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Journal of Occupational & Environmental Medicine:
doi: 10.1097/JOM.0b013e31821ad724
Epidemiologic Research: Original Article

Identification of Systemic Markers from A Pulmonary Carbon Nanotube Exposure

Erdely, Aaron PhD; Liston, Angie BS; Salmen-Muniz, Rebecca AAS; Hulderman, Tracy BS, MT; Young, Shih-Houng PhD; Zeidler-Erdely, Patti C. PhD; Castranova, Vincent PhD; Simeonova, Petia P. MD, PhD

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Author Information

From the Toxicology and Molecular Biology Branch (Dr Erdely, Dr Simeonova, Ms Liston, Ms Salmen-Muniz, Ms Hulderman), Pathology and Physiology Research Branch (Dr Young, Dr Zeidler-Erdely, Dr Castranova), and Laboratory of Occupational Cardiovascular Toxicology (Dr Erdely, Dr Castranova, Ms Salmen-Muniz, Ms Hulderman), Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WVa.

Deceased.

Address correspondence to: Aaron Erdely, PhD, NIOSH/HELD/TMBB, 1095 Willowdale Rd, MS-3014, Morgantown, WV 26505 (efi4@cdc.gov).

The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the National Institute for Occupational Safety and Health.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.joem.org).

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Abstract

Objective: Interest exists for early monitoring of worker exposure to engineered nanomaterials. Here, we highlight quantitative systemic markers of early effects after carbon nanotube (CNT) exposure.

Methods: Mice were exposed by pharyngeal aspiration to 40-μg CNT and harvested 24 hours, 7 days, and 28 days postexposure for measurements of whole blood, lung and extrapulmonary tissue gene expression, blood and bronchoalveolar lavage (BAL) differentials, and serum protein profiling.

Results: Early effects included increased inflammatory blood gene expression and serum cytokines followed by an acute phase response (eg, CRP, SAA-1, SAP). Beyond 24 hours, there was a consistent increase in blood and BAL eosinophils. At 28 day, serum acute phase proteins with immune function including complement C3, apolipoproteins A-I and A-II, and α2-macroglobulin were increased.

Conclusions: Carbon nanotube exposure resulted in measurable systemic markers but lacked specificity to distinguish from other pulmonary exposures.

Inhalation of airborne particles results in adverse cardiovascular outcomes in humans. In fact, epidemiological data shows that increased cardiovascular morbidity and mortality correspond to high levels of airborne particulate matter (PM), and at-risk populations appear to be more susceptible to these effects.1 In humans and animals, pulmonary exposure to PM results in increased atherosclerosis, impaired fibrinolysis, and reduced vascular function.1 Evidence also suggests that the smaller the particle, from PM10 to PM2.5 to PM less than 0.18 μm, the greater the cardiovascular risk.1,2 Consequently, these findings have led to the assessment of cardiovascular effects of other inhaled particles, particularly nanoparticles.

Carbon nanotubes (CNT) are engineered nanomaterials. Because of their small size, large surface area, and high reactivity, CNT are hypothesized to potentially elicit systemic effects if inhaled. Studies have shown significant endpoint effects directly related to cardiovascular disease, including vascular oxidative stress, increased prothrombotic potential, and progression of atherosclerosis, occur after exposure.3,4 Carbon nanotubes–related immune effects have also been described.5,6 A key mechanism proposed to contribute to these observed downstream effects of CNT is the release of soluble mediators from the lung into the circulation.6,7 To date, the pulmonary response to CNT is well described and is characterized by a granulomatous or interstitial fibrosis, dependent on the particle dispersion, inflammation, and biopersistence.814 Therefore, the potential exists not only to measure markers of the lung response but also to identify those that could promote endpoint extrapulmonary effects.

Currently there is expanding interest, from the perspectives of occupational health surveillance and future epidemiological research, in early monitoring of worker exposure to engineered nanomaterials including CNT.15,16 Recently, we showed that within 4 hours after a CNT exposure, systemic inflammation as indicated by whole blood cell gene expression occurred along with elevated inflammatory and procoagulant serum proteins.7 A generalized stress response in various extrapulmonary tissues, including acute sensitivity in the aorta, was also found. The systemic markers measured directly reflected the ongoing lung response to CNT.7 Here, we highlight quantitative systemic markers of early effects in mice from 4 hours to 28 days after a single CNT exposure. Results from NIOSH indicate that pulmonary and systemic responses are qualitatively similar in mice exposed to single-walled CNT (SWCNT) or multiwalled CNT (MWCNT). However, we observed that the MWCNT produced a greater magnitude of response than SWCNT at an equal mass dose.7 Therefore, our studies focused primarily on MWCNT.

