MWCNTs. Plasma SAA3 levels correlated strongly with pul- 37

monary Saa3 levels. Analysis of global gene expression revealed perturbation of the same biological processes 38

and pathways in liver, including the HMG-CoA reductase pathway. Both MWCNTs induced similar histological 39

hepatic changes, with a tendency towards greater response following CNTLarge exposure. 40

Overall, we show that pulmonary exposure to two different MWCNTs induces similar systemic and hepatic re- 41

sponses, including changes in plasma APR, lipid composition, hepatic gene expression and liver morphology. 42

The results link pulmonary exposure to MWCNTs with risk of cardiovascular disease. 43

© 2015 Published by Elsevier Inc.

Introduction

Abbreviations: APR, Acute phase response; BET, Brunauer-Emmett-Teller; CNT, Carbon nanotube; CRP, C-Reactive protein; CVD, Cardiovascular disease; FDR, False discovery rate; GO, Gene ontology; HDL, High density lipoprotein; LDL, Low density lipoprotein; MWCNT, Multi-walled carbon nanotube; Nano-CB, Nano-carbon black; Nano-TiO2, Nano-sized titanium dioxide; SAA, Serum amyloidA; SEM, Scanning electron microscopy; TEM, Transmission electron microscopy; VLDL, Very low density lipoprotein.

* Corresponding author at: National Research Centre for the Working Environment, Lerso Parkalle 105, DK-2100 Copenhagen, Denmark

E-mail addresses: spo@nrcwe.dk (S.S. Poulsen), ats@nrcwe.dk (A.T. Saber), almo@food.dtu.dk (A. Mortensen), szarek@uwm.edu.pl (J. Szarek), dongmei.wu@hc-sc.gc.ca (D. Wu), andrew.williams@hc-sc.gc.ca (A. Williams), oa@ruc.dk (O. Andersen), nrj@nrcwe.dk (N.R. Jacobsen), carole.yauk@hc-sc.gc.ca (C.L. Yauk), hwa@nrcwe.dk (H. Wallin), sabina.halappanavar@hc-sc.gc.ca (S. Halappanavar), ubv@nrcwe.dk (u. Vogel).

Cardiovascular disease (CVD), a broad term used for all diseases of 50 the cardiovascular system, is the leading cause of death worldwide, 51 being responsible for 3 in every 10 deaths in 2008 (World Health 52 Organization et al., 2011). Retrospective and prospective epidemiologi- 53 cal studies show that pulmonary exposure to respirable air particulates 54 increases the risk of CVD (Chen and Nadziejko,2005; Clancy et al., 2002; Q5 Dockery et al., 1993; Erdely et al., 2011a; Li et al., 2007; Mikkelsen et al., 56 2011; Pope et al., 1995,2004). Recent increases in the development and 57 use of nanomaterials will inevitably increase their presence in the envi- 58 ronment and thus enhance the risk of human exposure. Concern has 59 been raised that this exposure may lead to increased risk of CVD 60 (Saber et al., 2014). 61

http://dx.doi.org/mi 016/j.taap.2015.01.011 0041-008X/© 2015 Published by Elsevier Inc.

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Several studies have linked pulmonary exposure to different types of multi-walled carbon nanotubes (MWCNTs) via inhalation, instillation or aspiration to lung inflammation, sustained interstitial fibrosis, and granuloma formation in rodents (Ma-Hock et al., 2009; Pauluhn, 2010a,b; Porter et al., 2010; Reddy et al., 2010; Wang et al., 2011; Poulsen et al., 2013). In addition, extrapulmonary effects, such as plaque progression in apoE knock-out mice, increased levels of acute phase response (APR) proteins in the serum, and adverse developmental effects in offspring, have been reported in mice following pulmonary exposure to CNT (Li et al., 2007; Hougaard et al., 2013; Erdely et al., 2011b).

Systemic effects of MWCNTs may occur by direct translocation from the target tissue. Indeed, several studies have reported translocation of MWCNTs to different organs, suggesting that they are capable of crossing the air-blood barrier. For example, MWCNTs are found in the lymph nodes following instillation (Aiso et al., 2011), in the brain, kidney, heart and liver following inhalation (Mercer et al., 2013; Stapleton et al., 2012), and in the spleen, liver and bone marrow following pharyngeal aspiration (Czarny et al., 2014). It is also possible that secondary effects result from MWCNT-induced release ofcytokines and APR proteins into the systemic circulation during pulmonary inflammation. Increased concentrations of plasma APR proteins have been reported by many epidemiological studies investigating the cardiovascular effects of air pollution (Lowe, 2001; Mezaki et al., 2003; Libby et al., 2010; Estabragh and Mamas, 2013; Pussinen et al., 2007). Increased APR has been recognized as an important risk factors for CVD (Ridker et al., 2000; Saber et al., 2013; Kaptoge et al., 2012; Taubes, 2002; Estabragh and Mamas, 2013; Rivera et al., 2013; Johnson et al., 2004; Pai et al., 2004; Saber et al., 2014).

The APR is characterized by changes in plasma levels of APR proteins, including C-reactive protein (CRP), serum amyloid A (SAA) and fibrino-gen, and changes in cholesterol homeostasis following acute and chronic inflammatory states (Bourdon et al., 2012a; Gabay and Kushner, 1999). SAA is a family of conserved and highly homologous high density lipoprotein (HDL) apolipoproteins, which in mice are the predominant APR proteins (Meek et al., 1992). Several tissues, including the lungs, express the Saa3 gene. The two other isoforms, Saal and Saa2, are considered liver-specific but are also expressed in lungs (Bourdon et al., 2012a; Husain et al., 2013; Uhlar and Whitehead, 1999; Halappanavar et al., 2011; Halappanavar et al., 2014). The most studied APR protein in humans is CRP. Physiologically, the APR is a beneficial response to local or systemic disturbances (e.g. infections); however, a persistent chronic APR is suggested to alter blood lipids and cholesterol biosynthesis, thereby increasing the risk of developing CVD (Bourdon et al., 2012a; Lindhorst et al., 1997). In the circulation, SAA is primarily a part of HDL. During an APR the concentration of SAA can be induced over 1000-fold, whereby SAA replaces ApoA-1 as the major HDL protein. HDL-SAA is cleared faster from systemic circulation than regular HDL (Hoffman and Benditt, 1983; McGillicuddy et al., 2009; Salazar et al., 2000), and SAA remodeling of HDL impairs HDL's ability to serve as an acceptor for macrophage cholesterol efflux mediated through ABCA1. The consequences are retaining peripheral cholesterol, reduced cholesterol biliary excretion from liver (Artl et al., 2000; Banka et al., 1995; Lindhorst et al., 1997), and macrophage transformation into foam cells (Artl et al., 2000; Lee et al., 2013). Foam cells are a major component of the fatty streak observed during development of atherosclerosis, a multigenic, endothelial disease. Consistent with this, viral vector-mediated overexpression of Saa1 in ApoE-/- mice leads to increased plaque progression (Dong et al., 2011). Interestingly, nano-sized titanium dioxide (nano-TiO2) from the same batch increased both pulmonary APR in C57BL/6 mice (Halappanavar et al., 2011; Husain et al., 2013), and induced plaque progression in ApoE-/- mice (Mikkelsen et al., 2011) following pulmonary exposure, thus linking nanoparticle exposure to CVD.

We recently reported that intratracheal instillation of two MWCNTs, a short, entangled CNTSman and a longer CNTLarge, caused similar increases in pulmonary inflammation and APR in mice, characterized by

global mRNA changes, increased infiltration of inflammatory cells into the lung lumen and changes in the lung morphology (Poulsen et al., 2014). CNTSmall and CNTLarge were selected by the OECD Working Party on Manufactured Nanomaterials and are available at the EU Joint Research Centre. We chose these two based on their physicochemical differences. In the present study we explore changes in various CVD biomarkers and in hepatic gene expression in mice from the above mentioned study at 1,3 and 28 days following intratracheal instillation of CNTSmall and CNTLarge.

