overhead 49
Begin 04/14/09
Bases are targets for primary (direct) damage by ROS, and guanine, which is the most easily oxidized of the nucleobases, is particularly a target for the formation of lesions by oxidizing species. The most reactive of the ROS, hydroxyl radical, can react either by addition to guanine or abstraction of anelectron. An electron abstraction is synonymous with 1-electron oxidation and the terms can be used interchangeably. Reactions initiated by ionizing radiation, which are not of course initially chemical processes, also serve as models for one-electron oxidations by chemical oxidantsbecause in either case the net result of the process is ejection of an electron from the base. The next slide shows some of the products of hydroxyl radical oxidation of guanine by electron abstraction
[OH: hydroxyl radical oxidations of guanine]
The predominant product of one-electron abstraction is 8-oxo-guanine, which is readily bypassed if not repaired and we have already discussed 8-oxo-Gua as a mutagenic lesion leading to G → T transversions. Because it is the major product of Gua oxidation, 8-oxo-Gua, is currently used as a biomarker of oxidative stress. A second oxidation pathway via 1-electron oxidation involves the transient dehydroguanidinohydantoin (DGh), arising from a sequence of reactions starting with the trapping of superoxide by the Gua radical from the one-electron abstraction and continues through two hydrolytic reactions to yield an isolable product diaminooxazolone, abbreviated dZ. dIz is stable enough for isolation and the mutagenicity of both dIz and dZ have been investigated by replication on a template containing the lesions. Both are mutagenic and blocking (i.e., bypassed inefficiently) with prokaryotic polymerases. Their significance as mutagenic lesions in vivo has yet to be verified, however, it is clear if present in vivo, they occur and much lower levels than 8-oxo-guanine.
The next overhead shows the products of hydroxyl radical addition to dGuo. Three of the products arising from one-electron abstraction are also products of hydroxyl radical addition. 8-Oxo-Gua is the major product of this pathway, andimidazolone (dIz) and diamino oxazolone are also products of hydroxyl radical addition via trapping of dioxygen by the initial hydroxyl radical adduct. In addition, FapyG has also been characterized.
While dGuo is the most easily oxidized of the bases, dCyd and dThyd are also modified by hydroxyl radical, which adds directly to the bases. The next slide shows the major products identified.
[OH: Products of HO∙ with dGuo, dCyd, dThyd]
5-OH dCyd, a cis-5,6-dCyd glycol are formed from dCyd, and cis-5,6-dThyd glycol from dThyd.
A great deal of in vitro work has also been done using singlet oxygen, which is the first excited electronic state of dioxygen. The rationale for this seems to be interest in the damage arising from photodynamic therapy, in which singlet oxygen is generated as the active cytotoxic species, although singlet oxygen can also be generated in some chemical reactions and in oxidative bursts by neutrophils. Photodynamic therapy is probably the most significant mechanism by which singlet oxygen would be generated in cells in vivo, and it is therefore probably not a major processes induced through community environmental exposures. The next slide summarizes the oxidations of Gua by singlet oxygen.
[OH: 8-oxo-dGuo, Gh, Sp,]
8-oxo-dGuo is the initial product, with the subsequent oxidized species–spiroiminohydantoin and guanidinohydantoin, as major products and the diamino oxazolone, which I haven’t shown on the overhead, as a minor product.
It turns out that 8-oxo-dGuo has a lower oxidation potential than dGuo, so there has been in the last few years a great deal of interest in characterizing products of the oxidation of 8-oxo-dGuo, based on the rationale that this is a very common lesion and there is enough of it for a secondary oxidation product to be of concern. In addition to the oxidants arising from the cascade initiated by dismutation of superoxide, a reactive nitrogen transient, peroxynitrite, may be pertinent to the oxidation of 8-oxo-G. Peroxynitrite is a product of the reaction of the ubiquitous neurotransmitter nitric oxide (NO) and superoxide (which is generated simultaneously with NO). The importance of peroxynitrite as a DNA damaging agent in vivo is at this juncture speculative, although its reactions have been studied intensively in vitro. Interest has been motivated by the fact that both NO and O2- can be present in cells at locally high concentrations, and peroxynitrite is therefore a potentially a significant genotoxic exposure.