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METHODS

C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) 10 weeks of age were used in this study. All mice were provided food (Teklad 7913) and tap water ad libitum in ventilated cages in a controlled humidity and temperature environment with a 12-hour light/dark cycle. Animal care and use procedures were conducted in accordance with the “PHS Policy on Human Care and Use of Laboratory Animals” and the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23, 1996). These procedures were approved by the National Institute for Occupational Safety and Health Institutional Animal Care and Use Committee.

MWCNT used in this study were obtained from Mitsui & Company, courtesy of Dr Endo, Shinshu University, Japan (MWNT-7; average 49 nm in diameter and 3.9 μm in length; 0.27% iron). Comparative data for SWCNT (Carbon Nanotechnologies, Inc, Houston, TX; 1 nm in diameter and 0.1 to 1 μm in length; 8.8% iron), at 24 hours postexposure only, will be included. Dispersion of CNT with the vehicle dispersion media (DM; phosphate buffered saline with 0.6 mg/mL serum albumin and 0.01 mg/mL 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and characterization (degree of dispersion and distribution of MWCNT lengths and widths) were described previously.7,12 Mice were exposed by pharyngeal aspiration17 with 40 μg of CNT in a total of 50 μl, and blood and tissues were harvested at 24 hour, 7 days, and 28 days postexposure. In our ongoing studies, both male and female mice have been studied with no observable sex differences. For the illustration of systemic markers, the data presented here utilized male mice at 24 hour (DM, n = 6; SWCNT, n = 5; MWCNT, n = 6), 7 days (DM, n = 6; MWCNT, n = 6) and in reference to 4 hour7 and female mice at 7 days (DM, n = 6; MWCNT, n = 6) and 28 days (DM, n = 6; MWCNT, n = 6) post-MWCNT exposure. Mice were sacrificed by carbon dioxide asphyxiation, and blood was collected for serum antigen analysis and whole blood messenger RNA expression. The lung, heart, aorta, and a consistent section of the liver were harvested and frozen in liquid nitrogen. All samples were stored at −80°C before analysis.

