Tangled Multi-Walled Carbon Nanotubes

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biochemical or cellular interactions

Adequacy of study:

other information

Principles of method if other than guideline:

Examination of the effects of MWCNT exposure on oxidative stress and monocyte adhesion to human umbilical vein endothelial cells (HUVECs), with emphasis on the interaction with palmitic acid (PA).

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: oxidative stress and monocyte adhesion

Species:

other: Human umbilical vein endothelial (HUVECs), monocytic THP-1 and type II pneumocytes (A549) cells

Details on test animals or test system and environmental conditions:

- Sources
HUVECs: Invitrogen (Carlsbad, CA), cultured in Endothelial Cell Growth Medium Kit with 2% serum
Monocytic cell line THP-1 and type II pneumocytes (A549): American Type Culture Collection (Manassas, VA), grown in RPMI or F-12 medium with 10% serum
THP-1 cells were differentiated into adherent macrophage-like cells (denoted THP-1a) by treatment with 10 ng/ml phorbol 12-myristate 13-acetate overnight. PMA-differentiated THP-1 macrophages resemble primary human monocyte-derived macrophages.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
MWCNT stock solution was prepared by sonicating a 2.56 mg/ml suspension of particles in double-distilled water containing 2% bovine serum, using a Branson Sonifier S-450D equipped with a disrupter horn before each experiment. The suspension was sonicated continuously for 16 min at amplitude of 10% and cooling on ice to avoid sample heating and evaporation. After sonication, the suspension was diluted and used only once.
On the day of use, 5mM
Palmintic acid (PA) suspension was made in Hanks solution containing 10% BSA at 37C for about 60 min including vortexing for 5–10 min, and then further diluted in HUVEC medium to a final concentration of 100 mM for exposure.

Details on analytical verification of doses or concentrations:

The structure of MWCNTs in suspension before and after sonication was investigated by scanning electron microscopy.
To determine an approximate size of the particles in suspension, analysis was performed with a NanoSight LM20 in triplicates on three independent days.

Remarks:

Doses / Concentrations:
0, 2, 4, 8, 16 and 32 µg/ml
Basis:

Examinations:

In this study, the effects of MWCNT exposure on oxidative stress and monocyte adhesion were examined on human umbilical vein endothelial cells
(HUVECs), with emphasis on the interaction with PA, whereas oxidative stress measured as intracellular ROS and glutathione (GSH) level was determined in HUVECs and A549 monocultures. In addition, an alveolar– endothelial Transwell co-culture model with two compartments separated by a porous membrane was used to assess the monocyte adhesion to HUVECs in the lower chamber with the presence of PA, and induced by MWCNT exposure to A549 epithelial cells and THP-1-derived macrophages in the upper compartment. The protein level of tumor necrosis factor (TNF), interleukin (IL)-6 and IL-8 was measured in the medium from the lower chamber to indicate inflammatory responses. The adhesion of THP-1 mono- cytes to HUVECs was used as a major endpoint since the recruitment of monocytes is an important characteristic in the pathogenesis of atherosclerosis, and increased monocyte adhesion to HUVECs has been observed by in vitro exposure to, e.g., wood smoke particles as well as MWCNTs.

Positive control:

None

Cytotoxicity and proliferation ability

There was very low cytotoxicity when cells were exposed to both NM400 and NM402, the detailed statistical analysis is shown and described in Supplemental Figures S5 (WST-1) and S6 (proliferation).

Intracellular ROS levels

The increase in intracellular ROS levels induced by MWCNT exposure is shown in Figure 2. The exposure to NM400 was associated with substantially increased ROS at all concentrations (p<0.05), whereas the NM402 only increased the ROS levels at 32 mg/ml (p<0.05) (Figure 2A), and this increase was far lower than for NM400.

The ROS level was further assessed by fluorescence microscopy of HUVECs exposed to 8 mg/ml MWCNT with or without PA in the cell medium (representative images are shown in Supplemental Figure S7). Only NM400 increased the ROS levels in the presence of PA (p<0.05), NM402 had no effect on the levels of ROS (Figure 2B). For A549 monocultures, the exposure to NM400 was associated with increased ROS levels at all concentrations (p<0.05), whereas the NM402 did not significantly affects ROS levels (Figure 2C).

Intracellular-reduced GSH

Figure 3 shows the effect of MWCNT exposure on the intracellular GSH concentrations. The GSH level was significantly reduced in HUVECs exposed to NM400 (8–32 and 16–32 mg/ml with or without PA, respectively, p<0.05). There was significantly reduced intracellular GSH concentration at all concentrations of NM402 (p<0.05), which was independent of the presence of PA (Figure 3A). For A549 cells, the exposure to both NM400 and NM402 significantly decreased the GSH concentration at all concentrations (p<0.01) (Figure 3B).

Adhesion assay

Figure 4(A) shows the THP-1 adhesion to HUVECs after 24 h direct exposure to MWCNTs. The adhesion followed a bell-shaped concentration–response curve with increased adhesion at the middle concentrations, whereas the high concentrations (16 and 32 mg/ml) showed lower adhesion.

The addition of the antioxidant NAC or IL-6 as a co-factor during exposure to the MWCNTs did not significantly change the adhesion of THP-1 cells to the HUVEC cells (Supplemental Figure S9).

Microscopy (Supplemental Figure S8) showed that the presence of PA increased the adhesion of THP-1 cells to HUVECs (p<0.05). The exposure to NM400 and NM402 also increased the adhesion (p<0.05), whereas there was no interaction between PA and particle treatment in the full-factorial ANOVA (Figure 4B).

Figure 4(C) shows the results from experiments of the Transwell co-culture system where the MWCNTs were applied to the inserts with or without A549 and/or THP-1a cells. The lower chamber contained HUVECs and THP-1 cells in medium with PA. The presence of A549 monolayer in the upper chamber did not affect the adhesion of THP-1 cells to HUVECs in the lower chamber, whereas there was an interaction between the presence of THP-1a cells and the exposure to MWCNTs (p<0.05). The presence of THP-1a cells increased the NM400- and NM402-mediated adhesion of THP-1 cells to HUVECs in the lower chamber (p<0.05). The addition of 100 ng/ml of TNF to the inserts with A549 monolayer was associated with a 70% increased THP-1 adhesion to HUVECs (p<0.05; results not shown).

