Assessment of primary and inflammation-driven genotoxicity of carbon black nanoparticles in vitro and in vivo

Figures & data

Table 1. Main physicochemical characteristics of carbon black nanoparticles included in this study.

Carbon black particle	Primary size (nm)	Specific Surface Area (m^2/g)	Z-average RPMI-1640 (nm)	Z-average water + 2% serum (nm)	24 h deposition RPMI-1640 (%)
Flammruss 101	95	24	474 ± 9	385 ± 6	43
Printex XE2B	30	1000	180 ± 2	190 ± 7	41
Printex 90	14	295	168 ± 3	81 ± 10	38^a

^a38% was obtained in THP-1a cells in complete RPMI-1640 cell medium, using the same method that has previously demonstrated 25% deposition on A549 cells by exposure to 160 ug/mL for 24 h exposure in F-12 cell medium (Di Ianni, Samulin, et al. 2021). FL101 and XE2B had polydispersity index of 0.33 ± 0.03 and 0.17 ± 0.02, respectively, in complete RPMI medium, and 0.22 ± 0.03 and 0.21 ± 0.01, respectively, in water with 2% serum.

Table 2. Effect of carbon black nanoparticle exposure on IL-8 gene induction in A549 and THP-1a cells for administered doses and effective doses quantified by TGA-MS.

A549
NM	IL-8	Administered dose (µg)	IL-8/µg administered	Effective dose (µg)	IL-8/µg effective
FL101	2.94	160	0.018	69	0.042
XE2B	16.23	160	0.101	66	0.246
THP-1a
NM	IL-8	Administered dose (µg)	IL-8/µg administered	Effective dose (µg)	IL-8/µg effective
FL101	0.83	160	0.005	69	0.012
XE2B	7.38	160	0.046	66	0.112

IL8: Fold IL-8 in exposed groups compared to controls (0 µg/mL).

Figure 1. A-cellular reactive oxygen species (ROS) production of (A) FL101, (B) P90 and (C) XE2B in the concentration range causing ROS steady increase. ROS production poorly correlated with CBNP concentration (D) (p > 0.05), whereas surface area was significantly correlated with ROS production (E) (p < 0.001 and R²:0.75). The lines represent the linear regression of ROS production as function of CBNP (D) concentration or (E) surface area.

Figure 2. Pro-inflammatory response in terms of IL-8 gene expression in (A) A549 and (B) THP-1a cells exposed to FL101 and XE2B for 6 and 24 h. The data represent mean ± SEM of three independent replicates. Stars represent significance at p < 0.05 from ANOVA test of significance (*:p < 0.05:**:p < 0.01).

Table 3. Effects of carbon black nanoparticle exposure on DNA strand breaks levels (fold) in A549 and THP-1a cells for administered doses and effective doses quantified by TGA-MS.

A549
NM	SB	Administered dose (µg)	SB/ µg administered	Effective dose (µg)	SB/µg effective
FL101	0.63	160	0.0039	69	0.0091
XE2B	1.58	160	0.0098	66	0.0239
THP-1
NM	SB	Administered dose (µg)	SB/ µg administered	Effective dose (µg)	SB/µg effective
FL101	1.7	160	0.010	69	0.024
XE2B	1.65	160	0.010	66	0.025

SB: Fold DNA strand breaks in exposed groups compared to controls (0 µg/mL)

Figure 3. DNA damage (% tail) in A549 (A) and THP-1a (B) cells exposed to FL101, P90 and XE2B. The data represent mean values ± SD from three independent replicates, the error bars represent the SD. Stars represent significance at p < 0.05.

Table 4. Inflammatory cell infiltrates in broncho-alveolar lavage fluid from mice exposed to vehicle and CB nanoparticles; XE2B, FL101 and Printex 90 (P90) (positive control in this study), at day 1, 28 and 90.