Measurements of blood and bronchoalveolar lavage (BAL) cell differentials by flow cytometry were done on separate groups of exposed mice (groups detailed in Results) by the following methods. Mice were sacrificed, and BAL was collected as previously described.18 Differential counts of BAL cells were done as previously described19 with the following modifications. The BAL cells were resuspended in 250 μL PBS, and 100 μL was added into a flow cytometry tube with 100 μL of 10% rat serum in FACS buffer for 10 minutes. Then, 50 μL of premixed antibodies in FACS buffer was added, and cells were stained for 30 minutes at room temperature with agitation. The mixture contained a final concentration of 5 μg/mL of the following antibodies: Fc block, Ly6G-FITC, Siglec-F-PE, CD45-PerCp, and CD11c-APC. All the antibodies were purchased from PharMingen (Becton Dickinson, San Diego, CA). The Caltag counting beads (PCB-100, Invitrogen, Carlsbad, CA) were added for cell enumeration before analysis in the FACSCalibur (BD Biosciences, San Diego, CA). Samples were acquired through a live gate without compensation. After collecting 4000 counting beads, the data of all cells were exported to the analysis software, FlowJo (Treestar, Costa Mesa, CA). The leukocytes were identified by cells that expressed CD45+. Neutrophils were defined as cells that expressed CD45+Ly6G+, eosinophils as CD45+Siglec-F+, and macrophages as CD45+CD11c+. For blood, collected in EDTA, 100 μL was added into a flow cytometry tube with 100 μL of 10% rat serum in FACS buffer for 10 minutes. Then, 50 μL of premixed antibodies in FACS buffer was added, and cells were stained for 30 minutes at room temperature with agitation. The mixture contained the following monoclonal antibodies in these final concentrations: MHC II-FITC (2.5 μg/mL, 2G9), Gr-1-APC (2 μg/mL, RBC-8C5), CCR3-PE (0.625 μg/mL, 83.101.111), CD3-Per-CP (10 μg/mL, 145–2C11), B220-Per-CP (2 μg/mL, RA3–6B2), and NK1.1-PE (2 μg/mL, PK136). All the antibodies were purchased from PharMingen (Becton Dickinson, San Diego, CA) except CCR-3, which was purchased from R&D Systems (Minneapolis, MN). To prevent nonspecific binding to Fc receptors, 2.4G2 blocking reagent (6 μg/mL) was added to the monoclonal antibody mix. Red blood cells were lysed with 100 μL of Caltag Cal-lyse lysing solution (GAS-010, Invitrogen, Carlsbad, CA) for 10 minutes in the dark followed by 1 mL of deionized water. The Caltag counting beads (PCB-100, Invitrogen, Carlsbad, CA) were added for cell enumeration before analysis in the FACSCalibur (BD Biosciences). Samples were acquired through a predefined gate in Cellquest, and the compensation was done afterward by FlowJo (Treestar, Costa Mesa, CA) analysis software. After collecting 3500 counting beads, the data of all cells were exported to FlowJo. The data were then analyzed according to the following gating strategy. First, leukocytes were separated by side scattering and forward scattering into three gates: lymphocytes, monocytes, and eosinophils plus neutrophils. Lymphocytes were identified by FSC/SSC and expression of CD3 or B220. B cells were distinguished from T cells by MHC-II expression in the lymphocyte gate. Eosinophils were defined as cells expressing the CCR3 receptor. Neutrophils were defined as those cells expressing the myeloid differentiation antigen Gr-1 and lacking CCR3. Monocytes were identified by FSC/SSC and expression of Gr-1.

Gene expression changes were measured as previously described utilizing the same custom designed TaqMan array profile (Supplemental Digital Content, Table S1, http://links.lww.com/JOM/A52).7 Serum antigen measurements were determined by Rules Based Medicine (Austin, TX) using the multiplex immunoassay RodentMAP v2.0. Total plasminogen activator inhibitor 1 (PAI-1) levels were determined by ELISA (Molecular Innovations). For PAI-1, male C57BL/6J mice (n = 6 vehicle and n = 6 MWCNT) were sacrificed 24 hour postexposure, and blood was collected into 3.2% sodium citrate at a 9 to 1 ratio, respectively. After centrifugation at 1500 g for 12 minutes, plasma samples were collected and frozen for PAI-1 determination.

Proteomics and subsequent analysis were performed by Protea Biosciences (Morgantown, WV) utilizing Isobaric Tags for Relative and Absolute Quantitation technology. Given the volume required for the analysis, a pooled serum sample from the sham (n = 6) was compared with serum from MWCNT treated mice (n = 6). The P value is representative of the effect of contributing peptide ratios (treated/sham) for a specific protein. This method was chosen not only because of sample volume limitations but also as a pilot approach to initially find treatment effects.20

All data are presented as means ± standard errors. Analyses were performed using JMP Statistical Discovery Software. Serum protein analysis and quantitative real-time reverse transcriptase polymerase chain reaction confirmation of the Taqman arrays and any additional genes were analyzed by one-way analysis of variance generating a least squares mean table by Student t test. Analysis of Taqman arrays was done by Student t test comparing only control to treatment. Differences were considered statistically significant at P < 0.05.