Release of cytokines

The concentration of secreted IL-6 and IL-8 was higher in the lower chamber of the Transwell co-culture system when THP-1a cells were present in the upper chamber (Figure 5). The statistical analysis indicated that the IL-8 response depended on the type of cell culture on the inserts, with strongest responses with THP-1a cells present (p<0.05, nested ANOVA). The exposure to NM400 with THP-1a cells present was associated with a 33% increase in IL-8 content in the lower chamber (41% (95% confidence interval: 16–66%) higher IL-8 when based on the fold difference in the nested ANOVA) (Figure 5B). There was a similar response on the level of IL-6 as for the level of IL-8, although this was only of borderline statistical significance in the overall nested ANOVA (p=0.06). Nevertheless, the post hoc test indicated that exposure to NM400 resulted in 11% (95% CI: 1–21%) higher IL-6 level in the lower chamber of the experimental system when only THP-1a cells were on the insert. The TNF concentrations in medium from the lower chamber were all below the detection limit (1.2 pg/ml).

Epithelial barrier function

The transepithelial electrical resistance (TEER) development was followed during 8 d of culturing of A549 cells in the Transwell co-culture system to assess the barrier function. It reached a plateau at day 7 of the culture (Supplemental Figure S2). This was further evidenced by assessment of the permeability of FITCdextran after 8 d of culturing where the percentage of translocation of FITC-dextran was 2.4±0.18% and 0.6±0.05% for inserts without and with A549 monolayer, respectively (p<0.001, Supplemental Figure S3). The TEER value of the A549 monolayer on the inserts increased during the co-culturing with HUVECs and THP-1 cells (p<0.001), whereas the exposure to MWCNTs or TNF had no significant effect on the TEER value (p=0.58) (Figure 6).

Executive summary:

Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particlemediated cardiovascular health effects. The presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. The interaction between direct exposure to MWCNTs and PA was investigated on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar–endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs (NM400 (Nanocyl) and NM402 (Graphistrength C100)) type of entangled fibers, was associated with elevated levels of reactive oxygen species (MN400) as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures (both MWCNTs). Both effects were found to be independent of the presence of PA. NM 400 exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.

Reference 2

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The potential of Graphistrength C100 to induce cytotoxicity, measured by mitochondrial function using the WST-1 assay, and to induce an inflammatory response, measured by the release of pro-inflammatory cytokines (IL-6, IL-8 and TNF-a) was investigated in the C3A and primary human hepatocytes. Albumin was measured following exposure of the cells to the Graphistrength C100 as an indicator of hepatic functionality. The interaction of the Graphistrength C100 with the cells was investigated by the use of transmission and scanning electron microscopy (TEM and SEM).

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

other: hepatotoxicity

Vehicle:

other: MilliQ de-ionised water with 2% FCS. The MWCNT were sonicated for 16 min without pause

Details on exposure:

The human hepatoblastoma C3A cell line was obtained from the American Type Culture Collection (ATCC; Manassas, V A, USA). The cells were maintained in Minimum Essential Medium Eagle (MEM) (Life Technologies} with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillln/streptomycin, 1 mM sodium pyruvate and 1% non-essential acids, at 37"C and 5% C02. The primary human hepatocytes were purchased from Life Technologies. The cells were maintained in Wllliams E medium with 5% FCS, 10 µM dexamethasone in dimethyl sulfoxide (DMSO), 100 U/ml penicillin/streptomycin, 4 µg/ml bovine insulin, 2 nM glutamax and 15 mM of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), at 37°C and 5% C02.

NMs were dispersed in Milli-Q de-ionised water with 2% FCS. The NMs were sonicated for 16 min without pause following the protocol developed for ENPRA (Jacobsen et al. 2010). Following the sonication step, all samples were immediately transferred to ice water.

Analytical verification of doses or concentrations:

no

Remarks:

10 concentrations between 0.16 and 80 µg/cm² (corresponding to 0.5-256 µg/ml)

Details on results:

Graphistrength C100 was relatively non-toxic to the C3A cells and to primary human hepatocytes, LC50 was not reached up to 80 µg/cm2 after a 24 h exposure. Graphistrength C100 induces the production of the proinflammatory cytokine IL-8, but only at relatively high exposure concentrations (>= 20 µg/cm²). No change in the levels of IL-6 or TNF-a following exposure of the C3A cells and human hepatocytes was observed suggesting that the inflammatory response is limited. Albumin as measures of liver function was unaffected in primary human hepatocytes.

Impact of MWCNT on C3A cell and primary human hepatocytes viability:

MWCNT NM 402 was considered to be low toxicity material as the LC50 was not reached up to 80 µg/cm2 after a 24 h exposure to the C3A cells and primary human hepatocytes (Figure 1).

Impact of MWCNT on C3A cells and primary human hepatocytes IL-8 production:

Changes in cytokine production as a consequence of NM 402 exposure were assessed within the supernatant of exposed C3A cells and human hepatocytes and quantified via ELISA. The IL-8 production increased in a dose-dependent manner, reaching statistical significance compared with the control at high exposure concentrations (Figure 2).

Impact of the MWCNT on C3A cells and human hepatocytes IL-6 and TNF- a production:

Secretion of IL-6 and TNF-a into the supernatant of exposed C3A cells and human hepatocytes was quanti¿ed using ELISA analysis. There was no signi¿cant increase or decrease in the production of IL-6 or TNF-a after the exposure to NM 402 (data not shown).

Impact of MWCNT on albumin production by primary hepatocytes:

In order to establish whether any of the NM 402 affected albumin production, four exposure concentrations were chosen (80, 40, 20, and 10 µg/cm2). Following a 24 h exposure period, there was no significant decrease in levels of albumin secreted by hepatocytes (Figure 3).

 

Interaction of cells and the nanomaterials

NM 402 was found in the cytoplasm and the nucleus of both the cell line and the primary cells.

