Iron mobilization from crocidolite asbestos greatly enhances crocidolite-dependent formation of DNA single-strand breaks in phi X174 RFI DNA

Our plain-English summary of Iron mobilization from crocidolite asbestos greatly enhances crocidolite-dependent formation of DNA single-strand breaks in phi X174 RFI DNA, by L.G. Lund, A.E. Aust (Carcinogenesis, 1992).

The study

• Authors: L.G. Lund, A.E. Aust
• Journal: Carcinogenesis
• Published: 1992;13(4):637-42
• Cited by: 97 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The ability of the iron associated with asbestos to catalyze damage to phi X174 RFI DNA was determined and compared with iron mobilized from asbestos. Asbestos (1 mg/ml) suspended for 30 min in 50 mM NaCl containing 0.5 micrograms phi X174 RFI DNA, pH 7.5, did not catalyze detectable amounts of DNA single-strand breaks (SSB). However, addition of ascorbate (1 mM) resulted in 19, 26, 7 or 8% DNA with SSB for crocidolite, amosite, chrysotile or tremolite respectively. The percentage of DNA with SSB induced by each form of asbestos was directly related to its iron content. Inclusion of desferrioxamine B, which binds Fe(III) rendering it redox inactive, completely inhibited asbestos-dependent formation of DNA SSB, suggesting that iron was responsible for catalyzing the formation of DNA SSB. Mobilization of Fe(II) from crocidolite by citrate, EDTA or nitrilotriacetate (1 mM) in the absence of ascorbate resulted in 15, 33 or 63% DNA with SSB respectively. This activity was completely inhibited by compounds considered to be .OH scavengers, i.e. mannitol, 5,5-dimethyl-1-pyrroline N-oxide or salicylate (100 mM). Preincubation of crocidolite with citrate (1 mM) for 24 h resulted in mobilization of 52 microM iron and increased ascorbate-dependent induction of DNA SSB compared with crocidolite that was preincubated without citrate. Iron mobilized by citrate was entirely responsible for crocidolite-dependent formation of DNA SSB as evidenced by complete inhibition with desferrioxamine B. Therefore, the results of the present study strongly suggest that iron was responsible for asbestos-dependent generation of oxygen radicals, which resulted in the formation of DNA SSB. Mobilization of iron by chelators, followed by redox cycling, greatly enhanced crocidolite-dependent formation of DNA SSB. Thus, mobilization of iron in vivo by low mol. wt chelators may lead to the increased production of reactive oxygen species resulting in damage to biomolecules, such as DNA.

In plain English

This cell-free laboratory experiment tested whether iron associated with, or released from, asbestos could break strands of bacteriophage phi X174 RFI DNA. Asbestos (1 mg/ml crocidolite, amosite, chrysotile or tremolite, with 36, 36, 2 and 0.3% iron by weight) was incubated 30 minutes with 0.5 micrograms of DNA, with or without 1 mM ascorbate; the chelators citrate, EDTA and nitrilotriacetate (NTA) were tested with crocidolite. Asbestos alone produced no detectable single-strand breaks (SSB). With ascorbate, DNA with SSB was 19 plus or minus 4% (crocidolite), 26 plus or minus 8% (amosite), 7 plus or minus 2% (chrysotile) and 8 plus or minus 3% (tremolite), tracking iron content. Desferrioxamine B or ferrozine abolished the effect. Without ascorbate, crocidolite with citrate, EDTA or NTA gave 15, 33 or 63% DNA with SSB, citrate requiring 2 hours; hydroxyl radical scavengers blocked this. Preincubating crocidolite 24 hours with citrate mobilized 52 micromolar iron and, in a later 30-minute assay with ascorbate, raised SSB 7.7-fold over crocidolite preincubated without citrate; without ascorbate none were detected. The authors concluded that their results strongly suggest iron, especially mobilized and redox-cycled iron, was responsible.