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RESULTS

Previously, our laboratory reported that cytokines and chemokines involved in inflammation including IL-6, IL-5, CCL11, CCL22, and CXCL1 were elevated in the serum 4 hour after CNT exposure.7 By 24 hours, these proteins returned to baseline and others were reduced compared to sham. At 24 hours, levels of acute phase proteins including C-reactive protein (CRP), haptoglobin, and serum amyloid P (SAP) were increased in the serum (Table 1). Further analysis showed significant elevations of serum amyloid A1 (SAA-1), SAP, and haptoglobin gene expression in the liver (Fig. 1), which confirmed an acute phase response. At 24 hours, proteins associated with activation and recruitment of macrophages such as CCL7 and colony stimulating factor 1 (CSF1—macrophage) and neutrophil and lymphocyte chemoattractants, CXCL2 and lymphotactin, respectively were reduced with MWCNT exposure (Table 1). Plasma levels of plasminogen activator inhibitor 1 (PAI-1), a procoagulant cardiovascular risk marker that inhibits plasminogen activator thereby reducing the conversion of plasminogen to plasmin and resultant fibrinolysis,21 were shown to be elevated 4 hours post-CNT exposure7 and remained increased at 24 hours (1.12 ± 0.06 ng/mL DM vs 1.74 ± 0.12 MWCNT; P < 0.01). Lung particulate exposure data have shown examples of both increased PAI-1 and reduced plasminogen activator systemically,7,2224 indicating that this pathway is acutely affected. At 4 hours postexposure, the ratio of matrix metalloproteinase 9 (MMP-9), an extracellular matrix remodeling protein, to tissue inhibitor of metalloproteinase 1 (TIMP-1) showed an increasing trend in the MWCNT-exposed mice (160 ± 29 DM; 104 ± 7 SWCNT; 268 ± 81 MWCNT) because of increased levels of MMP-9.7 At 24 hour, MMP-9 levels had returned to control levels in the MWCNT group while TIMP-1, a primary inhibitor of MMP-9,25 was elevated in both the SWCNT and MWCNT groups (Table 1), likely in a compensatory mechanism. This significantly reduced the ratio of MMP-9 to TIMP-1 (187 ± 43 DM; 99 ± 16* SWCNT; 50 ± 2* MWCNT; *P < 0.05). There was a significant time-dependent effect with respect to MMP-9, TIMP-1, and the ratio from 4 to 24 hours in mice exposed to MWCNT. Alterations in circulating levels of MMP-9, TIMP-1, and/or the MMP-9/TIMP-1 ratio are implicated in the pathogenesis of cardiovascular disease including left ventricular remodeling, atherosclerotic plaque stability, and inflammatory cytokine production.2631

Table 1
Table 1
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Figure 1
Figure 1
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Utilizing a custom designed TaqMan array (Supplemental Digital Content, Table S1, http://links.lww.com/JOM/A52), aorta gene expression levels, elevated at 4 hours, were reduced or returned to baseline by 24 hours (Supplemental Digital Content, Table S2, http://links.lww.com/JOM/A52). Levels of metallothionein 1 (MT-1) and hypoxia inducible factor 3 alpha (Hif-3α) remained elevated at 24 hours after MWCNT exposure. TIMP-4 was increased at both 4 and 24 hours in SWCNT exposed mice (Supplemental Digital Content, Table S2, http://links.lww.com/JOM/A52). In the MWCNT groups, TIMP-4 showed further induction at 24 hours compared to 4 hours. Analysis of gene expression from the heart and liver at 24 hours also showed reduced levels for genes elevated at 4 hours (Supplemental Digital Content, Table S2, http://links.lww.com/JOM/A52).

Previous data from isolated whole blood cell RNA showed that at 4 hours after MWCNT exposure, several stress response and inflammation-related genes were increased.7 We applied the same custom-designed TaqMan array and found that none of the ∼100 genes tested were elevated at 24 hours (data not shown). Additional analysis of blood differentials was examined at all time points, and a consistent feature was an increase in eosinophils. This occurred after 24 hours lasting through 28 days and was most prominent at 3 to 7 days postexposure (Fig. 2). In the BAL, increased eosinophils were found at 24 hour (data not shown), which could explain the initial decline in blood eosinophils. Reflecting the consistent increase in blood eosinophils, BAL analysis by flow cytometry showed at 7 days eosinophils comprise 50% of the lavage cells by differential counts (Fig. 2). This was confirmed by manually counted cytospins, which also showed more than 50% of cells were eosinophils (data not shown). At 28 days post MWCNT exposure eosinophils in the BAL remained elevated, ∼15 fold greater than sham mice (Fig. 2). Regarding other cell types, at 24 hours there was a significant decrease in total lymphocytes and monocytes that returned to sham levels by 3 days (Table 2). Blood neutrophils were increased in males at 4 hours,7 3 days and in 7 days females, but not in the other groups (Table 2), suggesting this measurement was not a consistent marker of exposure.