Materials and methods

Materials. The two MWCNTs used in this study have been described previously (Poulsen et al., 2014). Briefly, the first MWCNT (NRCWE-026) is small and entangled, and was purchased from Nanocyl, Belgium. In this study NRCWE-026 will be referred to as CNTSmall. The other MWCNT (NM-401) was donated by the EU Joint Research Centre and is longer and thicker than CNTSmall. In this study it is referred to as CNTLarge, and it is physicochemically similar to Mitsui XNRi-7, which was recently classified as possibly carcinogenic to humans (Group 2B) by IARC (Grosse et al., 2014). Another batch of CNTSmall was donated to the EU Joint Research Centre repository; so both MWCNTs are included in the materials of the OECD Working Party on Manufactured Nanomaterials. The physicochemical characterization of CNTSmall and CNTLarge, including thermal gravimetric analyses (TGA), surface area analysis (BET), light microscopy imaging, scanning electron microscopy (SEM) imaging, transmission electron microscopy (TEM) imaging and elemental composition, has been conducted previously (Jackson et al., 2014; Kobler et al., in press; Poulsen et al., 2014), and the data are summarized in the Results section.

Preparation of instillation medium and exposure stock. MWCNTs were suspended to a concentration of 3.24 mg/ml by sonication using a Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a disruptor horn (model number: 101-147-037) in NanoPure water containing 2% serum collected from C57BL/6 mice. Total sonication time was 16 min at 40 W with continuous cooling on ice. Vehicle controls contained NanoPure water with 2% serum and were sonicated as described for the MWCNT suspensions.

Animals, exposure and tissue collection. All handling, care taking and experimental procedures involving live animals have been reported previously (Kobler et al., 2014, in press). Briefly, female C57BL/6 mice (6 per group) aged 5-7 weeks were allowed to acclimatize for 1-3 weeks before exposure. The mice were anesthetized with 4% isoflurane until fully relaxed and with 2.5% during the instillation. They were exposed to 18, 54 or 162 ^g/animal of either CNTSmall or CNTLarge via a single intratracheal instillation. Intratracheal instillation was chosen since it allows for control of the deposited doses; this would be difficult with inhalation exposure. Although instillation bypasses the upper respiratory system and results in a rapid bolus deposition, it is a valuable tool for understanding the potential systemic tox-icity following MWCNT exposure. Also, comparable inflammation levels following MWCNT administration by pharyngeal aspiration and by inhalation at a similar benchmark-deposited-dose has been demonstrated (Porter et al., 2013), indicating that non-inhalation administration may predict the response following inhalation. The doses used were selected for studying systemic and hepatic mechanisms following a pulmonary exposure to MWCNTs and they are within the dose ranges of other instillation/aspiration studies (Kim et al., 2014; Park et al., 2009; Porter et al., 2010; Shvedova et al., 2008; Snyder-Talkington et al., 2013). They correspond to 1,3, and 9 days of exposure (8 h/day) to CNT, assuming 33% deposition rate (Ma-Hock et al., 2009; Jackson et al., 2011) and a ventilation rate of 1.8 l/h for mice, at the current Danish occupational exposure level for carbon black (3.5 mg/m3). When considering the recommended exposure limit for CNTs of 1 |ag/m3 per

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8 hour work shift (NIOSH, 2013), the lowest dose of 18 |ag/mouse corresponds to the expected human work life exposure assuming a 10% deposition (Ma-Hock et al., 2009), a ventilation rate of 1.8 l/h, a 40 h working week and 40 year work life. The dose of 56 |ag corresponds to 3 times the life-long dose and 162 |ag/mouse corresponds to 9 times the proposed life dose. Work place exposure to CNT are reported in the range of 10-300 |jg/m3 (Birch et al., 2011; Dahm et al., 2013; Erdely et al., 2013; Han et al., 2008; Lee et al., 2010; Maynard et al., 2004; Methner et al., 2010b, 2012), thus 10-300 times above the proposed exposure limit. At an air concentration of 10 |ag/m3,162 ^g/mouse would correspond to the total dose during a 40-year working life, whereas 162 ^g/mouse corresponds to pulmonary deposition during 1.5 work years at 300 |ag/m3. Control animals were instilled with vehicle (NanoPure water with 2% serum). The mice were terminated 1, 3 or 28 days after exposure by exsanguination via intracardiac puncture. Immediately after withdrawal of heart blood (800-1000 |i), liver tissue was collected and samples were snap-frozen in cryotubes in liquid N2 and stored at — 80 °C. Whole blood was fractionated by centrifugation and plasma was collected and stored at — 80 °C. Additional liver specimens were taken from 12 to 24 vehicle control mice and from 5 to 6 mice from groups treated with either CNTSman or CNT^,-^. Tissues were fixed in 4% neutral buffered formaldehyde, paraffin-embedded and sections 4-6 |jm thick were stained with hematoxylin and eosin (HE) for histological examination.

All animal procedures followed the guidelines for the care and handling of laboratory animals established by Danish law. The Animal Experiment Inspectorate under the Ministry of Justice approved the study (#2010/561-1779).

Plasma protein measurements. ELISA analysis specifically targeting plasma SAA3 levels was conducted in accordance with the manufacturer's instructions (Mouse Serum Amyloid A-3, Cat.#EZMSAA3-12K, Millipore). All of the time points and doses were evaluated. Samples were pooled to a final N of 3 per group (representing 6 mouse samples in total). Plasma haptoglobin was determined by ELISA (mouse haptoglobin (Hpt/HP) ELISA kit, Cat. #CSB-E08586m, Cusabio) as described by the manufacturer. The high dose only from all time points was evaluated. The samples were pooled to a final N of 3 per group.

The statistical analyses were performed in SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Statistical significance was calculated using a parametric two-way ANOVA with a post-hoc Tukey-type experimental comparison test. In case of interaction between dose and time, the data were separated in time points and a one-way ANOVA with a post-hoc Tukey-type experimental comparison test was performed. For the statistical analysis of haptoglobin protein levels, no statistically significant time-variance between controls was found; thus, controls from all time points were pooled.

Plasma lipid composition. Plasma levels of total cholesterol, HDL and low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) in mice exposed to CNTSman and CNTLarge were determined colorimetrical-ly with the EnzyChrom™ AF HDL and LDL/VLDL assay kit (EHDL-100, BioAssay Systems) according to the manufacturer's instructions. All time points and doses were evaluated with 6 animals per treatment group. Briefly, a standard sample was produced from a standard cholesterol reference supplied by the manufacturer. HDL was isolated from the supernatant following centrifugation of a 1:1 plasma-precipitating reagent solution. The LDL/VLDL fraction was separated by dissolution of the collected pellet from the 1:1 plasma-precipitating reagent solution in PBS. Plasma, HDL, and LDL/VLDL isolations from each sample and a standard cholesterol reference supplied by the manufacturer were placed in 50 aliquots as duplicates in a 96-well plate. Sixty microliters of a NAD-enzyme buffer mix was added, and the plate was incubated at room temperature for 30 min. Fluorescence measurements were recorded on Victor2 1420 Multi label counter (Wallac, Perkin-Elmer)

at OD 340 nm and cholesterol concentrations were determined by comparison to the standard sample.

Plasma triglyceride levels were determined using the EnzyChrom™ AF Triglyceride assay kit (BioAssay Systems, ETGA-200) according to the manufacturer's instructions. All time points and doses were evaluated. There were 6 animals per treatment group. Briefly, a 10-fold serial diluted standard curve was produced from a standard cholesterol reference supplied by the manufacturer. Ten microliter aliquots of standard and sample were placed in duplicate in a 96-well plate, and 100 of a dye reagent-enzyme mix was added. The plate was incubated at room temperature for 30 min. The color intensity of the reaction product was determined spectrophotometrically at OD 570 nm on a Victor2 1420 Multi label counter (Wallac, Perkin-Elmer), and total triglyceride concentrations were determined by a standard curve.

