Three chemically distinct types of oxidants formed by iron-mediated Fenton reactions in the presence of DNA.

Abstract

Exposure of Escherichia coli to H202 leads to two kinetically distinguishable modes of killing: mode I killing occurs maximally near 2 mM H202, whereas mode II killing is essentially independent of H202 concentrations up to 20 mM.A major portion of H202 toxicity is attributed to DNA damage caused by the iron-mediated Fenton reaction. By studying DNA damage during Fenton reactions in vitro, the same complex kinetics were observed and three types of oxidants were distinguished based upon their reactivities toward H202 and alcohols and upon iron-chelator effects. but the unusual biphasic dose-response results to H202 observed for both killing and DNA nicking could indicate that there might be at least two types of oxidants. The objective of this study is to further investigate this possibility.MATERIALS AND METHODS H202 (30% solution) and 2-methyl-2-propanol ("t-butanol") were from Fisher Scientific. FeSO4, 1,10-phen, 4,7-phenanthroline (4,7-phen), 2,9-dimethyl-1,10-phenanthroline (dmphen), and 2,2'-dipy were from Aldrich. Ethanol was from Quantum Chemical (Tuscola, IL). Bacteriophage PM2 DNA labeled with [methyl-3H]thymidine (5) was isolated by detergent and phenol extraction, followed by density gradient centrifugation. The DNA was dialyzed against EDTA for at least 2 days and was not sensitive to H202 alone.Killing and Mutagenesis Analysis of E. coli. Strain AB1157 (WT) was exposed to H202 for 15 min and then plated. Where indicated, 1 mM 1,10-phen or 1 mM 2,2'-dipy was added to the cells 5 min prior to the challenge with H202.DNA Nicking Determination. PM2 [3H]DNA (17 AM nucleotide residues) was mixed with 0.8% NaCl/80 nM FeSO4 and 320 nM chelator ethanol and/or H202 as indicated. Reactions were stopped after 7.5 min at 25°C by the addition of catalase at 2 ,g/ml. Nicking of the DNA was quantitated by nitrocellulose filter binding as described (6).Other Methods. H202 concentrations were measured by UV absorption at 240 nm by assuming 8240 = 39.4 M-1 cm-1 (7) or by a peroxidase dye assay (8). Ferrous concentrations were determined by colorimetry with 1,10-phen by assuming that the Fe2+(1,10-phen)3 complex has an E510 = 1.11 X 104 M-1 cm-1 (9). The concentration of PM2 DNA was determined by UV absorption at 260 nm by assuming -260 = 6.5 mM-'-cm-'.RESULTS AND DISCUSSION Classification of Oxidants Formed by the Fenton Reaction in the Presence of DNA. In the presence of Fe2+ and H202, DNA nicking is maximal at 0.05 mM H202 then drops to about one-third that level at 3 mM H202, beyond which it remains relatively constant (Fig. 1). This dose-response result suggests that at least some species of the oxidant(s) is sensitive to H202.We were able to ascribe inhibition of aniline dye bleaching by higher concentrations of H202 to simple competition between H202 and the dye for -OH (4) in which H202 reacts with -OH to form the less active 'HO2 radical:H202 + 'OH -* 'HO2 + H20.[2]

Sequence-specific DNA Cleavage by Fe2+-mediated Fenton Reactions Has Possible Biological Implications

“…These results support the observation that RGGG is the preferential cleavage site for type II oxidations and provides further evidence for the prediction that the iron-DNA associations that lead to type II oxidations are due to iron imbedded in the base stack next to purines (9). The requirement for preincubation of the Fe 2ϩ and DNA would suggest a minimal contribution of the RGGG sequence acting as a sink of electrons (or electron holes) traversing the base stack, and favors specific Fe 2ϩ binding.…”

“…DNA is a major target in H 2 O 2 -mediated cell killing through such reactions (2,3), but the oxidizing species involved are probably not freely diffusible hydroxyl radicals ( ⅐ OH), but possibly a localized ⅐ OH and/or related iron-oxo species (4 -8) whose properties are apparently governed by the chelation state of the iron upon which they are generated (9). Because DNA can chelate Fe 2ϩ in several ways, DNA damage in vivo in prokaryotes (2) and eukaryotes (10,11), and in vitro (2,9) Hence, these oxidants appear to damage DNA only at lower H 2 O 2 concentrations; they are also scavenged by alcohols.…”

“…Because DNA can chelate Fe 2ϩ in several ways, DNA damage in vivo in prokaryotes (2) and eukaryotes (10,11), and in vitro (2,9) Hence, these oxidants appear to damage DNA only at lower H 2 O 2 concentrations; they are also scavenged by alcohols. The constant rate of damage observed with H 2 O 2 concentrations between 5 and 50 mM was defined to be due to type II oxidants.…”

“…The constant rate of damage observed with H 2 O 2 concentrations between 5 and 50 mM was defined to be due to type II oxidants. In this case DNA damage might be induced by the reaction of H 2 O 2 with Fe 2ϩ atoms that are intimately bound to DNA such that nascent oxidants react with the DNA before encountering exogenous scavengers such as H 2 O 2 or ethanol (9). Alternatively, one could propose that type II oxidants do not react with H 2 O 2 or ethanol.…”

Photoelectrochemical Detection of Oxidative DNA Damage Induced by Fenton Reaction with Low Concentration and DNA-Associated Fe2+

“…[3][4][5][6][7][8] However, there has been no direct experimental evidence to support such hypothesis. The surface reaction mode in our photoelectrochemical sensor provided a unique opportunity to investigate the role of DNA-associated metals in the Fenton reaction.…”