Table 2
Table 2
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Figure 2
Figure 2
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Comparison of gene expression changes in the lung between 4 and 24 hours post-CNT exposure is shown in Supplemental Digital Content, Table S3 (http://links.lww.com/JOM/A52). Significant inflammation was observed at 4 hours and was maintained through 24 hours with a greater response in MWCNT-exposed compared with SWCNT-exposed mice. Several genes related to macrophage function (eg, CCL2, osteopontin, and arginase I) were increased at 24 hours compared with 4 hours. Macrophage-dependent gene expression was more prominent at 7 days when compared with 4 hours and 28 days (data not shown). Lactate dehydrogenase (LDH) activity, a marker of cellular toxicity, was significantly increased by CNT exposure in a time dependent fashion (Supplemental Digital Content, Fig. S1, http://links.lww.com/JOM/A52).

By 28 days, primary inflammatory serum proteins, PAI-1, and blood gene expression returned to baseline levels (data not shown). Subsequent serum proteomic analysis showed increased levels of acute phase proteins associated with inflammation and the innate immune response such as complement C3 (C3), apolipoproteins A-1 and A-II, hemoglobin subunits alpha and beta-1, alpha-2-macroglobulin (A2M), serotransferrin, and liver carboxylesterase N (LCN) (Table 3). The same proteins were elevated following MWCNT exposure in a separate ongoing study, thus strengthening these initial observations (data not shown).

Table 3
Table 3
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DISCUSSION

Rapidly following a pulmonary exposure to CNT, we found that the response of the lung was translocated to the periphery via the blood. This response was measured by increased inflammatory whole blood gene expression and increased circulating factors including primary cytokines, chemokines, and markers of coagulation. Many observed changes returned to baseline by 24 hours with a subsequent rise in systemic inflammatory markers, such as acute phase proteins. This also occurred in the extrapulmonary tissues, which showed an early stress response followed by a resolution. The presence of eosinophils was a consistent feature in the BAL and blood following exposure. Beyond the acute systemic inflammatory response, serum proteomics data revealed markers of an ongoing systemic inflammatory response related to an innate immune response 1 month after a single exposure. Taken together, our data suggest that a systemic signature results from a single CNT exposure. The early effects we measured, however, were not unique in comparison to other exposures, such as PM.

To date, several studies have shown systemic endpoint effects following pulmonary CNT exposure. These effects include vascular oxidative stress, increased progression of atherosclerosis, enhanced prothrombotic potential, and immunosuppression.36 These studies, along with the known pulmonary fibrotic and allergic effects,814,3234 could provide the ability to systemically monitor effects of CNT exposure. Our initial studies exposed mice to 40 μg of CNT. This is equivalent to approximately 4 months of exposure12 utilizing peak measurements from a research laboratory of 400 μg/m3;35 thus, it was a high dose exposure, but representative of that currently used in the literature. Specifically, this dose was used as a positive control to verify the initiation of a systemic response and potential markers of exposure. Furthermore, because of the biopersistence of CNT, it cannot be assumed that lower doses over a longer period of time would not initiate similar responses.

Early effects of CNT exposure increased serum proteins of well-established markers of systemic inflammation and cardiovascular disease.7 These included IL-6 with subsequent elevated levels of acute phase proteins, such as CRP and SAA-1. Although serum SAA-1 levels were not determined because of the lack of a specific SAA-1 ELISA, it was the most prominent of the measured acute phase genes expressed in the liver and, therefore, circulating levels were likely increased. The systemic inflammatory markers, although CNT-nonspecific because they are also increased after PM exposure,1 could directly promote negative cardiovascular outcomes. For example, all of these markers are known to be associated with the development, progression, and/or stability of atherosclerotic plaques.36 In addition, both vascular dysfunction and prothrombotic potential were eliminated following PM exposure in mice lacking IL-6.37,38 C-reactive protein directly quenches nitric oxide thereby promoting vascular dysfunction.39,40 Also, serum amyloid A induced endothelial dysfunction by increasing reactive oxygen species and decreasing endothelial nitric oxide synthase.41 Therefore, the endpoint measurements of vascular oxidative stress, increased progression of atherosclerosis, and enhanced coagulation potential following CNT exposure could be proposed from the systemic inflammatory response markers.