All statistical analyses on lipid levels were performed in SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). Statistical significance was calculated using a parametric one-way ANOVA with a post-hoc Tukey-type experimental comparison test.

Total lipid extraction and total hepatic cholesterol analysis. Total lipids were extracted from liver tissue according to the Folch method (Folch et al., 1957). In brief, approximately 4-5 mg of liver tissue was collected from the sample and the weight was noted. The liver tissue was homogenized in 250 methanol and 250 water. A double volume of 5:1 chloroform/methanol was then added, and phases were separated by centrifugation. The lower layer containing chloroform/lipid was collected, and the chloroform removed under nitrogen gas flow. Lipids were resuspended in 100 EnzyChrom assay buffer and stored at — 20 °C until analysis.

Colorimetric quantification of hepatic cholesterol levels was determined with the EnzyChrom™ AF Cholesterol assay kit (BioAssay Systems, E2CH-100) according to the manufacturer's instructions. All time points and doses were evaluated with 6 animals per treatment group. Briefly, a 10-fold serial diluted standard curve was produced from a standard cholesterol reference supplied by the manufacturer. Fifty microliter aliquots of standard and sample were placed in duplicate in a 96-well plate, and 50 of a dye reagent-enzyme mix was added. The plate was incubated at room temperature for 30 min. The color intensity of the reaction product was spectrophotometrically measured at OD 570 nm on a Victor2 1420 Multi label counter (Wallac, Perkin-Elmer), and total cholesterol concentrations were determined by comparison to the standard curve, with normalization to extracted tissue weight.

Total RNA extraction. Total RNA was isolated from liver tissue of 144 mice in total (N was 6 mice per dose group and time point). The RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using RNeasy MiniKits (Qiagen, Mississauga, ON, Canada) as described by the manufacturer (for details see supplementary materials). On-column DNase treatment was applied (Qiagen, Mississauga, ON, Canada). All RNA samples showing A260/280 ratios between 2.0 and 2.15 were further analyzed for RNA integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). RNA integrity numbers above 7.0 were used in the experiment. If the RNA samples did not fulfill the criteria, new RNA extractions from the liver tissue were performed. Total RNA was stored at — 80 °C until analysis.

Microarray hybridization. We have found changed hepatic gene expression only after pulmonary exposure to high doses of nano-TiO2 or nano-carbon black (nano-CB) (Bourdon et al., 2012a; Husain et al., 2013). Therefore, microarray analysis was done only for the 0 and 162 |ag dose groups in this study. The two lower doses (18 and 54 |ag) were included for time points 1 and 3 days in the subsequent RT-PCR confirmation of the microarray results. A total of 200 ng of RNA from each sample (6 per treatment group) was analyzed by microarray

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315 hybridization on Agilent 8 x 60 K oligonucleotide microarrays (Agilent

316 Technologies Inc., Mississauga, ON, Canada) as described previously

317 (Poulsen et al., 2013). Data were acquired using Agilent Feature Extrac-

318 tion software version 9.5.3.1.

319 Statistical, functional and pathway analysis of microarray data. The

320 microarray data were analyzed as described previously (Poulsen et al.,

321 2013, 2014). Genes showing expression changes of at least 1.5-fold in

322 either direction compared to their matched controls and having false

323 discovery rate adjusted p-values of less than or equal to 0.05 (FDR

324 p < 0.05) were considered significantly differentially expressed and

325 were used in the downstream analysis. We used the Database for

326 Annotation, Visualization and Integrated Discovery (DAVID) v6.7 and

327 Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood City,

328 CA, USA) for functional and pathway analysis, as previously described

329 (Poulsen et al., 2014). The gene ontology (GO) classification of the

330 differentially expressed genes was explored; GO consists of three struc-

331 tured controlled vocabularies (ontologies) that describe gene products

332 in terms of their associated biological processes, cellular components

333 and molecular functions in a species-independent manner. In the

334 present study GO biological processes were utilized.

335 qRT-PCR validation. Eighteen genes were selected for further validation

336 by qRT-PCR in liver using custom RT2 Profiler PCR Arrays and a BioRad

337 CFX96 real-time PCR detection system at doses 0, 18, 54 and 162 |ag

338 for post-exposure days 1 and 3, and at doses 0 and |ag at post-

339 exposure day 28 (for details see supplementary materials). The selected

340 differentially expressed genes (FDR p < 0.05, FC 1.5 in at least one

341 condition) showed large changes in expression following MWCNT ex-

342 posure, and/or were associated with lipid homeostasis, or inflammatory

343 and acute phase responses. A custom RT2 Profiler PCR Array plate, the

344 RT2 First Strand Kit and RT2 SYBR® Green qPCR Mastermix (QIAGEN

345 Sciences, Maryland, USA) was used. Hypoxanthine-guanine

346 phosphoribosyltransferase (Hprt), actin (3 (Actb) and glyceraldehyde

347 3-phosphate dehydrogenase (Gapdh) were used as reference genes

348 for normalization and were selected based on their stable expression

349 levels in the treated and control samples in the microarray results. A

350 threshold value was set to 102. The final qRT-PCR validation group

351 consisted of a sample size of 3 per treatment condition.

352 Results

353 MWCNT

354 Detailed physicochemical characterization of the materials has been

355 published elsewhere (Kobler et al., 2014; Jackson et al., 2014; Kobler

356 et al., in press; Poulsen et al., 2014). In brief, CNTSmall were 847 ±

357 102 nm long and 11 (6-17) nm wide. They contained 87% carbon and

358 had a BET surface area of 245.8 m2/g. A chemical analysis of CNTSman

359 from the same batch showed that main components of CNTSman

360 (NRCWE-026) included the following: C (84.4%), Al2O3 (14.97%),

361 Fe2O3 (0.29%) and CoO (0.11%) (Jackson et al., 2014). CNTSmall appeared

362 curly and highly entangled when visualized by TEM and SEM. CNTLarge

363 were 4048 ± 366 nm in length and had an average width of 67

364 ( 24-138) nm. CNTLarge consisted of 97% carbon and the BET surface

365 area was 14.6 m2/g. The chemical composition of CNTLarge from the

366 same batch showed that the main components of CNTLarge (NM-401) in-

367 cluded the following: C (99.7%), P2O5 (0.14%), CO3 (0.08%) and Fe2O3

368 ( 0.05%) (Jackson et al., 2014). CNTLarge appeared large and straight

369 when visualized by TEM and SEM.

370 Plasma protein analysis

371 Plasma levels of SAA3 were statistically significantly increased in

372 mice exposed to both types of MWCNTs. This was observed following

CNTSmau exposure in the high dose group on post-exposure day 1 and 373 day 28, and at all doses on post-exposure day 3 (4.3-, 7.0-, 2.5-, 10.4-, 374 and 32.8-fold increase, respectively) (Fig. 1A). CNTLarge exposure result- 375 ed in increased SAA3 levels following high dose exposure on day 1, and 376 at the medium and high dose on post-exposure day 3 (6.9,3.2 and 61.0 377 fold increase, respectively) (Fig. 1A). There were no changes at 28 days 378 after CNTLarge exposure. One observation in the control group on day 28 379 was an outlier (more than 2 SD difference from the other values), 380 resulting in a relatively high control SAA3 plasma protein content. 381 If this observation was excluded, the difference was statistically signifi- 382 cant for both medium and high dose exposures 28 days post-exposure. 383 Interestingly, we observed large increases in pulmonary Saa3 mRNA 384 levels in the same animals after pulmonary exposure to CNTSman and 385 CNTLarge (Poulsen et al., 2014). Pulmonary Saa3 mRNA levels correlated 386 strongly with SAA3 protein levels in the plasma (Fig. 1B) (linear regres- 387 sion; p < 0.0005 across time points and MWCNT dose). The haptoglobin 388 plasma levels were statistically significantly increased 3 days after expo- 389 sure to 162 |ag CNTSman and CNTLarge compared to vehicle controls 390 (Fig. 1C) but were not changed at other time points. 391

