Effect of size fractionation on the toxicity of amosite and Libby amphibole asbestos

Our plain-English summary of Effect of size fractionation on the toxicity of amosite and Libby amphibole asbestos, by K.E. Duncan, A.J. Ghio, L.A. Dailey, et al (Toxicological Sciences, 2010).

The study

• Authors: K.E. Duncan, A.J. Ghio, L.A. Dailey, A.M. Bern, E.A. Gibbs-Flournoy, D.J. Padilla-Carlin, V.L. Roggli, R.B. Devlin
• Journal: Toxicological Sciences
• Published: 2010;118(2):420-34
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Abnormally high incidences of asbestos-related pulmonary disease have been reported in residents of Libby, Montana, because of occupational and environmental exposure to asbestos-contaminated vermiculite. The mechanism by which Libby amphibole (LA) causes pulmonary injury is not known. The purpose of this study is to compare the cellular stress responses induced in primary human airway epithelial cells (HAECs) exposed to a respirable size fraction (≤ 2.5 μm) of Libby amphibole (LA(2.5)) to a similar size fraction of a reference amphibole sample amosite (AM(2.5)). HAEC were exposed to 0, 2.64, 13.2, or 26.4 μg/cm(2) AM(2.5) or LA(2.5) or to equivalent doses of unfractionated amosite (AM) or LA for 2 or 24 h. Comparable messenger RNA transcript levels were observed for interleukin-8, cyclooxygenase-2, and heme oxygenase-1 in HAEC following a 24-h exposure to AM or LA. Conversely, exposure to AM(2.5) resulted in a 4- to 10-fold greater induction in these proinflammatory mediators compared with LA(2.5) after 24 h. Evaluation of the expression of 84 additional genes involved in cellular stress and toxicity responses confirmed a more robust response for AM(2.5) compared with LA(2.5) on an equal mass basis. Differences in total surface area (TSA) by gas adsorption, total particle number, or oxidant generation by the size-fractionated particles did not account for the observed difference in response. In summary, AM(2.5) and LA(2.5) are at least as potent in stimulating production of proinflammatory cytokines as unfractionated AM and LA. Interestingly, AM(2.5) was more potent at inducing a proinflammatory response than LA(2.5). This difference could not be explained by differences in mineral contamination between the two samples, TSA, or oxidant generation by the samples.

In plain English

This was a laboratory study of cultured cells, not people. The authors mainly compared Libby amphibole from near Libby, Montana, with reference amosite; crocidolite was an extra reference in some experiments. Airway lining cells were grown from eight healthy nonsmoking adult volunteers. Libby amphibole and amosite were each tested whole and as a fraction 2.5 micrometers or less in aerodynamic diameter. Doses reached 26.4 micrograms per square centimeter, for 2 or 24 hours. After 24 hours, whole amosite and whole Libby amphibole raised inflammation gene activity (messenger RNA) similarly. For the small fractions at equal dose, amosite raised IL-8 gene activity about fourfold more and COX-2 about tenfold more than Libby amphibole. Differences in particle numbers, surface area and oxidant generation did not account for this. The doses did not significantly reduce cell survival, and cells took up the small particles. The authors concluded that the small fractions were at least as potent as the whole samples in stimulating the cells' proinflammatory cytokine production. They called the findings preliminary, with cells from three or four donors per experiment.