A response of interest was the marked eosinophil influx into the lung. This response was predicted by increased markers of eosinophil recruitment and activation, which included IL-5 and CCL11 in the lung and serum.7 Most particle exposures, such as PM, silica, and welding fume, induce a neutrophil-dominated lung response, although some studies have shown an increase in eosinophils.4246 Similar to CNT, asbestos exposure can induce a marked eosinophil response,47,48 which may be the result of a similarity in physical properties. Recent studies have shown that eosinophils play an important role in the early development of allergic airway inflammation.49 In addition, CNT pulmonary exposure enhanced an allergic inflammatory response.3234 Therefore, the data suggests that CNT exposure may not only exacerbate, but potentially induce allergic airway inflammation.

Within hours after CNT exposure, alterations in inflammatory blood gene expression were evident.7 By 24 hours, blood gene expression changes from our panel had returned to baseline. This suggests a rapid and transient effect, but however, does not include changes that could have been discovered by global gene expression analysis. In parallel, a reduction of acute stress response genes seen in various extrapulmonary tissues was evident when comparing the response at 4 and 24 hours. Interestingly, we found increased TIMP-4 in the aortas of CNT-exposed mice at both 4 and 24 hours. TIMP-4, with suggested specificity to cardiovascular tissues,50 was recently proposed as a systemic marker for vascular inflammation.51 Cardiovascular disorders in both human and animal models including atherosclerosis, arterial balloon injury, and heart allograft rejection all showed increased TIMP-4.50,51 Therefore, the early and sustained expression of TIMP-4 in the aorta following CNT exposure was likely a surrogate marker for a vascular inflammatory response.

In this study, we found a select group of acute phase proteins linked to activation of the immune response at 28 days after MWCNT exposure. With regard to biomedical applications, studies have shown direct complement activation by CNT.52,53 Therefore, in the lung, CNT have the potential to directly activate complement in a similar manner especially if translocation occurs. Also, it is possible that the systemic inflammatory response was the result of increased C3 levels. While the mechanisms regarding changes in C3 should be explored, increased levels were found in the serum of individuals exposed to high levels of PM5456 and were associated with the development of diabetes and cardiovascular disease.5759 Furthermore, increased C3c, a marker of subclinical inflammation and a cleavage product resulting from activation of C3, was an independent predictor of PM associated risk of diabetes.60 Apolipoproteins A-I and A-II have anti-inflammatory actions on circulating leukocytes and protect endothelial cells lining the vascular wall from complement activation.6163 Also, if serum SAA-1 levels were increased, as predicted by liver gene expression, SAA-1 could displace apolipoproteins A-I and A-II creating an acute phase HDL resulting in a proatherogenic state.64,65 The hemoglobin subunits were increased possibly as a reflection of the hemolytic activity of complement. Liver carboxylesterase N and A2M also have immune functions related to surfactant. Liver carboxylesterase N cleaves surfactant protein B converting more active large to less active small aggregate surfactant. This action is considered pathologic in acute inflammation.66 Alpha-2-macroglobulin represents a conserved arm of the innate immune system that inactivates proteinases (eg, MMP-9) and decreases surfactant protein D degradation to increase innate immune function.67,68 Lastly, transferrin has a well-characterized immune function of iron binding. Therefore, at 28 days postexposure, a group of acute phase proteins were increased that suggested immune activation.

In summary, exposure to CNT results in a measurable systemic inflammatory response. As summarized in Table 4, early effects include increased serum levels of primary cytokines and inflammatory gene expression in blood cells. This was followed by a reduction in the initial inflammatory markers and a predicted acute phase response. Beyond 24 hours postexposure, a consistent eosinophilic response as well as a series of proteins related to immune activation was evident. The markers correlated well with existing literature showing endpoint measurements of pulmonary CNT exposure mainly related to adverse cardiovascular effects. Of note is the general lack of specificity of the markers. Many of the markers (eg, IL-6, acute phase proteins, PAI-1) would not be easily separated from other pulmonary exposures. Therefore, additional studies are underway to determine the potential of a specific systemic signature of CNT exposure, which will aid in the early monitoring of human exposure.

Table 4
Table 4
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