Detection of surface free radical activity of respirable industrial fibres using supercoiled phi X174 RF1 plasmid DNA

Our plain-English summary of Detection of surface free radical activity of respirable industrial fibres using supercoiled phi X174 RF1 plasmid DNA, by P.S. Gilmour, P.H. Beswick, D.M. Brown, et al (Carcinogenesis, 1995).

The study

• Authors: P.S. Gilmour, P.H. Beswick, D.M. Brown, K. Donaldson
• Journal: Carcinogenesis
• Published: 1995;16(12):2973-9
• Cited by: 50 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The ability of a number of respirable industrial fibres, amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man-made vitreous fibres (MMVFs) to cause free radical injury to plasmid phi X174 RFI DNA was assessed. The oxidative DNA damage was observed as depletion of supercoiled DNA after fibre treatment was quantified by scanning laser densitometry. The mechanism of fibre-mediated damage was determined by the use of the specific hydroxyl radical scavenger mannitol and the iron chelator desferrioxamine-B. The amosite and crocidolite asbestos caused substantial damage to DNA that was dose-related. The free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals, as determined by inhibition with mannitol. Asbestos fibre-mediated damage to DNA was completely ameliorated by the chelation of fibre-associated iron with desferrioxamine-B. The amount of Fe(II) and Fe(III) released by equal numbers of the different fibre types at equal fibre number was determined. The fibres released very small amounts of Fe(II) and there were no significant differences between the fibre types. The fibres released substantial amounts of Fe(III); MMVF 21 released significantly more Fe(III) than any of the other fibres and short fibre amosite also released more Fe(III) than three of the MMVFs and two of the RCFs. When ability to release Fe(II) and Fe(III) was compared with ability to cause DNA damage there was not a good correlation, because only the long amosite and crocidolite caused substantial free radical injury to DNA; this contrasts with MMVF 21 and short amosite being the two fibres that released the greatest amounts of iron. The loss of ability to damage DNA in DSF-B-treated asbestos fibres shows that iron at the surface of asbestos fibres definitely has a role in generating hydroxyl radicals. However, it is clear that some fibres, such as short amosite and MMVF 21, release large quantities of iron without causing free radical damage, whilst neither long amosite nor crocidolite released more iron than the other fibres. The exact role of iron in fibre reactivity therefore remains unresolved, but fibre-bound iron not released from the surface of asbestos could be important. Further research is under way to investigate this possibility.

In plain English

In this laboratory experiment, the authors tested whether industrial fibres damage DNA through free radicals. They mixed plasmid DNA with long and short amosite asbestos, crocidolite, four refractory ceramic fibres (RCFs) and four man-made vitreous fibres (MMVFs); fibres were generally used at equal fibre number, but long versus short amosite was compared at equal mass. Long amosite reduced intact (supercoiled) DNA by a mean of 55.5% and crocidolite by 43.0%. Damage rose with fibre number, and long amosite damaged DNA significantly more than short amosite. Mannitol, which removes hydroxyl radicals, partly restored supercoiled DNA (15.7% to 47.9% with long amosite); the authors reported that chelating fibre iron with desferrioxamine-B completely ameliorated the asbestos damage. MMVFs caused no statistically significant damage; RCF damage was small and significant only when the four were pooled. MMVF 21 released the most iron yet barely damaged DNA. The authors concluded that asbestos free radical activity was much greater than that of RCFs and MMVFs, that surface iron was involved for amosite and crocidolite, and that iron release alone was not sufficient.