		FL101			XE2B			P90
	Vehicle	18 µg^a	54 µg^a	162 µg^a	6 µg^b	18 µg^b	54 µg^b	162 µg^c
Day 1
Neutrophils (10^c)	10.8 ± 19.3	1.4 ± 1	7.5 ± 5.1	34.8 ± 20.7**	4.6 ± 3.2	8.0 ± 11.8	86.7 ± 17.9***	123.5 ± 40.9***
Macrophages (10^c)	81.1 ± 136.2	47.8 ± 8.4	47.4 ± 19	37.1 ± 6.6	60.0 ± 35.1	28.1 ± 22.6	33 ± 6.3	14.5 ± 2.4***
Lymphocytes (10^c)	2.8 ± 7.3	0.3 ± 0.2	0.4 ± 0.6	2.1 ± 1.1	0.4 ± 0.3	0.3 ± 0.5	1.0 ± 1.6	3.2 ± 2.2
Eosinophils (10^c)	4.5 ± 14.1	0.0 ± 0.1	0.8 ± 1.2	10.2 ± 8.3*	0.1 ± 0.2	0.7 ± 1.6	4.8 ± 2.5	13.9 ± 12**
Epithelial cells (10^c)	2.0 ± 1.7	1.6 ± 1.1	1.6 ± 0.6	2.2 ± 0.8	0.9 ± 1.1	0.8 ± 0.7	2.4 ± 1.2	1.9 ± 1.2
Total BAL cells(10^c)	101.4 ± 175.6	51.4 ± 7.3	58 ± 20.8	86.8 ± 24.1	66.3 ± 35.8	55.2 ± 11	128.2 ± 20.6*	157.1 ± 54.9**
Day 28
Neutrophils (10^c)	0.7 ± 0.7	0.8 ± 0.7	0 ± 0	0.6 ± 0.7	0.7 ± 0.6	2.3 ± 1.9	14.2 ± 5.9**	14.7 ± 8.1***
Macrophages (10^c)	38.3 ± 15	49.7 ± 23.7	46.9 ± 8.3	61.7 ± 22.7	34.4 ± 10.8	49.9 ± 14.1	51.5 ± 14.9	59 ± 27.3
Lymphocytes (10^c)	1.6 ± 1.3	2 ± 2.1	1.7 ± 1.2	2.2 ± 1.7	1 ± 0.5	3.7 ± 3.8	24.9 ± 16.4***	31.2 ± 18.3***
Eosinophils (10^c)	1.8 ± 3.4	1.5 ± 3.8	0.9 ± 1.2	0.8 ± 1.2	0 ± 0	0 ± 0	0 ± 0	0.1 ± 0.2
Epithelial cells (10^c)	2.4 ± 2.6	2.4 ± 1.8	2 ± 0.6	3.1 ± 2.7	1.3 ± 0.6	2 ± 1.1	2.9 ± 3.3	2.3 ± 1.7
Total BAL cells(10^c)	48 ± 11.6	56.8 ± 24.6	51.7 ± 8.9	68.6 ± 25.9	37.6 ± 11.7	58.1 ± 16.1	93.7 ± 30**	107.6 ± 49.7**
Day 90
Neutrophils (10^c)	1 ± 1.3			1.6 ± 1.8			6.6 ± 9.1*	12.9 ± 7***
Macrophages (10^c)	47.7 ± 10.2			41.5 ± 4.6			46.4 ± 19	56.3 ± 13
Lymphocytes (10^c)	1.3 ± 0.7			1.7 ± 1.2			13.1 ± 11.4**	32.8 ± 16.9***
Eosinophils (10^c)	0.1 ± 0.1			0.8 ± 2.1			0.2 ± 0.2	0.3 ± 0.3
Epithelial cells (10^c)	2.4 ± 1.5			1.7 ± 0.6			1.8 ± 1.7	2.3 ± 1.6
Total BAL cells(10^c)	52.6 ± 11.2			47.5 ± 5			68.3 ± 38.5	104.9 ± 28.9***

^a18, 54 and 162 µg corresponding to 4, 13, 39 cm², respectively, of FL101 dose.

^b6, 18, and 54 µg corresponding to 61, 181 and 541 cm², respectively, of XE2B dose.

^c162 µg corresponding to 470 cm² of P90 dose.

Stars represent significance at p < 0.05 from ANOVA test of significance (*:p < 0.05; **:p < 0.01; ***:p < 0.001).

Figure 4. Correlation of administered CBNP surface area with IL-8 mRNA induction in (A) A549 cells and (B) THP-1a macrophages following 24 h exposure in submerged conditions. Correlations assessed by linear regression analysis with dose and CBNP type as predictor, and IL-8 induction as outcome (p < 0.001 and R²: 0.61 in both A and B). Lines in A and B show trends with SSA dose on a logarithmic scale. C) Correlation of DNA tail (%) and administered dose as surface area in THP-1a cells (p = 0.292 and R²:0.02). (D) Correlation of DNA tail (%) and administered doses as mass in THP-1a cells (p < 0.01 and R²:0.58).

Table 5. Histological data including the numbers of lymphocyte infiltrates and macrophage aggregate separately and together.

Particle	Post-exposure day	Lymphocyte infiltrates (range)	Macrophage aggregates (range)	Lymphocyte and macrophage (range)	Cytoplasmic material present
VC	28	0	0	0	0/2
FL101	28	1.4 (0–4)	1.6 (0–4)	3.0 (0–8)	3/5
XE2B	28	3.2 (0–7)	4.0 (2–6)	7.2 (4–12)	5/5
VC	90	0	0	0	0/2
FL101	90	1.0 (0–3)	0.5 (0–2)	1.5 (0–5)	1/5
XE2B	90	4.0 (3–8)	2.8 (2–4)	6.8 (5–12)	4/5

In addition, the proportion of animals where cytoplasmic material is seen in macrophage cytoplasm is presented; VC: vehicle control.

