Cytotoxicity of oxidants and asbestos fibers in cultured human mesothelial cells

Our plain-English summary of Cytotoxicity of oxidants and asbestos fibers in cultured human mesothelial cells, by V.L. Kinnula, K. Aalto, K.O. Raivio, et al (Free Radical Biology & Medicine, 1994).

The study

• Authors: V.L. Kinnula, K. Aalto, K.O. Raivio, S. Walles, K. Linnainmaa
• Journal: Free Radical Biology & Medicine
• Published: 1994;16(2):169-76
• Cited by: 48 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The authors investigated the mechanisms caused by oxidants (superoxide and hydrogen peroxide) and asbestos (amosite) fibers in human mesothelial cells. Immortalized human pleural mesothelial cells (MET 5A) were exposed in vitro to one of the following: hypoxanthine (100-200 microM) plus xanthine oxidase (10-20 mU/ml) as a superoxide-generating system, H2O2 (50 microM-5 mM); or amosite (1-100 micrograms/cm2). Cellular adenine nucleotide depletion, DNA single strand breaks, extracellular release of nucleotides, and their catabolites and lactate dehydrogenase (LDH) were assessed as markers of cell damage after 4-6 h exposure to the oxidants or fibers. The effect of intracellular antioxidant enzymes and exogenous antioxidants on cell damage were investigated during oxidant and amosite exposure. Superoxide radical and H2O2 exposure resulted in the depletion of adenine nucleotides, accumulation of the products of nucleotide catabolism, induction of DNA single strand breaks, and extracellular LDH release. Amosite exposure did not cause nucleotide depletion or induction of DNA single strand breaks. Inactivation of the intracellular antioxidant enzymes glutathione reductase or catalase augmented cell damage during H2O2 exposure but not during amosite exposure.

In plain English

In this laboratory study of cells grown in dishes, the authors exposed an immortalized line of human pleural mesothelial cells (MET 5A) to amosite asbestos fibers (1 to 100 micrograms per square centimetre) or, for comparison, to hydrogen peroxide or a superoxide-generating system. Most tests used 4-hour exposures; for adenine nucleotide tests, cells were exposed for 1 hour, then incubated a further 16 or 6 hours. Both oxidants depleted adenine nucleotides, which are energy-carrying molecules. Hydrogen peroxide also caused DNA single strand breaks and release of LDH, a cell-damage marker; 5 mM released 87% of cellular LDH. Amosite did not deplete adenine nucleotides or cause DNA single strand breaks; only the highest dose produced marginal LDH release. Blocking the cells' antioxidant enzymes increased hydrogen peroxide damage but made no difference with amosite. The authors concluded that short-term amosite exposure did not cause free-radical damage to these cells, suggesting other mechanisms, such as mechanical membrane injury, may be involved. They noted that cultured cells may differ from cells in the body and that oxidant injury in living bodies was not excluded.

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) cell culture experiment. The adenine nucleotide tests used a 1-hour exposure followed by a further 16 hours of incubation, or 6 hours in the higher-dose amosite experiments.
• Who was studied: MET 5A, an immortalized human pleural mesothelial cell line made from a primary mesothelial cell culture transformed with SV40 antigen. The authors describe the cells as near-diploid and nontumorigenic.
• Exposure: Amosite fibers (UICC standard) at 1 to 100 micrograms per square centimetre. For comparison, cells were exposed to hydrogen peroxide (50 micromolar to 5 mM) and to hypoxanthine plus xanthine oxidase, which generates superoxide. The DNA strand break and LDH experiments used 4-hour exposures. The adenine nucleotide experiments used a 1-hour exposure followed by further incubation.
• Main results:
  • In cells exposed to hydrogen peroxide (50 to 100 micromolar) or to xanthine oxidase plus hypoxanthine, intracellular adenine nucleotide levels fell in all cases.
  • Amosite at 1 to 5 micrograms per square centimetre caused no changes in adenine nucleotides or their breakdown products. At concentrations 10 times higher, results did not differ significantly from controls.
  • Hydrogen peroxide induced DNA single strand breaks in the cells.
  • Amosite did not cause DNA single strand breaks, even at high concentrations. This held both in intact cells and in cells whose antioxidant enzymes had been inactivated.
  • Exposure to 5 mM hydrogen peroxide released 87% of the cells' LDH.
  • Only very high amosite concentrations (100 micrograms per square centimetre) produced marginal LDH release compared with control cells, at p less than 0.1.
  • Basal glutathione reductase activity in MET 5A cells was 18.5 plus or minus 6.2 mU per mg protein (n = 4). Basal catalase activity was 18.2 plus or minus 7.6 nmol oxygen produced per minute per mg protein (n = 6).
  • Inactivating glutathione reductase significantly increased hydrogen peroxide injury. Inactivating catalase caused significantly more injury at higher than at lower hydrogen peroxide concentrations.
  • When cells were exposed to amosite after these enzymes were inactivated, there was no difference between untreated and treated cells.
  • With hydrogen peroxide, added catalase, glutathione or desferoxamine prevented DNA strand breaks and LDH release, and 3-aminobenzamide gave partial protection. Amosite caused no DNA strand breaks or LDH release in untreated or pretreated cells.
• The authors' conclusion: The authors concluded that acute amosite exposure does not induce free radical damage in mesothelial cells in vitro. They suggested that other mechanisms, such as mechanical membrane injury rather than acute oxidant-type damage, may be involved.
• Limitations the authors state:
  • Cell characteristics may change in culture, so the cells may not reflect the same cells in the body.
  • Antioxidant enzymes in these cells may have changed compared with cells in their natural location.
  • The incubation time was short, 4 to 6 hours.
  • The results do not exclude the possibility that amosite and other asbestos fibers may cause oxidant-type injury in the living body.
  • The authors note that amosite doses even as low as 2 micrograms per square centimetre have been shown to be unphysiological.

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelial Cells, Superoxide, Amosite, Lactate Dehydrogenase (LDH), LDH Release, Glutathione (GSH).

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