Alterations in cholesterol homeostasis

Compared to the controls, total cholesterol levels were greatly in- 393 creased after exposure to either MWCNTs on day 3 at the high dose 394 (58% and 51% for CNTSmau and CNTLarge, respectively) (Fig. 2A) and on 395 post-exposure day 1 for CNTLarge (28%). Significantly higher LDL/VLDL 396 plasma levels were found for the high dose 3 days post-exposure groups 397 for both MWCNTs (153% and 128% for CNTSmall and CNTLarge respective- 398 ly) (Fig. 2B). HDL levels were increased following high dose exposure to 399 CNTSmau on day 3 (42%). A similar but statistically non-significant in- 400 crease was observed for CNTLarge under the same exposure conditions 401 (31%) (Fig. 2C). The ratios between plasma HDL and LDL/VLDL levels 402 in the controls (CNTSmau: 3.56, CNTLarge: 3.22) compared to the high 403 dose exposed mice at day 3 (CNTSmau: 1.92, CNTLarge: 1.94) showed 404 that the increase in the LDL/VLDL level was greater than the increase 405 in HDL following exposure. Plasma triglyceride levels were unaffected 406 by CNTSman and CNTLarge exposure (results not shown). 407

No change in total hepatic cholesterol levels was observed for 408 CNTSmall, but a statistically significant 47% increase was observed for 409 CNTLarge on post-exposure day 3 (Fig. 2D). 410

Microarray analysis

The experiments and analyses adhered to MIAME standards (Edgar Q11

and Barrett, 2006). All microarray data have been deposited in the 413

NCBI Gene Expression Omnibus database and can be accessed through 414

the accession number GSE61366. Q12

Alterations in global hepatic gene expression 416

Global hepatic gene expression was assessed for the highest dose of 417

both MWCNTs for all three time points. For CNTSmall exposed mice, a 418

total of4028 of the 60,000 probes were differentially expressed in he- 419

patic tissue. On day 1, day 3 and day 28 the expression of 2505, 2401, 420

and 255 genes, respectively, was changed (Supplementary Table 1). 421

Fig. 3A shows the overlap of differentially expressed genes across time 422

points. CNTLarge had a slightly smaller effect on hepatic gene expression 423

than CNTSmau exposed mice, but still caused a significant effect; a total of 424

3089 probes were differentially expressed compared to controls. On day 425

1, the expression of 2128 genes was changed, and on day 3,1667 gene 426

expressions were altered. No genes were significantly differentially 427

expressed on day 28. As observed after exposure to CNTSmall, many of 428

the same genes were differentially expressed on day 1 and day 3 429

(Fig. 3B). 430

GO classification analysis in DAVID (Huang et al., 2009a,b) revealed 431

statistically significant perturbations in biological processes in the liver 432

at the early time points (day 1 and 3) only. The common GO biological 433

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4,5 4 3,5 3 2,5 2 1,5 1 0,5 0

rfl M i a

0 ug 18 54 162

ug ug ug

Day 28

18 54 162 ug ug ug

Oug 18

ug ug Day 3 CNTsmaii

54 162 "g

Oug 18

y= 0.5148X-0.0018 R2 = 0.5435

log(Fold change microarray data)

ug ug Day 28

162 0 ug "g

ug ug Day 1

162 ug

ug ug Day 3 CNTIarge

162 ug

162 ug 162 ug 162 ug 0 ug 162 ug 162 ug 162 ug

Day 1 Day 3 Day 28 Day 1 Day 3 Day 28

CNTsmaii CNTIarge

Fig. 1. Plasma protein levels ofSAA3 and haptoglobin following exposure to CNTSmall orCNT^^ (A) Plasma levels ofSAA3 protein following intratracheal instillation of 0,18,54 or 162 |jg CNTsmaii or CNTLarge at post-exposure day 1,3 or 28. (B) Linear regression analysis of pulmonary Saa3 mRNA fold changes after microarray analysis and systemic SAA3 protein fold changes following ELISA analysis- Both microarray and protein level data have been log transformed- (C) Plasma levels of haptoglobin protein following intratracheal instillation of 0 or 162 |g CNTSmall or CNTLarge at post-exposure day 1,3 or 28. 'Statistically significantly different from vehicle instilled mice, p < 0.05. "Statistically significantly different from vehicle instilled mice, p < 0.01. '''Statistically significantly different from vehicle instilled mice, p < 0.001.

434 processes affected following CNTSman and CNTLarge exposure are shown

435 in Fig. 3C. We observed a high degree of concordance between the two

436 MWCNTs for enriched GO biological processes, especially for oxidation

437 reduction [G0:0055114], steroid metabolic process [G0:0008202],

438 lipid biosynthetic process [G0:0008610], fatty acid metabolic process

439 [G0:0006631], cofactor metabolic process [G0:0051186] and immune

440 response [G0:0006955], which were differentially enriched both 1

441 and 3 days following CNTSmau and CNTLarge exposure. The uniquely dif-

442 ferentially regulated biological processes are shown in Supplementary

443 Table 2 and revealed few major differences. Several of the highly

444 enriched processes in common for the two MWCNTs were associated

445 with lipid homeostasis. Remarkably similar and pronounced effects on

446 gene expression for both MWCNTs were observed by functional annota-

447 tion clustering (Supplementary Table 3), where lipid metabolic process

448 was the top scoring cluster at the early time points. Across both MWCNT

449 types at the early points, oxidation reduction [G0:0055114] was the

450 most enriched regulated biological process. Several of the differentially

451 regulated genes under this process were involved in other biological

452 processes as well, including lipid homeostasis processes.

453 IPA was employed to relate the functional significance of the G0

454 changes to biological functions and pathways. The individual enriched

455 functions in IPA were filtered by 1) removing redundant functions

456 with overlapping genes, and 2) removing functions that were not

457 directly relevant to the present study (e.g. renal diseases, ophthalmic

458 diseases etc.). By using these criteria, we identified the top 5 most signif-

459 icantly affected functions after CNTLarge exposure, and we compared

460 these to the corresponding functions following CNTSmall exposure

461 (Supplementary Fig. 1). The top-regulated functions were similar for

462 both MWCNTs, with 3 out of 5 of the most regulated functions being

the same. 'Lipid metabolism' was the most enriched function, in agree- 463

ment with the observed changes in the G0 biological processes involv- 464 ing lipid homeostasis. The third most perturbed function after CNTSmall 465

exposure was 'cardiovascular disease', and the fifth was 'carbohydrate 466

metabolism'. 467

Inflammation and acute phase response signaling 468

Inflammatory processes were among the most perturbed processes 469

in the liver. These were in part driven by changes in the mRNA levels 470

of several cytokines, including the following: Cxcl1, Cxcl9, Cxcl10, 471

Cxcl13, Ccl6, Ccl27a and Ccl25. We also found differential expression 472

of APR genes in the liver following MWCNT exposure including the 473

following: Saa1, Saa2, Saa3, Saa4, Orm1, Orm2, Orm3, Mt1 and Mt2 474

(Supplementary Table 4). As recently reported, strong pulmonary in- 475

flammatory and APR, both as increased neutrophil influx and increased 476

expression of cytokines and APR genes, were found in these mice 477

following same exposure to CNTSmau and CNTLarge (Poulsen et al., 2014). 478

Regulation of cholesterol homeostasis. 479 Analysis of the global hepatic gene expression revealed consistent 480

perturbation of lipid homeostasis related functions and pathways. 481 Genes involved in the HMG-CoA reductase pathway were substantially 482

down-regulated in the liver for both MWCNTs, which is consistent with 483

perturbations in lipid processes identified using DAVID (Fig. 3C) and 484 functional analysis in IPA (Supplementary Fig. 1 and Supplementary 485