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, cell-free biochemical experiment measuring DNA single-strand breaks in phi X174 RFI DNA exposed to asbestos, chelators and ascorbate
• Who was studied: No people or animals; purified closed-circular superhelical phi X174 RFI bacteriophage DNA (0.5 micrograms per assay) in buffer, each experiment repeated three times, in a laboratory at Utah State University
• Exposure: Crocidolite, amosite, chrysotile and tremolite asbestos at 1 mg/ml (iron contents 36, 36, 2 and 0.3% by weight), with or without 1 mM ascorbate; chelators citrate, EDTA and NTA (1 mM) were used with crocidolite, plus inhibitors desferrioxamine B, ferrozine and the hydroxyl radical scavengers mannitol, DMPO or salicylate (100 mM)
• Main results:
  • With 1 mM ascorbate and 30 minutes' incubation, the percentage of DNA with single-strand breaks was 19 plus or minus 4 for crocidolite, 26 plus or minus 8 for amosite, 7 plus or minus 2 for chrysotile and 8 plus or minus 3 for tremolite
  • Asbestos alone, without ascorbate, produced no detectable DNA single-strand breaks
  • Desferrioxamine B or ferrozine completely inhibited the formation of DNA SSB by all four asbestos types
  • Without ascorbate, mobilization of Fe(II) from crocidolite by citrate, EDTA or NTA (1 mM) gave 15, 33 or 63% DNA with SSB respectively
  • EDTA or NTA added to crocidolite and DNA produced breaks within a 30-minute incubation, whereas citrate required 2 hours for DNA SSB to be observed
  • 100 mM mannitol, salicylate or DMPO completely inhibited crocidolite-dependent DNA SSB formation with citrate, EDTA or NTA in the absence of ascorbate; salicylate mobilized iron at 6.7 nmol Fe/mg crocidolite/h while mannitol and DMPO mobilized no detectable iron
  • Preincubating crocidolite 24 h with 1 mM citrate mobilized 52 micromolar iron and, when breaks were then initiated with 1 mM ascorbate over 30 minutes, increased DNA with SSB 7.7-fold compared with preincubation without citrate; no SSB were detected without ascorbate
  • In the 24-hour preincubation experiment, no DNA SSB were detected in the absence of ascorbate
  • The supernatant containing only mobilized iron (crocidolite removed) produced the DNA breaks, indicating mobilized iron was responsible
  • A control of freshly prepared 50 micromolar Fe(III) with citrate catalyzed 52% DNA with SSB, within 4% of the crocidolite-plus-citrate sample with comparable mobilized iron
  • Anaerobically, 42% of total iron mobilized by NTA and 19% of that mobilized by citrate was Fe(II)
• The authors' conclusion: The authors concluded that the results strongly suggest iron was responsible for asbestos-dependent DNA single-strand break formation, and that iron mobilized from crocidolite by citrate or EDTA followed by redox cycling catalyzed DNA breaks much more readily than iron remaining on the fiber; they suggested this could lead to reactive oxygen species and damage to biomolecules such as DNA
• Limitations the authors state:
  • The authors stated the in vivo relevance of their findings was not clear
  • The authors noted that because ascorbate can itself mobilize iron from asbestos, it was unclear whether the observed activity came from iron associated with the fiber or iron mobilized by ascorbate
  • For the iron-mobilization experiment (Table III) the authors reported percentages from a single experiment, repeated at least twice

Topics

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

Medical terms in this study, explained in our Lexicon: Crocidolite, Amosite, Chrysotile, Tremolite, Desferrioxamine B, Redox Cycling, Reactive Oxygen Species (ROS).

Related studies

• Iron associated with asbestos bodies is responsible for the formation of single strand breaks in phi X174 RFI DNA — L.G. Lund, M.G. Williams, et al. (1994)
• The effect of iron binding on the ability of crocidolite asbestos to catalyze DNA single-strand breaks — J.A. Hardy, A.E. Aust (1995)
• Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals — D.W. Kamp, V.A. Israbian, et al. (1995)
• Iron-dependent formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA102 induced by crocidolite — S.P. Faux, P.J. Howden, et al. (1994)
• Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers — A. Puhakka, T. Ollikainen, et al. (2002)
• Mobilization of iron from urban particulates leads to generation of reactive oxygen species in vitro and induction of ferritin synthesis in human lung epithelial cells — K.R. Smith, A.E. Aust (1997)

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