 

Executive summary:

The potential of NM 402 (Graphistrength C100° to induce cytotoxicity, measured by mitochondrial function using the WST-1 assay, and to induce an inflammatory response, measured by the release of pro-inflammatory cytokines (IL-6, IL-8 and TNF-a) was investigated in the C3A and primary human hepatocytes. Albuminwas measured following exposure of the cells to the NM 402 as an indicator of hepatic functionality. The interaction of the NM 402 with the cells was investigated by the use of transmission and scanning electron microscopy (TEM and SEM). MM 402 was relatively non-toxic to the C3A cells and to primary human hepatocytes, LC50 was not reached up to 80 µg/cm² after a 24 h exposure. NM 402 induces the production of the proinflammatory cytokine IL-8, but only at relatively high exposure concentrations (>= 20 µg/cm²). No change in the levels of IL-6 or TNF-a following exposure of the C3A cells and human hepatocytes was observed suggesting that the inflammatory response is limited. Albumin as measures of liver function was unaffected in primary human hepatocytes.

Reference 3

Endpoint:

hepatotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

In vitro investigation of the potential of Graphistrength C100 to induce cytotoxicity, measured by mitochondrial function using WST-1 and Alamar Blue assays, and to induce an inflammatory response in the hepatoblastoma cell line C3A, measured by the release of proinflammatory cytokines (IL-6, IL-8 an d TNF-a). C-reactive protein (CRP) levels were also measured following hepatocyte exposure to the Graphistrength C100. As indicators of hepatocyte function, the effects of Graphistrength C100 exposure were investigated on albumin and urea production.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: hepatotoxicity

Vehicle:

other: Milli-Q de-ionised water with 2% FCS. The NMs were sonicated for 16 min without pause following the protocol developed for ENPRA

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The human hepatoblastoma C3A cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were maintained in Minimum Essential Medium Eagle (MEM, Invitrogen, Paisley, UK) with 10% foetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate and 1% nonessential amino acids (termed complete medium), at 37C and 5% CO2.

NMs were dispersed in Milli-Q de-ionised water with 2% FCS. The NMs were sonicated for 16 min without pause following the protocol developed for ENPRA (Jacobsen et al. 2010; Jensen et al. in preparation). Following the sonication step, all samples were immediately transferred to ice water.
To ascertain the toxicity of NMs to C3A cells 10 concentrations between 0.16 and 80 mg/cm2 were utilised (corresponding to 0.5–256 mg/ml) by diluting in medium containing 10% FCS.

Remarks:

10 concentrations between 0.16 and 80 µg/cm2 (corresponding to 0.5–256 µg/ml)

Details on results:

Graphistrength C100 was relatively non-toxic to the C3A cells at the times and concentrations tested. Graphistrength C100 induces the production of the proinflammatory cytokine IL-8, but only at relatively high exposure concentrations (>= 20 µg/cm²). No change in the levels of IL-6, TNF-a or CRP following exposure of the C3A cells was observed suggesting that the inflammatory response is limited. Both urea and albumin as measures of liver function were unaffected.

Impact of MWCNT on C3A cell viability:

MWCNT NM 402 was considered to be low toxicity material as the LC50 was not reached up to 80 µg/cm2 after a 24 h exposure to the C3A cells (Figure 1).

Impact of the engineered MWCNT on C3A hepatocyte IL-8 production:

Changes in cytokine production as a consequence of NM 402 exposure were assessed within the supernatant of exposed hepatocytes and quantified via ELISA. The IL-8 production increased in a dose-dependent manner, reaching statistical significance compared with the control at high exposure concentrations (Figure 1).

Impact of the MWCNT on C3A hepatocyte IL-6, TNF-a and CRP production:

Secretion of IL-6, TNF-a and CRP into the supernatant of exposed C3A hepatocytes was quanti¿ed using ELISA analysis. There was no signi¿cant increase or decrease in the production of IL-6, TNF-a or CRP after the exposure of the C3A cells to NM 402 (data not shown).

Impact of MWCNT on urea and albumin production by C3A hepatocytes:

In order to establish whether any of the NM 402 affected urea and albumin production, four exposure concentrations were chosen (80, 20, 2.5 and 0.31 µg/cm2). Following a 24 h exposure period, there was no significant modification of the urea production and decrease in levels of albumin secreted by hepatocytes (Figure 2).

Conclusions:

MM 402 was relatively non-toxic to the C3A cells at the times and concentrations tested. NM 402 induces the production of the proinflammatory
cytokine IL-8, but only at relatively high exposure concentrations (>= 20 µg/cm²). No change in the levels of IL-6, TNF-a or CRP following exposure of the C3A cells was observed suggesting that the inflammatory response is limited. Both urea and albumin as measures of liver function were unaffected.

Executive summary:

The potential of NM402 (Graphistrength C100) to induce cytotoxicity, measured by mitochondrial function using WST-1 and Alamar Blue assays, and to induce an inflammatory response, measured by the release of proinflammatory cytokines (IL-6, IL-8 and TNF-a) was investigated in the hepatoblastoma cell line C3A. C-reactive protein (CRP) levels were also measured following hepatocyte exposure to the NM402. As indicators of hepatocyte function, the effects of NM402 exposure were investigated on albumin and urea production. NM 402 was relatively non-toxic to the C3A cells at the times and concentrations tested. NM 402 induces the production of the proinflammatory cytokine IL-8, but only at relatively high exposure concentrations (>= 20 µg/cm²). No change in the levels of IL-6, TNF- a or CRP following exposure of the C3A cells was observed suggesting that the inflammatory response is limited. Both urea and albumin as measures of liver function were unaffected.

Reference 4

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

The proliferative response to Graphistrength C100 was assessed on mouse primary lung fibroblasts. Cell proliferation was assessed by counting the number of living cells after 24 h exposure.

GLP compliance:

no

Type of method:

in vitro

Species:

mouse

Strain:

other: C57BL/6

Sex:

female

Details on exposure:

Fibroblast exposure:
After 24 h at 37°C in complete culture medium (10% FBS), cells were washed once with their basal medium (no FBS, no AA) and then supplied with 200 µl (96-well culture plate) or 1 ml (24-well culture plate) medium with 1% AA containing 1/10 material dilutions. Cells were exposed during 24 h to 1 to 37.5 µg MWCNT or equivalent/cm2.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

24 hours

Remarks:

Doses / Concentrations:
1 to 37.5 µg MWCNT/cm2
Basis:

Details on study design:

