Free radical scavenging and formation by multi-walled carbon nanotubes in cell free conditions and in human bronchial epithelial cells

Our plain-English summary of Free radical scavenging and formation by multi-walled carbon nanotubes in cell free conditions and in human bronchial epithelial cells, by P. Nymark, K.A. Jensen, S. Suhonen, et al (Particle and Fibre Toxicology, 2014).

The study

• Authors: P. Nymark, K.A. Jensen, S. Suhonen, Y. Kembouche, M. Vippola, J. Kleinjans, J. Catalán, H. Norppa, J. van Delft, J.J. Briedé
• Journal: Particle and Fibre Toxicology
• Published: 2014;11:4
• Cited by: 43 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Certain multi-walled carbon nanotubes (MWCNTs) have been shown to elicit asbestos-like toxicological effects. To reduce needs for risk assessment it has been suggested that the physicochemical characteristics or reactivity of nanomaterials could be used to predict their hazard. Fibre-shape and ability to generate reactive oxygen species (ROS) are important indicators of high hazard materials. Asbestos is a known ROS generator, while MWCNTs may either produce or scavenge ROS. However, certain biomolecules, such as albumin - used as dispersants in nanomaterial preparation for toxicological testing in vivo and in vitro - may reduce the surface reactivity of nanomaterials.

Methods: Here, we investigated the effect of bovine serum albumin (BSA) and cell culture medium with and without BEAS 2B cells on radical formation/scavenging by five MWCNTs, Printex 90 carbon black, crocidolite asbestos, and glass wool, using electron spin resonance (ESR) spectroscopy and linked this to cytotoxic effects measured by trypan blue exclusion assay. In addition, the materials were characterized in the exposure medium (e.g. for hydrodynamic size-distribution and sedimentation rate).

Results: The test materials induced highly variable cytotoxic effects which could generally be related to the abundance and characteristics of agglomerates/aggregates and to the rate of sedimentation. All carbon nanomaterials were found to scavenge hydroxyl radicals (•OH) in at least one of the solutions tested. The effect of BSA was different among the materials. Two types of long, needle-like MWCNTs (average diameter >74 and 64.2 nm, average length 5.7 and 4.0 μm, respectively) induced, in addition to a scavenging effect, a dose-dependent formation of a unique, yet unidentified radical in both absence and presence of cells, which also coincided with cytotoxicity.

Conclusions: Culture medium and BSA affects scavenging/production of •OH by MWCNTs, Printex 90 carbon black, asbestos and glass-wool. An unidentified radical is generated by two long, needle-like MWCNTs and these two CNTs were more cytotoxic than the other CNTs tested, suggesting that this radical could be related to the adverse effects of MWCNTs.