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: Laboratory (in vitro) experiment on cultured primary human airway epithelial cells
• Who was studied: Primary human airway epithelial cells obtained by brush biopsy of the mainstem bronchus of 8 healthy, nonsmoking adult volunteers (6 male, 2 female). Dose-response experiments used cells from 3 donors (whole samples) or 4 donors (size-fractionated samples).
• Exposure: Libby amphibole (a mixture mainly of winchite, richterite and tremolite) from the Rainy Creek Complex near Libby, Montana, and a reference amosite sample. Each of these two was tested unfractionated and as a water-elutriated fraction with aerodynamic diameter of 2.5 micrometers or less. UICC crocidolite was used, unfractionated only, as an extra reference in parts of the study. Doses were 0, 2.64, 13.2 or 26.4 micrograms per square centimeter for 2 or 24 hours.
• Main results:
  • At 24 hours and 26.4 micrograms per square centimeter, whole amosite and whole Libby amphibole raised IL-8 messenger RNA 50 plus or minus 7.5 fold and 46 plus or minus 8.3 fold, respectively, relative to unexposed cells.
  • The responses to the whole samples were also comparable to those of cells exposed to crocidolite at the same concentration.
  • The small amosite fraction caused a fourfold greater induction of IL-8 messenger RNA and a 10-fold greater induction of COX-2 messenger RNA than the small Libby amphibole fraction at equal concentration. At 26.4 micrograms per square centimeter, IL-8 messenger RNA rose 120 plus or minus 25 fold with amosite versus 37 plus or minus 7.8 fold with Libby amphibole. COX-2 messenger RNA rose 16 plus or minus 2.8 fold versus 1.6 plus or minus 0.3 fold.
  • HO-1 messenger RNA, an oxidative stress marker, was raised about equally by the two small fractions: 4.5 plus or minus 0.3 fold for amosite and 5.1 plus or minus 0.6 fold for Libby amphibole.
  • After only 2 hours, the small fractions caused minimal increases in IL-8, COX-2 and HO-1 messenger RNA.
  • Measured IL-8 protein rose much less than expected from the messenger RNA changes. Because of likely particle interference with the protein assay, only messenger RNA changes were reported.
  • In an 84-gene stress and toxicity panel at 13.2 micrograms per square centimeter, the small amosite fraction raised CSF2 (GM-CSF) messenger RNA 54 plus or minus 35 fold, versus 6.0 plus or minus 3.7 fold for the small Libby amphibole fraction.
  • In the same panel, IL-6 messenger RNA rose 13-fold with the small amosite fraction but was not changed by the small Libby amphibole fraction.
  • The two small fractions had similar numbers of amphibole particles per milligram: 7.1 x 10^8 for amosite and 7.9 x 10^8 for Libby amphibole.
  • Libby amphibole samples, both whole and small fraction, had higher total surface area than the corresponding amosite samples.
  • Both amosite samples generated more hydroxyl radicals than both Libby amphibole samples. However, whole amosite and whole Libby amphibole produced equivalent inflammatory responses.
  • Amphibole particles in the small amosite fraction were longer on average than those in the small Libby amphibole fraction: a mean of 6.9 plus or minus 12 micrometers versus 1.9 plus or minus 2.1 micrometers.
  • The doses used did not significantly reduce cell viability over exposures of up to 24 hours.
  • By 24 hours, nearly all of the small particles had been taken up by the cells and were clustered around the nucleus.
  • No significant increase in TGF-beta1 messenger RNA, a pro-fibrotic factor, was seen after 24-hour exposure to the small fractions.
• The authors' conclusion: The authors concluded that the small size fractions of Libby amphibole and amosite were at least as potent as the unfractionated samples in stimulating epithelial cell production of proinflammatory cytokines. They also concluded that the small amosite fraction was more potent than the small Libby amphibole fraction. They said this difference was not explained by mineral contamination, surface area or oxidant generation.
• Limitations the authors state:
  • The authors described the findings as preliminary. They said the work needs repeating with cells from more donors, a wider range of concentrations and additional asbestos samples.
  • Experiments used cells from only 3 or 4 donors.
  • The study did not address whether the different size fractions differ in genotoxic potential, or the role of alveolar macrophages.
  • It is uncertain whether these in vitro results reflect what happens in exposed animals or humans.
  • Non-amphibole minerals, which differed between the samples, could have skewed the surface area measurements. Chemical differences in these minerals could not be excluded as a cause of the different responses.
  • IL-8 protein measurements were likely affected by particle interference, so only messenger RNA changes were reported.

Mines named here

Asbestos mines this study is about. Each mine's page says who ran it, when it worked, and what it produced.

• Libby vermiculite mine, Montana

All asbestos mines

Topics

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

Medical terms in this study, explained in our Lexicon: Amosite, Libby amphiboles, Environmental Exposure, vermiculite, Amphibole Group, Cytokine.

Related studies

• In vitro determinants of asbestos fiber toxicity: effect on the relative toxicity of Libby amphibole in primary human airway epithelial cells — K.E. Duncan, P.M. Cook, et al. (2014)
• Persistent effects of Libby amphibole and amosite asbestos following subchronic inhalation in rats — S.H. Gavett, C.U. Parkinson, et al. (2016)
• Long-term response of rats to single intratracheal exposure of Libby amphibole or amosite — J.M. Cyphert, D.J. Padilla-Carlin, et al. (2012)
• Comparative long-term toxicity of Libby amphibole and amosite asbestos in rats after single or multiple intratracheal exposures — J.M. Cyphert, D.J. Carlin, et al. (2015)
• Mechanisms of oxidative stress and alterations in gene expression by Libby six-mix in human mesothelial cells — J.M. Hillegass, A. Shukla, et al. (2010)
• Trees as reservoirs for amphibole fibers in Libby, Montana — T.J. Ward, T. Spear, et al. (2006)

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