Primary mouse lung fibroblasts were obtained from eight to twelve-week old female C57BL/6 mice (local breeding facility, Animalerie Centrale, Université catholique de Louvain, Brussels, Belgium), housed in positive pressure air-conditioned units (25°C, 50% relative humidity) on a 12 h light/dark cycle with free access to water and laboratory animal food. Mice were euthanized with an injection of pentobarbital (15 mg/mouse intraperitoneally, Certa, Braine-l'Alleud, Belgium). Whole lungs were perfused with phosphate buffered saline (PBS) via the right ventricle. Lungs were minced with scissors for 4 min and then digested in 9 ml Hank's buffered salt solution (HBSS), 0.8 ml Liberase TH (5 mg/ml) and 0.25 ml DNase (1 mg/ml) at 37°C during 35 min under gentle agitation. The resulting cell suspension was filtered (70 µm), resuspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibioticantimycotic (AA), transferred into a flat tissue culture flask and incubated at 37°C under 5% CO2. After 1 day, cells were washed with 10 ml DMEM and supplemented with fresh DMEM, 10% FBS, 1% AA. Cells were harvested 6 days later by adding 3 ml of 0.25% trypsin-EDTA and incubated for 10 min at 37°C. Trypsin was neutralized by adjunction of DMEM, 10% FBS and 1% AA. Cell suspensions were centrifuged for 10 min at 400 g and resuspended in 5 ml fresh DMEM, 10% FBS, 1% AA. Cell number and viability were determined with Trypan Blue Solution. Cells were seeded into 96-well or 24-well culture plates in complete medium (10% FBS) at a density of 30 000 or 180 000 cells/well, respectively, and incubated at 37°C under 5% CO2 for 24 h. Primary fibroblasts were sub-cultured for a maximum of 3 passages and used at passages 2 and 3 for cell purity).

Examinations:

Proliferation assays:
Cell proliferation was assessed by cell counting, WST-1 assay and propidium iodide staining. For cell counting, cells exposed for 24 h to test materials in 96-well plates were washed twice with their basal medium and then incubated with 25 µl 0.25% trypsin-EDTA for 10-15 min at 37°C. Trypsin was neutralized by adjunction of 100 µl of complete medium and cell suspensions were transferred and kept on ice. Cell number and viability were determined with Trypan Blue Solution on a B¿rker cell. Data are presented as relative cell count compared to the non-exposed cells.

Positive control:

Exposure to human platelet-derived growth factor (PDGF-BB) at the indicated final concentration was used as a positive control for proliferation assays.

Details on results:

Graphistrength C100 significantly increased cell number in a dose-dependent manner and the highest concentration induced an effect similar to PDGF.

Executive summary:

The proliferative response to Graphistrength C100 was assessed on mouse primary lung fibroblasts. Cell proliferation was assessed by counting the number of living cells after 24 h exposure to 1 to 37.5 µg/cm². Graphistrength C100 significantly increased cell number in a dose-dependent manner and the highest concentration induced an effect similar to human platelet-derived growth factor (positive control).

Reference 5

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

may 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

In order to evaluate the potential adverse effects of Graphistrength C100, a comprehensive approach was used, aimed to evaluate both cytotoxic effects and the underlying mechanisms (apoptosis, proliferation, oxidative stress, internalization).

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: cytotoxicity

Species:

other: A 549 Human alveolar epithelial cells and MT5A mesothelial cells

Remarks:

Doses / Concentrations:
0.1, 1, 10 and 100 µg/ml
Basis:

Details on study design:

The effects of MWCNTs were compared to those of two types of asbestos fibers-chrysotile (20 nm diameter) and crocidolite (80 nm diameter) and to nanosized carbon black (CB).
MWCNT were suspended at 10 mg/ml in aqueous (dipalmitoyl lecithin) DPL, PBS, or EtOH.

Cell Culture and Stimulation:
Human alveolar epithelial cells (A549 cell line) and mesothelial cells (MeT5A cell line) were cultured, seeded in 96-well plates at 60.000 cells/ml, and grown to confluence (48 to 72 h later). Cells were exposed for 6, 24, 48, or 72 h to serum-free medium, 0.1 to 100 µg/ml (0.02-20 µg/cm2) of MWCNT, or 100 µg/ml (20 µg/cm2) asbestos fibers or CB nanoparticles.

As MeT5A cells were used only to verify the main results obtained with A549 cells; these cells were exposed only to DPL-suspended MWCNT at different concentrations (0.1 to 100 µg/ml).

Examinations:

Assessment of Cell Viability:
Two methods were used to evaluate changes in cell viability: MTT, and neutral red assays.
Results of cell viability are expressed as the means of at least three independent experiments, each of six replicates, given as the ratio of the mean foreach condition to the mean of the control condition (cells cultured in media containing the respective suspension agent). Since nanomaterials might interfere with cytotoxicity tests, the assays were performed with and without 100 µg/ml MWCNT during incubation with the dye and measured absorbance. No interference of MWCNT with MTT and neutral red assays was observed.

Assessment of Cell Number:
Cell number was assessed by quantifying DNA content with the fluorescent dye bisbenzimide H33258 (Hoechst 33258). The dye was added to cells at the end of the stimulation period (24, 48, or 72 h) and fluorescence was measured at 460 nm (excitation: 360 nm). Interference of MWCNT with the fluorescent dye was evaluated by the incubation of a known concentration of DNA in the presence or absence of 100 µg/ml of MWCNT. No interference was observed.

Assessment of Apoptosis:
- DAPI Staining:
Cells were seeded onto sonic seals and were exposed for 24, 48, or 72 h to 100.tg/ml of particles or suspension medium alone. At the end of stimulation, the cells were fixed with 4% paraformaldehyde in PBS for 25 min at room temperature. DAPI solution (1 µg/ml) was added for 5 min at 37°C. Observations involved use of a fluorescence video microscope. For each condition of stimulation, 10 fields (magnification 63x) were selected from top to bottom across the vertical diameter of the culture well. The percentage of apoptotic cells was calculated as follows: Percent apoptotic cells=(total number of cells with apoptotic nuclei/total number of counted cells)x 100.

- DNA Laddering:
Genomic DNA (250 ng) of cells exposed for 72 h to DPL at 1% or 100 µg/ml MWCNT, crocidolite, or etoposide as a positive control was extracted and purified. Finally, degradation of DNA was visualized with SYBR green on a 1.5% agarose gel.