In plain English

This laboratory study tested, in cell-free solutions and human bronchial epithelial BEAS 2B cells, five multi-walled carbon nanotubes (MWCNTs): two long needle-like (MWCNT-7, NM-401), long tangled, short purified and short non-purified, plus Printex 90 carbon black, UICC crocidolite asbestos and MMVF-10 glass wool. Radical formation and scavenging were measured by electron spin resonance in buffer and culture medium with and without bovine serum albumin (BSA); cytotoxicity by trypan blue exclusion at 4, 24 and 48 hours. Considering all treatment times, asbestos was most cytotoxic (IC50 45, 16 and 9 micrograms per square centimetre at 4, 24 and 48 hours), though at 4 hours the short purified MWCNT had the lowest IC50 (20). Asbestos produced the most hydroxyl radicals in buffer; glass wool none in buffer with BSA. All carbon nanomaterials scavenged hydroxyl radicals in at least one solution. Both long, needle-like MWCNTs generated an unidentified radical (g-value 2.025) dose-dependently, with and without cells; for MWCNT-7 in cell-free buffer with BSA, the iron chelator deferoxamine did not remove it. The authors concluded this radical could relate to adverse effects of such MWCNTs.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory study: cell-free and cellular electron spin resonance (ESR) spectroscopy with spin trapping, cytotoxicity testing, and physicochemical characterisation of the test materials in exposure medium
• Who was studied: Transformed human bronchial epithelial BEAS 2B cells in culture, plus cell-free dispersions of eight test materials
• Exposure: Five MWCNTs (MWCNT-7 long needle-like; NM-401 long needle-like; MWCNT 8-15 nm OD long tangled; Baytubes C 150 HP short purified; NM-400 short non-purified), Printex 90 carbon black, UICC crocidolite asbestos and MMVF-10 glass wool, dispersed with or without 0.6 mg/ml BSA; cytotoxicity doses 5-350 micrograms per square centimetre, cell-free ESR at 1 mg/ml, cellular ESR at 10-80 micrograms per square centimetre
• Main results:
  • Cytotoxicity (IC50) in BEAS 2B cells: asbestos 45, 16 and 9 micrograms per square centimetre at 4, 24 and 48 hours; glass wool 90, 48, 66; MWCNT-7 (long needle-like 1) 128, 33, 27; NM-401 (long needle-like 2) 46, 60, 39; long tangled MWCNT 152 at 24 h only; short purified MWCNT 20, 174, 201; IC50 was not reached for short non-purified MWCNT or carbon black
  • Considering all treatment times, asbestos was the most cytotoxic, followed by the two long needle-like MWCNTs
  • The short purified MWCNT had the highest acute (4 h) cytotoxicity, which decreased with longer treatment
  • In cell-free conditions, hydroxyl radical formation was highest with asbestos in buffer with and without BSA; glass wool induced much less in buffer and none in buffer with BSA
  • No increase in hydrogen peroxide-induced radicals was seen with asbestos in culture medium alone, or with asbestos or glass wool in medium with BSA
  • All carbon nanomaterials scavenged hydroxyl radical induction in at least one solution; carbon black, short purified MWCNT and NM-401 scavenged in all four solutions, while MWCNT-7 scavenged only in buffer
  • The two long, needle-like MWCNTs produced a unique unidentified radical with a g-value of 2.025 and half-width of 1.7 mT
  • The deferoxamine (iron chelator) experiment was shown for MWCNT-7 (long needle-like 1) in cell-free buffer with BSA; the radical was not scavenged by deferoxamine, indicating it was not attributable to iron impurities
  • In the presence of cells, the unique radical from both long needle-like MWCNTs increased dose-dependently from 10 to 80 micrograms per square centimetre and coincided with the cytotoxicity of both materials
  • With cells present, asbestos at 10 micrograms per square centimetre produced a slight but significant level of hydroxyl radicals, while glass wool at the same dose did not
  • Materials with a high abundance of large (up to about 100 micrometre), open-structured agglomerates/aggregates and very high sedimentation rates also had strong cytotoxic effects
• The authors' conclusion: The authors concluded that specific ROS formation and material sedimentation rates affect cytotoxicity in BEAS 2B cells, that culture medium and BSA influence hydroxyl radical scavenging and production, and they speculated that the unique radical formed by the long, needle-like MWCNTs may be involved in the adverse effects of this type of MWCNT
• Limitations the authors state:
  • The authors stated the results should not be translated as such to the in vivo situation, that the relevance of large agglomerates during inhalation can be questioned, and that limitations of in vitro studies need to be considered
  • The authors stated that physicochemical characteristics need to be understood and controlled in greater detail to clarify the role of carbon and transition metal impurities and structural defects in radical formation and scavenging
  • The authors stated that possible chemical reactions between CNTs and the spin trap DMPO need to be addressed
  • The authors noted that time-dependent effects remain to be studied, since protein binding may be temporary and degradation of the protein corona may change radical formation and scavenging
  • At the highest dose the unique radical appeared more intense in cellular than cell-free settings, but variance between replicates was high and the difference was not statistically significant

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Reactive Oxygen Species (ROS), Surface Reactivity, Crocidolite, Hydroxyl Radical.

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