Table 3). CNTSmau was the most effective in perturbing the HMG-CoA re- 486

ductase pathway (Hmgcr, Mvk, Pmvk, Mvd, Fdps, Sqs, Sqle, Dhcr7) in the 487

liver. The changes in gene expression were similar on days 1 and 3 488

(Table 1). In addition to the HMG-CoA reductase pathway, other genes 489

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□ CNTsmall ■ CNTIarge

Fig. 2. Changes in systemic and hepatic cholesterol levels following exposure to CNTSmaii or CNTLarge. (A) Plasma total cholesterol, (B) plasma LDL, (C) plasma HDL and (D) hepatic total cholesterol in C57BL/6 mice exposed to 0,18,54 or 162 |og CNTSmall or CNTLarge at post-exposure day 1,3 or 28. 'Statistically significantly different from vehicle instilled mice, p < 0.05. •'Statistically significantly different from vehicle instilled mice, p < 0.01. '''Statistically significantly different from vehicle instilled mice, p < 0.001.

490 involved in lipid homeostasis were also affected by MWCNT exposure.

491 Low density lipoprotein receptor (Ldlr) was down-regulated after

492 CNTSmall exposure on day 1 ( — 1.79-fold) and day 3 ( — 1.7-fold), and

493 after CNTLarge exposure at day 3 (— 1.56-fold) (Table 1). Gene expres-

494 sion of another membrane protein, scavenger receptor class B, member

495 1 (Scarbl), was also down-regulated for CNTSmall day 1 ( — 1.53-fold),

496 whereas expression of low density lipoprotein receptor-related protein

497 1 (Lrpl) was up-regulated on day 3 for both MWCNTs (CNTSmall 1.84498 fold, CNTLarge 2.09-fold). Scarbl and Lrpl are involved in the transport

499 of HDL and LDL, respectively, over the hepatocyte cell membrane. A

500 small up-regulation in the expression of Abcal was identified 3 days

501 post-exposure for CNTSmall (1.68-fold).

502 Besides the HMG-CoA reductase pathway, analysis of canonical

503 pathways in IPA (Supplementary Fig. 2) also revealed enrichment of

504 LXL/RXR activation, glutathione-mediated detoxification, acute phase

505 response signaling, nicotine degradation III, hepatic cholestasis and

506 xenobiotic metabolism signaling pathways. Analysis of the LXL/RXR

507 activation pathway on day 3 for both MWCNTs revealed that most up-

508 stream and downstream genes related to the LXL/RXR heterodimer

509 complex show changes in expression (Supplementary Fig. 3), including

510 cholesterol transporter Abcal. As observed for the HMG-CoA pathway,

511 CNTSmall exposure induced the largest change in gene expression of

512 genes in this pathway.

513 qRT-PCR validation

514 We validated 18 differentially expressed genes in liver tissue follow-

515 ing pulmonary exposure to 162 |g CNTSmall or CNTLarge at post-exposure

516 day 1, 3 or 28. These genes were chosen due to their involvement in

517 lipid homeostasis (Hmgcr, Pmvk, Mvd, Fdps, Dhcr7, Ldlr, Lrpl, Cyp7a1,

Abcal), inflammatory and APR (Cxcll, S100a9, Saal, Saa2, Saa3, Illrl) 518 or due to large changes in their expression following MWCNT exposure 519 (Sultlel, Scdl, Dbp). In addition to this validation, we evaluated the 18 520 and 54 |g exposure groups on post-exposure days 1 and 3 in order to 521 assess hepatic changes following lower dose MWCNT exposure. The 522 qRT-PCR results are provided in Table 2 and are consistent with the 523 DNA microarray results. The lipid homeostasis genes Hmgcr, Ldlr and 524 Cyp7al were differentially expressed at all doses on days 3 and 28 525 after CNTSmall exposure, but not following CNTLarge exposure. In con- 526 trast, there was a tendency towards greater enrichment of inflammato- 527 ry and APR genes following exposure to CNTLarge compared to CNTSmall. 528 The expression of Dbp, a PAR leucine zipper transcription factor 529 involved in circadian rhythm regulation, was consistently down- 530 regulated at all doses and time points following exposure to CNTSmall, 531 whereas CNTLarge exposure resulted in up-regulation of the expression 532 at the high and medium dose on post-exposure day 1. 533

Liver histology 534

Different doses of CNTSmall or CNTLarge induced histological changes 535

in the liver at different times (Fig. 4 and Supplementary Table 5) with 536

no apparent dose- or time-dependence. No translocation of MWCNTs 537

from lungs to liver was observed. Changes such as vacuolar degenera- 538

tion (Figs. 4C-J), granulomas (Figs. 4C and G-J), necrosis of hepatocytes 539

(Figs. 4D and I-J), increased number and/or hypertrophy of Kupffer cells 540

(Figs. 4A and I-J) were frequent in CNT-treated mice. For both CNT types 541

the incidence of lesions was higher 3 and 28 days after exposure than on 542

post-exposure day 1. The sites of vacuolar degeneration in the cyto- 543

plasm of hepatocytes within the hepatic lobule differed between 544

CNTSmall and CNTLarge exposure. Whereas the vacuolar degeneration 545

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Day 28

Oxidation reduction Steroid metabolic process Lipid biosynthetic process Fatty acid metabolic process Fat cell differentiation Monosaccharide metabolic process Cofactor metabolic process Cellular amino acid derivative metabolic process Response to wounding Immune response Vitamin metabolic process Nitrogen compound biosynthetic process

I 1051 564 632 1

■ CNTIarge day 3

□ CNTIarge day 1

■ CNTsmall day 3

□ CNTsmall day 1

20 40 60 80 100 120

No. of hepatic genes with regulated expression

Fig. 3. Hepatic transcriptomic changes. (A) Venn diagram of differentially expressed genes following exposure to 162 |og CNTSmall. p < 0.05 and fold change ±1.5. (B) Venn diagram of differentially expressed genes following exposure to 162 |g CNTLarge. p < 0.05 and fold change ±1.5. (C) Changes in GO biological processes in the liver following exposure to CNTSmall and CNTLarge. Determined through DAVID Bioinformatics Resources 6.7.

546 after CNTLarge exposure was distributed throughout the whole area of

547 the hepatic lobule regardless of the time after instillation, after CNTSmall

548 exposure it was located in the centrilobular zone (i.e. near the central

549 vein) 1 day after exposure, mid-zonal three days after exposure and in

550 the periportal zone 28 days after exposure. Although liver granulomas

551 were observed after exposure to both MWCNTs, they were larger and

552 more frequent following CNTLarge exposure (Figs. 4C and G-J). Microfoci

553 of necrosis, eosinophilic necrosis and hepatocytes with pyknotic nuclei

554 were seen following exposure to either MWCNTs and were located

close to granulomas; however, they were more frequent after CNTLarge 555 exposure. Eosinophilic necrotic hepatocytes surrounding the central 556 vein were observed in the livers of mice 1 or 3 days after exposure to 557 162 |ag CNTLarge. Increased number and hypertrophy of Kupffer cells 558 were observed more frequently in livers from mice exposed to CNTLarge 559 (Figs. 4D and I-J). Binucleate hepatocytes were more frequent in the 560 livers of exposed mice than in the controls. Overall, pulmonary expo- 561 sure to CNTLarge was associated with increased incidence and severity 562 of morphological liver lesions compared to exposure to CNTSmau. 563

tl.l Table 1

tl.2 Differentially expressed hepatic genes involved in lipid metabolism processes following exposure to CNTSmall or CNTLarge.