Assessment of Cell Proliferation:
The effects of nanomaterials on cell proliferation were determined by the bromodeoxyuridine (BrdU) cell proliferation enzyme-linked immunosorbent assay (ELISA).
The assay is based on the immunodetection of BrdU incorporated into the genomic DNA in place of thymidine of proliferating cells. Cells were cultured in 96-well plates. Upon confluence, they were exposed, in serum free medium, for 24, 48, or 72 h to particles from 0.1 to 100 µg/ml or to suspension medium alone. BrdU was added to the medium 20 h before the end of stimulation. At the end of incubation, the labeling solution was removed, and 200 µl of FixDenat solution was added and incubated for 30 min at room temperature. After removing FixDenat, 100 µl anti-BrdU-POD solution was added and incubated for 90 min at room temperature. After washing 3 times with 200 µl/well of washing buffer, 100 µl tetramethylbenzidine (TMB) substrate solution was added and incubated for 5 to 30 min at room temperature until color development was sufficient for photometric detection. The reaction was stopped with the addition of 25 µl/well H2SO4 (1 M). The absorbance of the samples was measured in a microplate reader at 450-690 nm within 5 min after adding H2SO4.

Assessment of Internalization of Nanomaterials:
Cells exposed for 48 h to different nanomaterials at 100 µg/ml were analyzed by transmission electronic microscopy (TEM).

Markers of Oxidative Stress:
Oxidative stress was evaluated by analyzing mRNA expression of the anti- and pro-oxidant systems HO-1, SOD2, GPx, and NOX4, respectively,
by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR), after exposure of cells for 6 or 24 h up to 100 µg/ml nanoparticles.
Expression of mRNA was normalized to that of ubiquitin. In previous experiments it was verified that ubiquitin mRNA expression did not change in the different experimental conditions.

Details on results:

MWCNTs formed agglomerates on top of both cell lines (surface area 15-35 m2) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 µg/ml MWCNTs induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNTs cellular internalization nor oxidative stress was observed. This study demonstrates that MWCNTs exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.

- Cell Viability:

Cell viability was analyzed with the MTT and neutral red assays, which determine mitochondrial metabolism and plasma membrane permeability, respectively.

Compared with respective controls (cells cultured in media containing the respective suspension agent without particles), MTT values decreased significantly with 10 µg/ml to 100 µg/ml incubation, reaching 60% of control values for 100 µg/ml incubation at 48 h postexposure (for 10 and 100 µg/ml). Similar results were obtained after exposure of MeT5A cells to DPL-suspended MWCNTs.

In both cell types, incubation with 100 µg/ml chrysotile or crocidolite elicited similar decreases in MTT, which were not different from that induced by 100 µg/ml MWCNTs after 24h. No change in MTT was observed when cells were incubated with CB nanoparticles at 100 µg/ml, except for significant decrease after incubation of MeT5A cells with CB for 48 or 72 h.

In contrast, results of the neutral red assay showed no significant alteration of A549 cell viability after incubation with MWCNTs whatever the time point considered. Similar results were observed with MeT5A cells.

Moreover, neither asbestos fibers nor CB nanoparticles induced significant diminution of viability as assessed by neutral red assay, whatever the cell type studied.

These results suggest that MWCNTs and asbestos fibers alter mitochondrial metabolism, with no apparent effect on cell membrane permeability.

- Cell Number, Apoptosis, and Proliferation:

This was first assessed by quantifying DNA content.

Incubation with 100 µg/ml MWCNTs resulted in a significant decrease in A549 cell number after 24, 48, and 72 h regardless of dispersion media, with areduction of 15 to 20% of control cell number.

No such effect was observed with MeT5A cells.

Moreover, neither asbestos fibers nor CB induced a significant decrease in total DNA content, except after 24 h of treatment of MeT5A cells with CB.

Furthermore, a significant increase in DNA content was observed after incubation of A549 cells with crocidolite for 48 h.

DAPI staining showed a small proportion of control cells with apoptotic nuclei (1 to 2% of total cells) at 48 and 72 h, and this fraction did not change significantly in the cells incubated with MWCNTs.

In contrast, a significant increase in the proportion of apoptotic cells was observed after 72 h incubation with crocidolite (approximately 5% of total cells). Similar results were obtained with MeT5A cells.

DNA laddering experiments confirmed those results in both cell types, except for an absence of modification induced by exposure to asbestos fibers.

Finally, no significant modification of proliferation was observed, whatever the nanomaterials, type point, and cell type.

- Cellular Internalization of Nanomaterials:

Internalization of the different materials in A549 cells was analyzed by TEM.

The morphological characteristics (lamellar body structure, microvilli, and tonofilament characteristics of alveolar epithelial type II cells in control cell culture) were similar in cells incubated with 100 µg/ml MWCNT for 24 h. No nuclear change revealing apoptosis was observed. No evidence of MWCNT internalization, whatever the dispersion media used, was found.

In contrast, CB particles and chrysotile and crocidolite, fibers were observed inside cells incubated with these particles. Similar results were obtained after exposure of MeT5A cells to the different nanomaterials.

- Oxidative Stress:

Since MWCNTs were not internalized in cells, whether the observed decrease in viability could be related to oxidative stress was examined by analyzing mRNA expression of the different genes implied in an oxidative response: HO-1, SOD2, GPx, and NOX4.

No significant change in expression of HO-1 mRNA was observed in cells incubated with MWCNTs for 6 or 24 h or with asbestos fibers. The same results were obtained for SOD2 and GPx genes. Finally, no change was observed in the mRNA of NOX4.

Conclusions:

Graphistrength C100 formed agglomerates on top of both cell lines (surface area 15-35 m2) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 µg/mlGraphistrength C100 induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither Graphistrength C100 cellular internalization nor oxidative stress was observed. this study demonstrates that Graphistrength C100 exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.

Executive summary:

The aim of an in vitro study was to evaluate adverse effects of MWCNT Graphistrength C100, on the human epithelial cell line A549 (Tabet et al., 2009). MWCNTs were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in two other media: ethanol (EtOH) and phosphate-buffered saline (PBS). Effects of MWCNTs were also compared to those of two asbestos fibers (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells but also in mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. MWCNTs formed agglomerates on top of both cell lines (surface area 15-35 µm²) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 µg/ml MWCNTs induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNTs cellular internalization nor oxidative stress was observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects. In conclusion, this study demonstrates that MWCNTs exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.