Figure 5. Correlation of surface area with the inflammatory responses in terms of neutrophil influx in mice 1 (A), 28 (B) and 90 days post-exposure (C), by linear regression analysis with specific surface area (SSA) as predictor, and neutrophil influx as dependent variable. The data shown include data from the present study as well as previously published studies. D1: R-squared = 0.64 (p < 0.001); D28: R-squared = 0.35 (p < 0.001); D90: R-squared = 0.29(p = 0.06). Study 1: Saber et al. (2015): Study 2: Kyjovska et al. (2015); Study 3: Modrzynska et al. (2018); Study 4: Billing et al. (2020); Study 5: Barfod et al. (2020); Study 6: Hadrup et al. (2021); Study 7: Bourdon, Saber, et al. (2012); Study 8: Poulsen et al. (2016); Study 9: Bengtson et al. (2017); Study 10: Hadrup et al. (2020) and Husain et al. (2015); Study 11: Bendtsen et al. (2019); Study 12: Bendtsen et al. (2020); Study 13: CBNPs tested in the present study; FL101 and XE2B.

Figure 6. Histological changes in lung tissue 90 days post-exposure at 20× magnification. (A) Tissue from mouse exposed to a total carbon black surface area of 540 cm² (corresponding to instilled mass dose of 54 ug) XE2B, (B) tissue from mouse exposed to 39 cm² (corresponding to 162 ug) FL101. Alveolar walls in A appear widened due to capillary congestion, probably related to the euthanasia.

Figure 7. DNA strand breaks levels (% DNA tail) quantified in BAL cells, lung and liver tissue samples from mice exposed to the three carbon black nanoparticles for 1, 28 and 90 days (controls n = 22; exposed n = 6).

Di Ianni, Emilio, Johanna Samulin Erdem, Peter Møller, Nicklas Mønster Sahlgren, Sarah Søs Poulsen, Kristina Bram Knudsen, Shan Zienolddiny, et al. 2021. “In Vitro-in Vivo Correlations of Pulmonary Inflammogenicity and Genotoxicity of MWCNT.” Particle and Fibre Toxicology 18 (1): 1–16. doi:10.1186/s12989-021-00413-2.

 PubMed Web of Science ®Google Scholar

Saber, Anne Thoustrup, Alicja Mortensen, Józef Szarek, Ismo Kalevi Koponen, Marcus Levin, Nicklas Raun Jacobsen, Maria Elena Pozzebon, et al. 2015. “Epoxy Composite Dusts with and without Carbon Nanotubes Cause Similar Pulmonary Responses, but Differences in Liver Histology in Mice following Pulmonary Deposition.” Particle and Fibre Toxicology 13 (1): 37. doi:10.1186/s12989-016-0148-2.

 Google Scholar

Kyjovska, Zdenka O., Nicklas R. Jacobsen, Anne T. Saber, Stefan Bengtson, Petra Jackson, Håkan Wallin, and Ulla Vogel. 2015. “DNA Damage Following Pulmonary Exposure by Instillation to Low Doses of Carbon Black (Printex 90) Nanoparticles in Mice.” Environmental and Molecular Mutagenesis 56 (1): 41–49. doi:10.1002/em.21888.

 PubMed Web of Science ®Google Scholar

Modrzynska, Justyna, Trine Berthing, Gitte Ravn-Haren, Nicklas Raun Jacobsen, Ingrid Konow Weydahl, Katrin Loeschner, Alicja Mortensen, Anne Thoustrup Saber, and Ulla Vogel. 2018. “Primary Genotoxicity in the Liver following Pulmonary Exposure to Carbon Black Nanoparticles in Mice.” Particle and Fibre Toxicology 15 (1): 2–12. doi:10.1186/s12989-017-0238-9.

 PubMed Web of Science ®Google Scholar

Billing, A. M., K. B. Knudsen, A. J. Chetwynd, L. A. Ellis, S. V. Y. Tang, T. Berthing, H. Wallin, I. Lynch, U. Vogel, and F. Kjeldsen. 2020. “Fast and Robust Proteome Screening Platform Identifies Neutrophil Extracellular Trap Formation in the Lung in Response to Cobalt Ferrite Nanoparticles.” ACS Nano 14 (4): 4096–4110. doi:10.1021/acsnano.9b08818.

