Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes

Ravanat, Jean‐Luc; Sauvaigo, Sylvie; Caillat, Sylvain; Martinez, Gláucia Regina; Medeiros, Marisa H. G.; Mascio, Paolo Di; Favier, Alain; Cadet, Jean

doi:10.1515/bc.2004.003

Cited by 75 publications

(60 citation statements)

References 37 publications

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“…Diphenylacetaldehyde was purchased from Aldrich. Ile 7 -angiotensin III (AngIII), human adrenocorticotropic hormone fragments [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]), ␣-cyano-4-hydroxycinnamic acid, sinapinnic acid, and trifluoroacetic acid were obtained from Amersham Biosciences (Upsala, Sweden). Sequencing grade trypsin was purchased from Promega (Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

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Changes in the Spin State and Reactivity of Cytochrome c Induced by Photochemically Generated Singlet Oxygen and Free Radicals

Estevam

Nascimento

Baptista

et al. 2004

Journal of Biological Chemistry

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This work compares the effect of photogenerated singlet oxygen (O 2 ( 1 ⌬ g )) (type II mechanism) and free radicals (type I mechanism) on cytochrome c structure and reactivity. Both reactive species were obtained by photoexcitation of methylene blue (MB ؉ ) in the monomer and dimer forms, respectively. The monomer form is predominant at low dye concentrations (up to 8 M) or in the presence of an excess of SDS micelles, while dimers are predominant at 0.7 mM SDS. Over a pH range in which cytochrome c is in the native form, O 2 ( 1 ⌬ g ) and free radicals induced a Soret band blue shift (from 409 to 405 nm), predominantly. EPR measurements revealed that the blue shift of the Soret band was compatible with conversion of the heme iron from its native low spin state to a high spin state with axial symmetry (g ϳ 6.0). Soret band bleaching, due to direct attack on the heme group, was only detected under conditions that favored free radical production (MB ؉ dimer in SDS micelles) or in the presence of a less structured form of the protein (above pH 9.3). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the heme group and the polypeptide chain of cytochrome c with Soret band at 405 nm (cytc405) revealed no alterations in the mass of the cytc405 heme group but oxidative modifications on methionine (Met 65 and Met 80 ) and tyrosine (Tyr 74 ) residues. Damage of cytc405 tyrosine residue impaired its reduction by diphenylacetaldehyde, but not by ␤-mercaptoethanol, which was able to reduce cytc405, generating cytochrome c Fe(II) in the high spin state (spin 2).

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Section: Methodsmentioning

confidence: 99%

“…This excited species, which can be generated by an energy transfer mechanism (6,7), exhibits high reactivity and can be produced in biological systems (8,9). In mammalian cells, O 2 ( 1 ⌬ g ) can be generated during oxidative stress, and it is able to attack DNA, protein thiol groups, and membrane lipids (11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

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confidence: 99%

Changes in the Spin State and Reactivity of Cytochrome c Induced by Photochemically Generated Singlet Oxygen and Free Radicals

Estevam

Nascimento

Baptista

et al. 2004

Journal of Biological Chemistry

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“…In contrast to the water soluble, thermolabile endoperoxide-derived 1 O 2 used in the above study, which is generated in cell culture medium, we would argue that 6-TGderived 1 O 2 is generated in relative close proximity to DNA. On this basis, levels of 8-oxodG and its secondary oxidation products would be expected to be much greater compared to the system used by Ravanat et al [40], and hence could account for the greater number of ALS we report. It has been reported that performing unwinding and electrophoresis at pH ~ 12 versus pH > 13 can discriminate between agents which induce SSB alone, and those that induce ALS [43].…”

Section: Discussionmentioning

confidence: 58%

“…Whether 1 O 2 can generate Azathioprine and UVA = 8-oxodG 14 SSB and AP sites in cellular DNA has been the subject of some debate. However, a thorough study by Ravanat et al [40], using a pure 1 O 2 -generating system, concluded that 1 O 2 does not produce significant levels of SSB or AP sites. The authors went on to suggest that low levels of ALS are the consequence of subsequent oxidation of 8-oxodG to products such as imidazolone, oxazolone [41] and oxaluric acid [42], some of which are alkali-labile [32].…”

Section: Discussionmentioning

confidence: 99%

“…The authors went on to suggest that low levels of ALS are the consequence of subsequent oxidation of 8-oxodG to products such as imidazolone, oxazolone [41] and oxaluric acid [42], some of which are alkali-labile [32]. It is also possible that SSB arise, transiently, from endogenous, cellular repair activity towards DNA damage [40] e.g. hOGG1.…”

Section: Discussionmentioning

confidence: 99%

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Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine

Cooke¹,

Duarte²,

Cooper³

et al. 2008

DNA Repair

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Thiopurine antimetabolites, such as azathioprine (Aza) and 6-thioguanine (6-TG), are widely used in the treatment of cancer, inflammatory conditions and organ transplantation patients. Recent work has shown that cells treated with 6-TG and UVA generate ROS, with implied oxidatively generated modification of DNA. In a study of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in renal transplant patients, we provided the first in vivo evidence linking Aza and oxidatively damaged DNA. Using the hOGG1 comet assay we herein demonstrate high levels of 8-oxodG and alkali-labile sites (ALS) in cells treated with biologically relevant doses of 6-TG, or Aza, plus UVA. This damage was induced dose-dependently. Surprisingly, given the involvement of 6-TG incorporation into DNA in its therapeutic effect, significant amounts of 8-oxodG and ALS were induced in quiescent cells, although less than in proliferating cells. We speculate that some activity of hOGG1 towards unirradiated, 6-TG treated cells, implies possible recognition of 6-TG or derivatives thereof. This is the first report to conclusively demonstrate oxidatively damaged DNA in cells treated with thiopurines and UVA. These data indicate that Aza-derived oxidative stress will occur in the skin of patients on Aza, following even low level UVA exposure. This is a probable contributor to the increased risk of non-melanoma skin cancer in these patients. However, as oxidative stress is unlikely to be involved in the therapeutic effects of Aza, intercepting ROS production in the skin could be a viable route by which this side effect may be minimised.Azathioprine and UVA = 8-oxodG 3

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Peroxides in Biological Systems

Cadet

Mascio

2009

Patai's Chemistry of Functional Groups

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Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes

Cited by 75 publications

References 37 publications

Changes in the Spin State and Reactivity of Cytochrome c Induced by Photochemically Generated Singlet Oxygen and Free Radicals

Changes in the Spin State and Reactivity of Cytochrome c Induced by Photochemically Generated Singlet Oxygen and Free Radicals

Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine

Peroxides in Biological Systems

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