t1.3 Gene name CNTsmall CNTLarge

t1.4 Day 1 Day 3 Day 28 Day 1 Day 3 Day 28

t1.5 FC p-Value FC p-Value FC p-Value FC p-Value FC p-Value FC p-Value

t1.6 HMG-CoA reductase (HMGCR) -2.18 0.013 -3.0 0.001 - - - - - 2.63 0.005 - -

t1.7 Mevalonate kinase (MVK) -1.98 0.006 - - - - - - - _ _ _

t1.8 Phosphomevalonate kinase (PMVK) -3.05 0.0 - 1.75 0.001 - - -2.27 0.0 - 2.66 0.0 - -

t1.9 Mevalonate-5-pyrophosphate (MVD) - - - 2.22 0.012 - - - - - 2.93 0.001 - -

t1.10 Farnesyl-PP synthase (FDPS) -2.31 0.000 - 1.89 0.017 - - -2.67 0.0 - 3.16 0.0 - -

t1.11 Squalene synthase (SQS) -1.75 0.009 - - - - - - - - - -

t1.12 Squalene epoxydase (SQLE) -2.05 0.025 - - - - - - - - - -

t1.13 7-Dehydrocholesterol reductase (DHCR7) - 2.22 0.0 - 1.99 0.0 - - -2.09 0.0 - 2.33 0.0 - -

t1.14 Low density lipoprotein receptor (LDLR) -1.79 0.002 - 1.70 0.006 - - - - -1.56 0.041 - -

t1.15 Low density lipoprotein receptor-related protein 1 (LRP1) - - 1.84 0.0 - - - - 2.09 0.0 - -

t1.16 Scavenger receptor class B, member 1 (SCARB1) - 1.53 0.0 - - - - - - - - - -

t1.17 Cytochrome P450, family 7, subfamily a, polypeptide 1 (CYP7A1) - - -20.16 0.0 - - - - - 4.0 0.007 - -

t1.18 Hepatic lipase (LIPC) - - - 1.50 0.002 - - - - - - - -

t1.19 ATP-binding cassette, sub-family A, member 1 (ABCA1) - - 1.68 0.0 - - - - - - - -

tl.20 FC: Fold change. -: Not significantly differentially expressed. Numbers are statistically significant at least at the p < 0.05 level.

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t2.1 Table 2

t2.2 Hepatic mRNA expression changes for selected genes in mice 1,3 and 28 days post-exposure to 18 |g, 54 |g and 162 |g CNT.

t2.3 CNTsmall

t2:28 t2:29 t2:30 t2:31 t2:32 t2:33 t2:34 t2.35 t2.36 t2:37 t2:38 t2:39 t2:40 t2:41 t2:42 t2:43 t2:44 t2.45

Day 28

PCR array

Microarray

162 Lg

PCR array

Microarray

162 Lg

PCR array

162 Lg

Microarray

162 Lg

Sult1e1

S100a9

Cyp7a1

CNTLarge

-2.1 -23.3 - 26.1 Day 1

1.5 - 4.6

51.3 - 2.5

2.4 2.6

5.2 - 2.2 - 3.1

- 2.2 - 1.8

- 1.8 - 3.4

- 6.6 - 33.8 Day 3

- 2.2 - 3.8 -11.0

58.6 - 227.5 63.3 17.1

- 2.5 1.9

- 39.3

- 47.1 18.6 12.2 8.2 6.1 2.2 10.1

- 1.8 - 2.2

- 1.7 1.8

- 20.2 1.7

- 10.1

- 2.0 - 2.1 - 22.7 Day 28

- 12.3

PCR array

Microarray

162 Lg

PCR array

Microarray

162 Lg

PCR array

162 Lg

Microarray

162 Lg

Sult1e1

S100a9

Cyp7a1

172.6 - 1.9

10.4 10.6

34.7 - 1.8 3.6 2.4 3.0 2.8

28.3 - 111.9 72.2 7.7

3839.4 2521.4 9.5 10.9

- 52.4 14.1 4.1

8.2 3.2 4.4

- 1.6 2.1

t2.46 Fold change compared to vehicle instilled control mice. -: Not significantly differentially expressed. Numbers are statistically significant at least at the p < 0.05 level.

564 Discussion

565 In this study we investigated extrapulmonary effects in female

566 C57BL/6 mice 1, 3 or 28 days following a single, intratracheal instillation

567 of either of two MWCNTs with very different physicochemical proper-

568 ties. We report increased plasma levels of the APR protein haptoglobin

569 and a large, sustained increase in plasma SAA3 protein levels following

570 exposure to both types of MWCNTs. Systemic increases in haptoglobin

571 and SAA levels have been observed previously following pulmonary

572 CNT exposure (Erdely et al., 2011b; Saber et al., 2013), with increased

573 SAA3 levels being detected up to one year post-exposure in male mice

574 (Kim et al., 2014). We recently reported that CNTSmall and CNTLarge ex-

575 posures, despite physicochemical fiber dissimilarities, elicit very similar,

576 strong pulmonary inflammation and APR (Poulsen et al., 2014). This

577 pulmonary APR was characterized by time- and dose-dependent

578 increases in the expression of a number of acute phase genes, with

579 Saa3 being the most differentially expressed gene. We have previously

580 reported similar findings for other nanomaterials and particles (Saber

581 et al., 2014). In the present study, we observed a strong and statistically

582 significant linear relationship between the pulmonary Saa3 mRNA

583 levels and SAA3 plasma levels (Fig. 1B), indicating a probable pulmo-

584 nary origin of plasma SAA3. The hepatic expression levels of Saa3

585 following CNTSmall and CNTLarge exposure were 10 to 100 times lower

than the expression levels of pulmonary Saa3, thus indicating that the 586 observed systemic APR may be a secondary response to the pulmonary 587 APR induced by MWCNTs. This is in agreement with previous reports 588 showing that pulmonary exposure to CB, diesel exhaust particle and 589 MWCNTs induced little or no hepatic APR, but a large pulmonary APR 590 (up to a 600-fold increase) (Bourdon et al., 2012a,b; Saber et al., 2009, 591 2013,2014). 592

Elevated plasma levels of APR proteins are a recognized risk factor 593 for CVD (Estabragh and Mamas, 2013; Johnson et al., 2004; Kaptoge 594 et al., 2012; Libby et al., 2010; Lowe, 2001; Mezaki et al., 2003; Pai 595 et al., 2004; Pussinen et al., 2007; Ridker et al., 2000; Rivera et al., 596 2013; Saber et al., 2013; Taubes, 2002). Epidemiological studies have 597 established associations between particulate air pollution exposures 598 and blood levels of CRP (Elliott et al., 2009; Barregard et al., 2006; 599 Allen et al., 2011; Hertel et al., 2010; Ohlson et al., 2010). Increased 600 blood levels of SAA and CRP were associated with future risk of CVD in 601 the Nurses' Health Study (Ridker et al., 2000). The Nurses' Study report- 602 ed that a 5-fold increase in blood SAA levels was associated with a 3-fold 603 increased risk of coronary heart disease. Comparable increases in SAA3 604 levels were reported in the present study (7-fold increase, 28 days fol- 605 lowing pulmonary CNTSmau exposure) and in male mice by (Kim et al., 606 2014) (4-fold increase, one year following pulmonary MWCNT expo- 607 sure), indicating that human exposure to doses comparable to those in 608