 PubMed Web of Science ®Google Scholar

Barfod, Kenneth Klingenberg, Katja Maria Bendtsen, Trine Berthing, Antti Joonas Koivisto, Sarah Søs Poulsen, Ester Segal, Eveline Verleysen, et al. 2020. “Increased Surface Area of Halloysite Nanotubes Due to Surface Modification Predicts Lung Inflammation and Acute Phase Response after Pulmonary Exposure in Mice.” Environmental Toxicology and Pharmacology 73: 103266. (July 2019): doi:10.1016/j.etap.2019.103266.

 PubMed Web of Science ®Google Scholar

Hadrup, Niels, Kukka Aimonen, Marit Ilves, Hanna Lindberg, Rambabu Atluri, Nicklas M. Sahlgren, Nicklas R. Jacobsen, et al. 2021. “Pulmonary Toxicity of Synthetic Amorphous Silica–Effects of Porosity and Copper Oxide Doping.” Nanotoxicology 15 (1): 96–113. doi:10.1080/17435390.2020.1842932.

 PubMed Web of Science ®Google Scholar

Bourdon, Julie A., Anne T. Saber, Nicklas R. Jacobsen, Keld A. Jensen, Anne M. Madsen, Jacob S. Lamson, Håkan Wallin, et al. 2012. “Carbon Black Nanoparticle Instillation Induces Sustained Inflammation and Genotoxicity in Mouse Lung and Liver.” Particle and Fibre Toxicology 9 (1): 5. doi:10.1186/1743-8977-9-5.

 PubMed Web of Science ®Google Scholar

Poulsen, Sarah S., Petra Jackson, Kirsten Kling, Kristina B. Knudsen, Vidar Skaug, Zdenka O. Kyjovska, Birthe L. Thomsen, et al. 2016. “Multi-Walled Carbon Nanotube Physicochemical Properties Predict Pulmonary Inflammation and Genotoxicity.” Nanotoxicology 10 (9): 1263–1275. doi:10.1080/17435390.2016.1202351.

 PubMed Web of Science ®Google Scholar

Bengtson, Stefan, Kristina B. Knudsen, Zdenka O. Kyjovska, Trine Berthing, Vidar Skaug, Marcus Levin, Ismo K. Koponen, et al. 2017. “Differences in Inflammation and Acute Phase Response but Similar Genotoxicity in Mice following Pulmonary Exposure to Graphene Oxide and Reduced Graphene Oxide.” PLoS One 12 (6): e0178355. doi:10.1371/journal.pone.0178355.

 PubMed Web of Science ®Google Scholar

Hadrup, Niels, Anne T. Saber, Zdenka O. Kyjovska, Nicklas R. Jacobsen, Minnamari Vippola, Essi Sarlin, Yaobo Ding, et al. 2020. “Pulmonary Toxicity of Fe2O3, ZnFe2O4, NiFe2O4 and NiZnFe4O8 Nanomaterials: Inflammation and DNA Strand Breaks.” Environmental Toxicology and Pharmacology 74: 103303. doi:10.1016/j.etap.2019.103303.

 PubMed Web of Science ®Google Scholar

Husain, Mainul, Zdenka O. Kyjovska, Julie Bourdon-Lacombe, Anne T. Saber, Keld A. Jensen, Nicklas R. Jacobsen, Andrew Williams, et al. 2015. “Carbon Black Nanoparticles Induce Biphasic Gene Expression Changes Associated with Inflammatory Responses in the Lungs of C57BL/6 Mice following a Single Intratracheal Instillation.” Toxicology and Applied Pharmacology 289 (3): 573–588. doi:10.1016/J.TAAP.2015.11.003.

 PubMed Web of Science ®Google Scholar

Bendtsen, Katja Maria, Anders Brostrøm, Antti Joonas Koivisto, Ismo Koponen, Trine Berthing, Nicolas Bertram, Kirsten Inga Kling, et al. 2019. “Airport Emission Particles: Exposure Characterization and Toxicity following Intratracheal Instillation in Mice.” Particle and Fibre Toxicology 16 (1): 23–23. doi:10.1186/s12989-019-0305-5.

 PubMedGoogle Scholar

Bendtsen, Katja Maria, Louise Gren, Vilhelm Berg Malmborg, Chandra Shukla, Martin Tunér, Yona J. Essig, Annette M. Krais, et al. 2020. “Particle Characterization and Toxicity in C57BL/6 Mice Following Instillation of Five Different Diesel Exhaust Particles Designed to Differ in Physicochemical Properties.” Particle and Fibre Toxicology 17: 38. doi:10.1186/s12989-020-00369-9.

 PubMed Web of Science ®Google Scholar

Data availability statement

Data are available upon reasonable request.