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 (cell-free, in vitro) experiment measuring fibre-caused damage to plasmid DNA
• Who was studied: phiX174 RF plasmid DNA exposed to long and short amosite asbestos, crocidolite asbestos, refractory ceramic fibres RCF 1, 2, 3 and 4 (kaolin-based, high purity, zirconium-containing, and high-temperature-treated RCF 1), and man-made vitreous fibres MMVF 10 and 11 (fibre glass) and MMVF 21 and 22 (insulation wool). The work was done at Napier University, Edinburgh, UK. Crocidolite, the RCFs and the MMVFs came from the TIMA Fibre Repository.
• Exposure: Plasmid DNA was incubated for 8 h at 37°C with each fibre type at equal fibre numbers of 616 600, 924 900 or 1 233 200 WHO-defined fibres; the comparison of short with long amosite was described by the authors as made at equal mass. In some tests the fibres were pretreated for 24 h with 10 mM desferrioxamine-B (an iron chelator). In others, 4 mM mannitol (a hydroxyl radical scavenger) was added. Release of Fe(II) and Fe(III) from the fibres was measured over 8 h.
• Main results:
  • Long amosite reduced supercoiled (intact) plasmid DNA by a mean of 55.5% and crocidolite by 43.0%. Both differed significantly from untreated DNA (P less than 0.001).
  • DNA damage from asbestos increased with fibre number (Table II; each data point was from a single experiment).
  • Long amosite caused a significantly greater reduction in supercoiled DNA than short amosite (P less than 0.01); the authors state this comparison was at equal mass.
  • With long amosite, supercoiled DNA was 15.7%, rising to 47.9% when 4 mM mannitol was added (partial protection; untreated control was about 70-80%).
  • With crocidolite, supercoiled DNA was 15.64%, rising to 54.8% when mannitol was added.
  • Pretreating crocidolite and amosite with the iron chelator desferrioxamine-B reduced the DNA damage they caused; in their summary the authors describe this damage as completely ameliorated.
  • The four MMVFs showed no significant overall treatment effect, despite an average 6.2% depletion of supercoiled DNA.
  • The RCFs showed no significant overall effect when analysed by individual fibre. When all RCF treatments were pooled, supercoiled DNA was 62.9 ± 1.7 versus 71.2 ± 2.5 in untreated controls, a significant difference (P less than 0.05).
  • Mannitol consistently reduced the small amount of DNA damage caused by MMVFs and RCFs. Desferrioxamine-B did not protect against it, unlike with asbestos.
  • No fibre type released substantial amounts of Fe(II), and there was no significant difference between fibres.
  • All fibres released Fe(III). MMVF 21 released the most, significantly more than any other fibre, but had negligible ability to cause DNA damage.
  • Compared at equal mass, short amosite released more Fe(III) than long amosite but had little ability to damage DNA. Table V gives mean Fe(III) values of 107.4 for short amosite and 43.2 for long amosite; units are not stated in the table.
• The authors' conclusion: The authors concluded that the free radical activity of asbestos is much greater than that of RCFs and MMVFs, and that surface iron was involved in generating hydroxyl radicals for amosite and crocidolite. They found that RCFs and MMVFs caused detectable but minimal hydroxyl radical damage that did not involve iron. They also concluded that release of iron alone was not sufficient to cause free radical DNA damage in the case of short amosite and MMVF 21.
• Limitations the authors state:
  • The fibre samples may have differed in surface area. The RCFs tended to be slightly longer, even though comparisons were made at equal fibre number.
  • Particles too small to meet the WHO fibre definition may have added surface area and affected the results.
  • The low activity of short amosite might partly reflect its many fibre 'ends', which may have different surface chemistry. The authors considered this unlikely.
  • Iron release was measured in buffer alone. In the body, natural chelators might release iron from fibres differently.

Topics

Medical terms in this study, explained in our Lexicon: Amosite, Crocidolite, Vitreous, Hydroxyl Radical.

Related studies

• Supercoiled plasmid DNA as a model target for assessing the generation of free radicals at the surface of fibres — K. Donaldson, P.S. Gilmour, et al. (1995)
• Free radical activity of natural and heat treated amphibole asbestos — C. Otero Areán, F. Barceló, et al. (2001)
• Free radical activity associated with the surface of particles: a unifying factor in determining biological activity? — K. Donaldson, P.H. Beswick, et al. (1996)
• Free radical activity of synthetic vitreous fibers: iron chelation inhibits hydroxyl radical generation by refractory ceramic fiber — D.M. Brown, C. Fisher, et al. (1998)
• Free radical activity of industrial fibers: role of iron in oxidative stress and activation of transcription factors — P.S. Gilmour, D.M. Brown, et al. (1997)
• Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles — A. Valavanidis, T. Vlachogianni, et al. (2009)

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