Reference 6

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Surface reactivitie of NM 402 (Graphistrength C2100) was studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with the MWCNT, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Carbonylated proteins were assessed by 1D immunoblotting.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: Toxicity mechanism

Vehicle:

other: H2O and DMEM cell culture medium supplemented with 10 % fetal calf serum

Examinations:

Surface reactivity analysis by ESR:
ROS activity of NPs was determined by ESR spectroscopy using two different methods. Employing the method of Papageorgiou et al., that uses CPH (1-hydroxy-3-carboxypyrrolidine) as spin probe, possible (surface) reactivity was investigated. Additionally, employing the method of Shi et al., potential hydroxyl radical (OH•) formation was determined in the presence of hydrogen peroxide (H2O2) and the spin trap 5,5-dimethyl-1-pyrroline-Noxide (DMPO). The surface reactivity was calculated as ratio between radical formation in the presence of the NPs and the response of deionized water (dH2O) as reference signal. For the solid materials, a suspension of 1 mg/mL was prepared, meaning that a final concentration of 0.25 mg/mL (DMPO) and 0.5 mg/mL (CPH), respectively, was analyzed.

Cell culture:
NRK-52E cells

Cell viability:
WST-1 assay

ROS generation:
DCFDA assay

Protein carbonylation:
1D immunoblotting

Details on results:

MWCT NM-402 was found reactive in the presence of DMPO indicating that a fenton-like reactions occured. No cytotoxicity by WST-1 assay and protein carbonylation by 1D immunoblotting was observecd up to 100µg/mL (30 µg/cm²) in NRK-52E cells. NM-402 showed an about 1.4 fold  increase of ROS generation by DCFDA assay at a concentration  of 34 µg/mL (10 µg/cm²).

Nanoparticle surface reactivity measured by ESR:

MWCT NM-402 was found reactive in the presence of DMPO indicating that a fenton-like reactions occured.

Surface reactivity NP (NP free supernatant):

ESR                     ESR                       ESR    

CPH/ dH2O       DMPO/dH2O       Overall

1.1                     3.4                         Positive

Cell viability:

No cytotoxicity up to 100µg/mL (30 µg/cm²)

ROS generation by DCFDA assay:

NM-402 showed an about 1.4 fold  increase  in  fluorescence  at  a  concentration  of 34 µg/mL (10 µg/cm²).

Protein carbonylation by 1D immunoblotting:

Negative in NRK-52E cells up to 100 µg/ml (30 µg/cm²).

Executive summary:

Surface reactivitie of NM 402 (Graphistrength C2100) was studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with the MWCNT, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Carbonylated proteins were assessed by 1D immunoblotting. MWCT NM-402 was found reactive in the presence of DMPO indicating that a fenton-like reactions occured. No cytotoxicity and protein carbonylation was observecd up to 100µg/mL (30 µg/cm²). NM-402 showed an about 1.4 fold  increase of ROS generation at a concentration  of 34 µg/mL (10 µg/cm²).

Reference 7

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Study the biological effects of MWCNT on macrophage and human lung adenocarcinoma cell lines with different endpoints, such as cell viability, phagocytic activity and pro-inflammatory M1 macrophage activation.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: cell viability, phagocytic activity and pro-inflammatory M1 macrophage activation

Specific details on test material used for the study:

MWCNT were heated at 230 °C for 4h to eliminate possible contamination from lipopolysaccharide (LPS).
Weight catalyst (%): 10.6±0.4
Main decomposition temperature (ºC): 555±9
24-hour dissolution ratio (0.05% dispersion) :
BSA in water: none
Gambles solution: 1.3 wt % Al; 1.6 wt % Fe
Caco-2 medium:none

Species:

other: MH-S murine alveolar macrophages; RAW264.7 murine peritoneal macrophages; Calu-3 human lung adenocarcinoma cells.

Details on test animals or test system and environmental conditions:

MH-S cells were obtained from prof. Dario Ghigo, University of Torino (Italy). RAW264.7 cells were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna (Brescia, Italy). Cells were routinely cultured in 10-cm diameter dishes in RPMI1640 medium supplemented with 10% FBS, streptomycin (100 µg/ml) - penicillin (100 U/ml), L-glutamine (2 mM) and (for MH-S cells only) ß-mercaptoethanol (0.05 mM) in a humidified atmosphere of 5% CO2 in air. For viability experiments, macrophages were seeded in 96-well plates, at the density of 30x103 cells. For microscopy analysis and phagocytosis assay, they were seeded in 4-chamber glass culture slides (BD Falcon,MA,USA), at the density of 20x104 and 25x104, respectively. For the other experiments, cells were seeded in 24-well plates, at the density of 20x104.
Calu-3 cells were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna (Brescia, Italy). Cells were routinely cultured in 10-cm diameter dishes in EMEM supplemented with 1 mM sodium pyruvate, 10 % FBS and streptomycin (100 µg/ml) and penicillin (100 U/ml) in a humidified atmosphere of 5% CO2 in air. BEAS-2B, human bronchial epithelial cells, and A549, human alveolar basal epithelial cells, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Euroclone, Italy), supplemented with Gln (4 mM) and 10% FBS in a humidified atmosphere of 5% CO2 in air. For the experiments, epithelial cells were seeded into culture inserts with permeable membrane filters (pore size of 0.4 µm) for Falcon 24-well-multitrays (BD Bioscience, USA), at a density of 75×103 cells/well.

Route of administration:

other: in vitro

Vehicle:

other: 0.05 wt% BSA in water

Details on exposure:

After cooling at room temperature, NM402 was dispersed in a stock solution at a concentration of 2.5 mg/mL by prewetting powder in 0.5% ethanol (96% purity) followed by dispersion in 0.05 wt% BSA in water. After 15 min of sonication, working concentrations of NM-402 were obtained by serial dilutions. For permeability experiments MWCNT dispersions were added to the growth medium at the apical side of the permeable filter on which cell monolayers had grown.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

72h

Examinations:

- Resazurin assay
Resazurin is a non-fluorescent molecule which is converted by intracellular reductases in the fluorescent compound resorufin (Lem = 572 nm). After its production, resorufin accumulates into the medium and can be readily determinated with a fluorometer[82]. For the measurement of the viability of Calu-3 cells monolayers, they were incubated for 2 hours with resazurin, added to both the basolateral and the apical compartments. Because the culture inserts did not allow direct fluorescence reading from the wells, the measurement has been performed on the medium of apical chamber transferred in a clean 96-well dish. Fluorescence was measured at 572 nm with a multimode plate reader Perkin Elmer Enspire (Waltham, Massachusetts, USA).
For macrophages, after the selected incubation periods in the presence MWCNT, medium was replaced with a solution of resazurin (44 µM) in serum-free medium. Fluorescence was read after 30 min. Since nanomaterials can interfere with viability tests, the dye was preliminarily incubated with the preparations of MWCNT (at the maximal dose used), and the fluorescence was then measured. No fluorescence signal was detected above the blank. Data were expressed as the % of the value obtained for the untreated control.