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Fig. 4. Histopathologic findings in the liver. (A) 1 day after instillation (a.i.) of 54 |ogCNTSmau: Hypertrophy of Kupffer cells (long arrows), numerous binucleate hepatocytes (short arrows). (B) 1 day a.i. of 162 |g CNTSmau: Macrophage (long arrow), central zonal vacuolar (hydropic) degeneration (short arrows). (C) 3 days a.i of 54 |g CNTSmau: Granuloma surrounded by eosinophilic necrotic hepatocytes (long arrow), vacuolar degeneration (short arrows); (D) 28 days a.i. of 162 |g CNTSmau: Foci of necrosis (asterisks), vacuolar degeneration (short arrows), hyperplasia of the bile ducts epithelium (long arrow). (E) 1 day a.i. of 162 |g CNTLarge: Eosinophilic necrotic hepatocytes surrounding central vein (long arrows), vacuolar degeneration of hepatocytes on the whole area of the liver lobule (head of arrows), and numerous binucleate hepatocytes (short arrows). (F) 3 days a.i. of 18 |g CNTLarge: Focus of eosinophilic necrotic hepatocytes (long arrow), vacuolar degeneration of hepatocytes on the whole area of the liver lobule (short arrows). (G) 3 days a.i. of 54 |g CNTLarge: Granuloma surrounded by eosinophilic necrotic hepatocytes (arrows), vacuolar degeneration (short arrows). (H) 3 days a.i. of 162 |g CNTLarge: Granuloma surrounded by eosinophilic necrotic hepatocytes (arrow), vacuolar degeneration (short arrows), numerous binucleate hepatocytes (head of arrows). (I) 28 days a.i. of 54 |g CNTLarge: Granuloma surrounded by eosinophilic necrotic hepatocytes (long arrow), foci of necrosis (asterisks), vacuolar degeneration (short arrows), hypertrophy of Kupffer cells (head of arrows). (J) 28 days a.i. of 162 |g CNTLarge: Granuloma surrounded by eosinophilic necrotic hepatocytes (long arrow), foci of necrosis (asterisks), vacuolar degeneration (short arrows), hypertrophy of Kupffer cells (head of arrows). (K) typical microscopic pattern of the mouse liver — the control group. Staining HE, magnification on the figures A-J as scale in K.

609 these two murine studies may increase the risk of CVD in humans.

610 Occupational studies have reported human CNT exposure levels of

611 10-300 |ag/m3 (Birch et al., 2011; Dahm et al., 2013; Erdely et al.,

612 2013; Han et al., 2008; Lee et al., 2010; Maynard et al., 2004; Methner

613 et al., 2010a, 2012). The highest dose in the present study corresponds

614 to pulmonary deposition during a 40 year working life at 10 |ag/m3, or

615 to pulmonary deposition during 1.5 work years at 300 |ag/m3. Further-

616 more, based on the observed strong correlation between plasma SAA3

617 levels and pulmonary Saa3 mRNA levels, and the reported correlation

618 between pulmonary neutrophil influx and pulmonary Saa3 mRNA

619 levels (Saber et al., 2013), pulmonary Saa3 expression or neutrophil

influx may be used as sensitive biomarkers of nanomaterial-induced 620 acute phase response and may be used to group and rank nanomaterials 621 in relation to possible CVD inducing potential. 622

The APR, and SAA in particular, affect blood lipid homeostasis and 623 regulation of cholesterol biosynthesis (Bourdon et al., 2012a; 624 Lindhorst et al., 1997; Saber et al., 2013, 2014). We show that pulmo- 625 nary exposure to two very different MWCNTs induces changes in 626 blood lipid composition that are similar to changes observed after sys- 627 temically induced APR (Lindhorst et al., 1997). The data indicate that 628 both MWCNTs cause perturbations in lipid-related processes as early 629 as 1 day post-exposure, reaching their peak increases on day 3 and 630

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completely reversed to basal levels by day 28 post-exposure. Thus, blood levels of HDL and LDL/VLDL cholesterol followed a time-course similar to what has been described for a strong APR in mice subcutane-ously injected with silver nitrate (Lindhorst et al., 1997). During APR, SAA becomes incorporated into HDL thereby replacing ApoA-1 (Feingold and Grunfeld, 2010). Acute phase HDL is defective in reverse cholesterol transport, resulting in reduced cholesterol efflux from macrophages and lowered hepatic cholesterol excretion (Lindhorst et al., 1997; Banka et al., 1995; Artl et al., 2000; Annema et al., 2010).

We found that pulmonary exposures to the two very different MWCNTs elicit similar hepatic gene expression patterns. Whereas Saa3 primarily drives the pulmonary APR (Bourdon et al., 2012a; Halappanavar et al., 2011; Saber et al., 2013, 2014), both Saal and Saa2 were differentially expressed as part of the hepatic APR. This is in agreement with previous observations (Uhlar and Whitehead, 1999). Functional analyses revealed that GO biological processes and IPA functions involved in cholesterol homeostasis and lipid metabolism were perturbed following exposure to both types of MWCNTs. This was driven, in part, by a uniform down-regulation of genes across the entire HMG-CoA reductase pathway (Hmgcr, Mvk, Pmvk, Mvd, Fdps, Sqs, Sqle, Dhcr7). Interestingly the opposite was observed in the lungs. In lungs, CNTSmall and CNTLarge induced consistent up-regulation of several genes involved in the HMG-CoA reductase pathway. The HMG-CoA re-ductase pathway is activated during low-sterol conditions and plays a central role both in the synthesis of cholesterol and in the production of intermediates for terpenoid synthesis, actin cytoskeleton remodeling, hormones and protein prenylation (Liao, 2002). However, the down-regulated expression levels of Sqle and Dhcr7 in the liver indicate that MWCNT specifically targets cholesterol synthesis. The HMG-CoA reduc-tase pathway is regulated by a negative feedback loop; the down-regulation is most likely due to the increased cholesterol levels in plasma and liver (both observed for CNTLarge only).

In the present study, the systemic effects of MWCNT exposure were evaluated in female mice. Similarly increased plasma levels of SAA were recently reported for male mice exposed to MWCNTs (Kim et al., 2014). Lipoprotein profiles among pre-menopausal women differ from men's, as they have lower LDL cholesterol levels while HDL cholesterol levels are higher. Also, LDL cholesterol levels in post-menopausal women are equal to, or higher than, those in age-matched males, and can be lowered by hormone replacement therapy (Skafar et al., 1997). Thus, a cardioprotective effect of female sex hormones, particularly estrogen, has been proposed (Skafar et al., 1997; De Marinis et al., 2008). It is therefore possible that the differences in systemic HDL levels following MWCNT exposure observed between the study of (Kim et al., 2014) and our study are related to the gender of the experimental animals. A study in rats reported gender-related differences in the HMG-CoA reductase pathway, with females having lower activity and expression of 3-hydroxy 3-methylglutaryl coenzyme A reductase (Hmgcr), the rate-limiting enzyme in the HMG-CoA reductase pathway, compared to age-matched males (De Marinis et al., 2008). Similar differences were observed when comparing non-treated males with males treated with 17-(-estradiol, indicating an estrogen-reduction of the HMG-CoA re-ductase pathway. Thus, the present study adds to the overall weight of evidence of systemic acute phase response in mice following MWCNT exposure in both genders.

Studies in rodents have shown that severe inflammation, initiated through intraperitoneal injection of LPS, induces the hepatic HMG-CoA reductase pathway (Feingold et al., 1993,1995; Memon et al., 1993). Perturbations in the hepatic HMG-CoA reductase pathways were also observed following pulmonary exposure to nano-CB (Bourdon et al., 2012a). However, the directions of the observed effects were opposite. HMG-CoA was up-regulated following nano-CB exposure, whereas, it was down-regulated following exposures to both types of MWCNTs. Another difference includes the down-regulation of Ldlr in the livers of MWCNTs exposed mice in the present study, but no effects following LPS and nano-CB exposure (Bourdon et al., 2012a; Feingold et al.,

1993, 1995). Ldlr and Hmgcr are regulated in a coordinated fashion with parallel increases or decreases in mRNA levels in response to stimuli (Goldstein and Brown, 1990), as observed in the present study. Ldlr primarily facilitates the transport of VLDL, intermediate-density lipoproteins and LDL across the membrane (Lagor and Millar, 2010), and the hepatic down-regulation of Ldlr could in part explain the increased plasma levels of LDL/VLDL. Thus, the observed dissimilarities between our results and those of others (Bourdon et al., 2012a; Feingold et al., 1993,1995) indicate that MWCNT exposure may elicit hepatic and systemic responses related to their structures, that differ from responses seen following LPS and nano-CB exposure.

