Calcium-Dependent Mitochondrial Formation of Species Mediating DNA Single Strand Breakage in U937 Cells Exposed to Sublethal Concentrations of Tert-Butylhydroperoxide

  1. Andrea Guidarelli1,
  2. Emilio Clementi2,3,
  3. Clara Sciorati3,
  4. Flaminio Cattabeni1 and
  5. Orazio Cantoni1
  1. 1Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale (A.G., F.C., O.C.), Università di Urbino, Urbino; 2Dipartimento di Farmacologia (E.C.), Università di Reggio Calabria, Catanzaro; 3Consiglio Nazionale delle Ricerche, Molecular and Cellular Pharmacology Centre (E.C., C.S.), DIBIT - San Raffaele Scientific Institute, Milano, Italy

    Abstract

    Treatment of U937 cells with a sublethal albeit DNA-damaging concentration of tert-butylhydroperoxide (tB-OOH) enhanced mitochondrial Ca++ uptake and ruthenium red (RR), a polycation that inhibits the calcium uniporter of mitochondria, significantly reduced the extent of DNA cleavage generated by the hydroperoxide. Release of Ca++ from the ryanodine(Ry)/caffeine(Cf)-sensitive stores further increased mitochondrial Ca++ uptake and elicited a parallel enhancement in DNA strand scission induced by tB-OOH that was prevented by both Ry and RR. DNA damage caused by tB-OOH alone or associated with either Cf or RR was prevented by iron chelators, insensitive to antioxidants and repaired with kinetics superimposable with those observed after treatment with H2O2. Cf enhanced the DNA-damaging effects of tB-OOH in permeabilized cells as well, and similar effects were observed upon addition of CaCl2. Cf did not further increase the formation of DNA lesions elicited by tB-OOH in the presence of CaCl2. The enhancing effects of Cf were prevented by RR and ryanodine, whereas those mediated by exogenous calcium were prevented only by RR. DNA strand scission caused by tB-OOH alone or associated with Cf in the permeabilized cell system was severely inhibited by ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid. The mechanism(s) whereby Ca++promotes the mitochondrial formation of species that will ultimately result in the formation of DNA lesions was subsequently analyzed using intact as well as permeabilized cells. Hydrogen peroxide was identified to be one of these species.

    Footnotes

    • Send reprint requests to: Dr. Orazio Cantoni, Istituto di Farmacologia e Farmacognosia, Università di Urbino, Via S. Chiara, 27, 61029 Urbino (PS), Italy.

    • Abbreviations:
      SSBs
      DNA single strand breaks
      tB-OOH
      tert-butylhydroperoxide
      EGTA
      ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid
      Tg
      thapsigargin
      Iono
      ionomycin
      Cf
      caffeine
      FCCP
      carbonyl cyanidep-(trifluoromethoxy)phenylhydrazone
      RR
      ruthenium red
      Ry
      ryanodine
      SOD
      superoxide dismutase, BHT, butylated hydroxytoluene
      DPPD
      N, N′-diphenyl-1,4-phenylene-diamine
      o-pt
      o-phenanthroline
      EDTA
      etylenediaminetetraacetic acid
      [Ca++]i
      intracellular free Ca++ concentration
      SERCA
      sarcoplasmic/endoplasmic reticulum Ca++- ATPase
      IP3
      inositol 1, 4, 5-trisphosphate
      ER
      endoplasmic reticulum
      • Received March 4, 1997.
      • Accepted June 23, 1997.
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    1. JPET October 1, 1997 vol. 283 no. 1 66-74
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