- Phagocytosis assay
Fluorescent yellow-green polystyrene latex beads (2 µm, Sigma-Aldrich, Milano, Italy) were opsonized with 50% human serum for 30 min at 37°C before the experiments. After the treatment in presence of MWCNT, macrophages were incubated for 2 h at 37 °C in complete growth medium in the presence of latex beads (20 microspheres/cell). Cell monolayers were then washed vigorously with PBS to remove extracellular beads, counterstained with 2 µM of the vital cytoplasmic dye CellTracker Red CMTPX (Invitrogen, Milano, Italy) and fixed with 2% paraformaldehyde for 10 min. The number of internalized latex particles was determined by counting intracellular fluorescent beads with a fluorescent microscope. For each culture, at least 3–5 fields containing about 100–150 cells were analysed. Percent of phagocytosis was calculated as the number of cells with at least one bead inside/total number of cells counted[83].

- Gene expression analysis
After the selected incubation periods in presence of ASNP or MWCNT, the expression of Nos2 (in macrophages treated with ASNP and MWCNT) and Hmox-1 (in macrophages treated with ASNP) was assessed with Real Time PCR. 1µg of total RNA, isolated with GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) was reverse transcribed. For Real-Time qPCR, cDNA was amplified with Go Taq PCR Master Mix (Promega, Italia, Milan, Italy), using the forward and reverse primers indicated in Table 1 (5pmol each). The expression of the gene of interest under each experimental condition was normalized to that of Gapdh and shown relative to its expression level control, untreated cells following the method of Relative Standard Curve Method[84].

- Western blot
After treatments with ASNP, macrophages were lysed in a buffer containing 20 mMTris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, 2 mM imidazole and a cocktail of protease inhibitors (Complete, Mini, EDTA-free, Roche, Milan, Italy). Lysates were sonicated for 15s and centrifuged at 12,000g for 20min at 4°C. After quantification with the Bio-Rad protein assay, aliquots of 40 µg of proteins were mixed with Laemmli buffer 4× (250 mMTris–HCl, pH 6.8, 8% SDS, 40% glycerol, and 0.4M DTT), warmed at 95°C for 10 min and loaded on a 8% gel for SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membranes (Immobilon-P, Millipore, Millipore Corporation, MA, USA). Non-specific binding sites were blocked with an incubation of 1h at room temperature in BSA 5% in TBS-Tween. The blots were then exposed at 4 °C overnight to anti-Nos2 (rabbit policlonal, 1:400, Santa Cruz Biotechnology) or anti-Actin (rabbit polyclonal, 1:30,000, Cell Signaling Technology diluted in the same solution). After washing, the blots were exposed for 1h at room temperature to HRP-conjugated anti-rabbit antibody (Cell Signaling Technology), diluted 1:20.000 in blocking solution. Immunoreactivity was visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Milan, Italy).

- Determination of NO production
After the selected incubation periods in presence of ASNP and MWCNT, Nitrite concentration in the culture media of macrophages treated with ASNP and MWCNT, as an indicator of NO production, was determined through a fluorometric approach, as previously described[85]. The method is based on the production of the fluorescent molecule 1H-naphthotriazole from 2,3-diaminonaphthalene (DAN) in acid environment. For nitrite determination, 100 µl of medium were put in wells of a black 96-well plate with a clear bottom (Corning, Cambridge, MA, USA). DAN (20 µl of a solution of 0.025 mg/ml in 0.31 M HCl) was then added and, after 10 min at room temperature, the reaction was stopped with 20 µl of 0.7 N NaOH. Standards were performed in the same medium from a solution of 1 mM sodium nitrite. Fluorescence was determined with an EnSpire plate reader (Perkin Elmer). Nitrite production was expressed in nmoles per ml of extracellular medium (µM).

- Trans-epithelial electrical resistance (TEER)
Measurements of the TEER of Calu-3 cells monolayers treated with MWCNT were made with an epithelial voltmeter (EVOM, World Precision Instruments Inc., Sarasota, FL, USA) that produces an AC current. Before the permeability experiments, cells were allowed to grow, usually for 10–14 days, until a value of TEER higher than 1,000 O cm2 was reached, indicating the formation of a tight epithelial layer. TEER changes were expressed as the percentage of the initial value adjusted for control cell layers.

- Confocal microscopy
. Confocal laser scanning microscopy on Calu-3 cells monolayers
Confocal analysis was carried out with a LSM 510 Meta scan head integrated with an inverted microscope (Carl Zeiss, Jena, Germany). Calu-3 cells monolayers treated with MWCNT-SA were observed through a 40x (1.3 NA) or a 63x (1.4 NA) oil objectives. Image acquisition was carried out in multitrack mode, i.e. through consecutive and independent optical pathways. Vertical sections were obtained with the function Display – Cut (Expert Mode) of the LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) software (Microscopy Systems, Hartford, CT). Reconstructions were performed from z-stacks of digital images (minimum 32 confocal sections, zaxis ,acquisition interval of 0.39 µm), processed with the Axiovision module inside 4D release 4.5, applying the shadow or the transparency algorithm.

Details on results:

NM402 induced a modest 40% viability decrease at the maximal dose of 80 µg/cm2 in both macrophage cell lines. NM402 did not affect phagocytosis of RAW264.7 cells. Nos2 expression, NO and nitrites production were significantly induced at 10 µg/cm². After 8 days of exposure, TEER measurements on Calu-3 cell monolayers showed a significant decrease in TEER but no significant alteration of the monolayer. Contact-mediated cytotoxicity was found in monolayers in close contact with the aggregates of NM402.

Effects on macrophage viability

The effects on macrophage viability of NM402 (range of doses from 0 to 80 µg/cm²; exposure time 72h), were assessed with resazurin assay (Figure 9). In both the macrophage cell lines, NM402 showed a modest 40% viability decrease at the maximal dose of 80 µg/cm2.