The dissimilarities observed between nano-CB relative to MWCNT exposures may result from greater and more prolonged APR (observed at both mRNA and protein levels) following MWCNT exposure than those observed following nano-CB. Plasma SAA levels were at least 30-fold increased following MWCNT exposure, whereas a maximum increase of 15.6% has previously been observed following a similar nano-CB exposure (Bourdon et al., 2012a). The greater and prolonged APR would lead to a higher proportion of circulating HDL-SAA. Besides having decreased cholesterol efflux ability, HDL-SAA is also able to transform peripheral macrophages into foam cells, and to promote plaque progression in APOE-/— mice (Artl et al., 2000; Lee et al., 2013). Thus, our results indicate that increased HDL-SAA levels may present a significant risk factor for CVD following MWCNT exposure.

Supplementary Fig. 4 depicts the observed pulmonary, systemic and hepatic changes following pulmonary MWCNT exposure. In particular, we note that down-regulation of hepatic expression of Abcg5 and Abcg8 occurs following exposure to both types of MWCNTs (Supplementary Table 1). Abcg5 and Abcg8 encode biliary transporters that facilitate cholesterol excretion into the bile (Yu et al., 2002). Biliary excretion is a major function in reverse cholesterol transport, and decreased expression of Abcg5 and Abcg8 has been linked to retention of cholesterol in the liver (McGillicuddy et al., 2009). Due to the perturbations observed for many steps in the reverse cholesterol transport, it is possible that the observed increased APR may have a direct or indirect effect on bile excretion of cholesterol, which, in part, could explain the observed increase in hepatic cholesterol levels. In addition, our toxicogenomics analysis of hepatic gene expression revealed a general inflammatory response in the liver, which is known to affect bile excretion (McGillicuddy et al., 2009).

Intratracheal instillation of either type of MWCNTs in mice caused histological changes in the liver. An increased number of binucleate hepatocytes in MWCNT treated mice were observed, indicating hepatocytic regeneration typically seen after a toxic insult (Kostka et al., 2000). Similar increases in binucleated hepatocytes have previously been reported following nanoparticle-exposure (Hougaard et al., 2013; Kostka et al., 2000; Saber et al., 2012). Other indications of systemic MWCNT-induced mild liver injury in our study include the following: microfoci of necrosis, eosinophilic necrosis of single hepatocytes, and hepatocytes with pyknotic nuclei (Fig. 4 and Supplementary Table 5), which all are frequent sequels to liver injury (Haschek et al., 2010). MWCNT also induces hepatic inflammatory changes. Small foci of inflammatory cells, polymorphonuclear cell foci, macrophages, and granulomas were observed. Although scattered inflammatory cell accumulations are commonly observed in untreated mice (Haschek et al., 2010), these were more frequent in the MWCNT-treated mice. Increased numbers and/or hypertrophy of Kupffer cells could be related to one of their functions in the liver, production of mediators of inflammation (Harrada et al., 1999). Hepatic focal necrosis, single cell necrosis, inflammatory changes and hyperplasia or hypertrophy of Kupffer cells have been observed in mice after intratracheal instillation of other nanoparticles (Hougaard et al., 2013; Saber et al., 2012) and in rats exposed by inhalation to nanoparticles (Sung et al., 2009; Ji et al., 2007). The histological hepatic effects of exposure to the two MWCNTs are similar in types of lesions induced, but CNTLarge exposure causes larger granulomas, and the incidence of vacuolar degeneration tended to be

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763 higher. Vacuolar degeneration was distributed throughout the whole

764 area of the hepatic lobule and its location did not change with time.

765 Also the eosinofilic necrosis of hepatocytes of a single row of hepato-

766 cytes surrounding the central vein was characteristic for livers from

767 the CNTLarge exposure groups. In general the histological changes were

768 not indicative of strong toxicity.

769 Although the gene expression patters were highly similar after expo-

770 sure to either CNT, our data also indicated subtle differences in gene ex-

771 pression and lipid concentrations following CNTSmall or CNTLarge

772 exposure. Only CNTSmall exposure resulted in changes in hepatic gene

773 expression at day 28 and the number of differentially expressed genes

774 was greater than that in the CNTLarge groups. CNTSmall caused an earlier

775 onset than CNTLarge in the down-regulation of the HMG-CoA reductase

776 pathway; the expression of Ldlr was differentially down-regulated

777 even at the lowest dose at days 1 and 3 following CNTSmall exposure

778 (Table 2). Exposure to CNTLarge, on the other hand, seemed to induce a

779 greater hepatic APR in accordance with the recorded morphological in-

780 flammatory changes. However, despite these functional differences and

781 different physicochemical properties, the responses following CNTSmall

782 and CNTLarge exposures were more similar than different.

783 MWCNTs are HARN (high aspect ratio nanoparticles) and have, as

784 such, been extensively discussed in relation to asbestos toxicity. Our re-

785 sults show that pulmonary exposure to MWCNT induces a pulmonary-

786 based systemic APR resulting in changes in cholesterol homeostasis and

787 liver morphology. We observed a close correlation between plasma

788 SAA3 levels and pulmonary Saa3 mRNA levels. We have previously

789 demonstrated a close correlation between pulmonary Saa3 levels and

790 neutrophil influx (Saber et al., 2013, 2014; Halappanavar et al., 2014;

791 Poulsen et al., 2014). The observed APR and increased cholesterol levels

792 link MWCNT exposure to risk of CVD. Since Teeguarden et al. (2011)

793 identified the same APR proteins in the lungs of mice following expo-

794 sure to MWCNTs or asbestos, this suggests that asbestos exposure is

795 likely to induce a similar pulmonary APR. In concordance with this, pro-

796 spective studies on smoking-adjusted, occupational asbestos exposure

797 have reported an association between exposure to asbestos and in-

798 creased risk of ischaemic heart disease (Harding et al., 2012; Sanden

799 et al., 1993). Thus, our findings in parallel with others suggest that

800 MWCNT exposure increases the risk of CVD. The present work has

801 established a correlation between pulmonary and systemic APR follow-

802 ing pulmonary exposure to MWCNTs, and suggests that pulmonary APR

803 can be used to group and rank different nanomaterials in relation to

804 CVD inducing potential.

805 Conclusion

806 We show that pulmonary exposure to two very different MWCNTs

807 induced very similar systemic APR, with induced alterations in serum

808 levels of APR proteins, cholesterol, LDL/VLDL and HDL, and changes in

809 hepatic gene expression and liver morphology. Furthermore, we found

810 a close correlation between plasma SAA3 levels and pulmonary Saa3

811 mRNA levels. Taken together, the results link pulmonary exposure to

812 MWCNTs with risk of CVD.

813 Supplementary data to this article can be found online at http://dx.

814 doi.org/10.1016/j.taap.2015.01.011.

815 Conflict of interests

816 The authors declare that they have no competing interests.

817 Funding

818 The project was supported by grants from the National Research

819 Centre for the Working Environment in Denmark and the Danish

820 NanoSafety Center, grant# 20110092173-3, the European Community's

821 Seventh Framework Programme (FP7/2007-2013) under grant agree-Q17;22 ment # 247989 (Nanosustain), and Health Canada's Chemical

Management Plan-2 Nano research funds and Genomics Research and 823 Development Initiative. The funders had no role in study design, data 824 collection and analysis, decision to publish, or preparation of the 825 manuscript. 826

Acknowledgments

The authors thank Michael Guldbrandsen, Lisbeth M Petersen, 828 Marianne Lauridsen, Lourdes Petersen and Elzbieta Christiansen for 829 their technical assistance. 830

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