Effects of MWCNT on the phagocytic activity

The effects of NM402 on phagocytic activity, an indicator of functional competence in RAW264.7 cells, is showed in Figure 10. For the experiment, RAW264.7 cells were treated for 24h with MWCNT at the dose of 10 µg/cm2 (non-cytotoxic dose). The results obtained showed that NM402 did not affect phagocytosis.

Effects of MWCNT on M1-macrophage activation

Figure 11A reports Nos2 mRNA expression after 48h of treatment of MH-S macrophages with noncytotoxic doses (5 or 10 µg/cm2) of NM402 preparations. Nos2 was significantly induced with NM402 (at 10 µg/cm²). This effect corresponded to a clear cut increase in NO production after 72h of treatment (Figure 11C). A significant increase of nitrites was registered with NM402 at 10 µg/cm2 .

Analyses in live cell monolayers

TEER measurement were carried out on Calu-3 cell monolayers to identify the responses of these cell cultures following exposure to NM402 (80 µg/cm2). After 8 days of exposure, TEER measurements showed that a significant decrease in TEER was detectable (Figure 12 A). Calu-3 cell monolayer integrity was biochemically assessed by resazurin assay. After exposure to NM402, no significant alteration of the monolayer was detectable by this assay (Figure12B).

Calcein/Propidium Iodide assay

Contact-mediated cytotoxicity was found in monolayers treated with NM402, in this case, propidium-positive cells were detectable in close contact with the aggregates (Figure 16).

Executive summary:

The biological effects of NM-402 (Graphistrength C100) on macrophage and human lung adenocarcinoma cell lines was evaluated with different endpoints, such as cell viability, phagocytic activity and pro-inflammatory M1 macrophage activation. NM402 induced a modest 40% viability decrease at the maximal dose of 80 µg/cm² in MH-S and RAW264.7 macrophage cell lines. NM402 did not affect phagocytosis of RAW264.7 cells. Nos2 expression, NO and nitrites production were significantly induced at 10 µg/cm². After 8 days of exposure, TEER measurements on Calu-3 cell monolayers showed a significant decrease in TEER but no significant alteration of the monolayer. Contact-mediated cytotoxicity was found in monolayers in close contact with the aggregates of NM402.

Description of key information

In vitro studies have used a variety of cell types but the toxic response is not consistently reproduced by different investigators, and may therefore be related to the experimental set up (specifically the cell investigated, MWCNTs concentration, or exposure time, and assays used to assess toxicity) used to determine their toxic potential.

 

In order to evaluate the potential adverse effects of Graphistrength C100, a comprehensive approach was used, aimed to evaluate both cytotoxic effects and the underlying mechanisms (apoptosis, proliferation, oxidative stress, internalization) (Tabet et al., 2009). Graphistrength C100 was dispersed in DPL, a component of pulmonary surfactant. Incubation with 100 µg/ml Graphistrength C100 induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither Graphistrength C100 cellular internalization nor oxidative stress was observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. Carbon black was internalized without any adverse effects. In conclusion, this study demonstrates that Graphistrength C100 exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.

 

The potential of Graphistrength C100 (named NM 402 in the publication) to induce cytotoxicity, measured by mitochondrial function using the WST-1 assay and Alamar Blue assays, and to induce an inflammatory response, measured by the release of pro-inflammatory cytokines (IL-6, IL-8 and TNF-a) was investigated in C3A hepatoblastoma cell and primary human hepatocytes (Kermanizadeh et al., 2012, 2013). As indicators of hepatocyte function, the effects of Graphistrength C100 exposure were investigated on albumin and/or urea production. The interaction of the Graphistrength C100 with the cells was investigated by the use of transmission and scanning electron microscopy (TEM and SEM). Graphistrength C100 was relatively non-toxic to the C3A cells and to primary human hepatocytes, LC50 was not reached up to 80 µg/cm² after a 24 h exposure. Graphistrength C100 induces the production of the proinflammatory cytokine IL-8, but only at relatively high exposure concentrations (>= 20 µg/cm²). No change in the levels of IL-6 or TNF-a following exposure of the C3A cells and human hepatocytes was observed suggesting that the inflammatory response is limited. Both urea and albumin as measures of liver function were unaffected.

 

The proliferative response to Graphistrength C100 (named NM 402 in the publication) was assessed on mouse primary lung fibroblasts (Vietty et al., 2013). Cell proliferation was assessed by counting the number of living cells after 24 h exposure to 1 to 37.5 µg/cm². Graphistrength C100 significantly increased cell number in a dose-dependent manner and the highest concentration induced an effect similar to human platelet-derived growth factor (positive control).

 

Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle mediated cardiovascular health effects (Cao et al., 2016). The presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. The interaction between direct exposure to MWCNTs and PA was investigated on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar–endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to Nanocyl MWCNT (named NM400) and Graphistrength C100 (named NM402), was associated with elevated levels of reactive oxygen species (MN400) as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures (both MWCNTs). Both effects were found to be independent of the presence of PA. NM 400 exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.

 

Surface reactivity of Graphistrength C100 (named NM 402) was studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with the MWCNT, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay) (Driessen et al., 2015). Carbonylated proteins were assessed by 1D immunoblotting. Graphistrength C100 was found reactive in the presence of DMPO indicating that a fenton-like reactions occurred. No cytotoxicity and protein carbonylation was observed up to 100µg/mL (30 µg/cm²). Graphistrength C100 showed an about 1.4 fold increase of ROS generation at a concentration of 34 µg/mL (10 µg/cm²).

 

The biological effects of Graphistrength C100 (named NM-402) on macrophage and human lung adenocarcinoma cell lines was evaluated with different endpoints, such as cell viability, phagocytic activity and pro-inflammatory M1 macrophage activation (Di Cristo, 2015). Graphistrength C100 induced a modest 40% viability decrease at the maximal dose of 80 µg/cm² in MH-S and RAW264.7 macrophage cell lines. Graphistrength C100 did not affect phagocytosis of RAW264.7 cells. Nos2 expression, NO and nitrites production were significantly induced at 10 µg/cm². After 8 days of exposure, TEER measurements on Calu-3 cell monolayers showed a significant decrease in TEER but no significant alteration of the monolayer. Contact-mediated cytotoxicity was found in monolayers in close contact with the aggregates of Graphistrength C100.