8-Oxo-dGuo from all sources of oxidation is present at relatively high levels in vivo (2/10+8 bases). It is important to note, however, thatof all the products of the chemistry that has been described to this point, only 8-oxo-Gua has been demonstrated in vivo. FapyG has been observed in cell culture and spiroiminodihydantoin has been generated in cell culture by treatment with K2Cr2O7 concentrations much higher than would occur physiologically.So the biological significance of the remaining lesions is described on the previous overheads is unknown. However, it is clear that any of the other lesions described here occur at much lower levels than 8-oxo-Gua.
Besides oxidation of the bases, hydroxyl radical also abstracts hydrogen from the deoxyribose backbone structure of DNA, resulting in lesions from reaction with sugars as well as nucleobases. In principle, all seven of the deoxyribose hydrogens are abstractable. Energetically, the order of reactivity has been calculated to be H1 >H3 H4 > H2, H2. The 5,5 hydrogens were not considered, but by virtue of their position to a hetero atom, which is the O of the 5-phosphate, they should be about as reactive as the 3 and 4 hydrogens. In double stranded DNA, accessibility as will as reactivity will determine the relative frequency of abstraction, and experimentally it has been determined that the 4 and 5,5 hydrogens are abstracted by hydroxyl radical, since they are the most exposed to solvent. Hydrogen abstraction at these positions results in strand breaks and the generation of small reactive fragments or reactive strand termini at strand breaks. The overhead summarizes the consequences of H4 and H5 or H5 abstraction:
[OH: Fig. 3, Tullius]
Left column gives products under aerobic conditions; right column, products under anaerobic conditions; center, products generated under both conditions. Above the dotted line are products of H4′ abstraction, and below the line, products of H5′, H5″ abstraction. “R” or “R′” represents the DNA chain. Of the scission products, the base propenal and furfural are recognized as biomarkers of oxidative stress and also are potential electrophiles forming promutagenic adducts with DNA.
The top panel of the next overhead shows the formation of a base propenal by abstraction of H4′ of the deoxyribose followed by trapping of the radical by O2. The bottom panel shows sthe base propenal derived from dAdo reacting with dGuo to give the tricyclic base 1,N2-propeno dGuo:
[OH: base propenal + dGuo M1G adduct]
On the next overhead, I have indicated in more detail the formation by Michael addition on the next slide. This mechanism is identical to the formation of M1G from the enol tautomer of malondialdehyde, where the leaving group from Michael addition would be OH-, rather than B-. (Define Michael addition
Furfural react is proposed to react with dAdo to give kinetin, the trivial name for N6-furfuryl dAdo.
[OH: Kinetin structure]
While both furfural and the dAdo adduct have been detected as products of oxidative stress, a correct mechanism for formation of this adduct has not been published. A paper in 1997 proposes coupling with the exocyclic amino group of dAdo via Schiff base formation, as shown on the next overhead.
[OH: kinetin formation]
but this product is oxidized by two electrons with respect to kinetin, and a reduction pathway is not suggested.
In addition to the hydroxyl radical generated from lipid peroxidation, radicals may also be generated by single electron oxidations of xenobiotics with low oxidation potentials like certain PAH. One electron oxidations are proposed to be mediated by peroxidases and even cytochrome P450. Abstraction of a single electron from a PAH generates a cation radical.
The next slide shows the product of a one-electron oxidation of PAH PB and 6-methyl BP. The oxidations yield fairly stable cation radical species, which have lifetimes sufficiently long to form covalent adducts with the purines in cell nuclei. PAH cation radicals have recently been of interest as contributors to PAH genotoxicity.
[OH: BP, 6-MePB cation radicals]
The cation radicals from several PAH have been demonstrated to form covalent adducts with dAdo and dGuo and several in vivo adducts that have been characterized are shown on the slide. The adducts have been detected in urine excreted by animals treated with the PAH.
[OH: cation radical adducts of BP and DMBA with dAdo and dGuo]
The adducts depurinate readily, as the overhead indicates, and the abasic sites are potentially mutagenic lesions. The fact that these adducts have been detected in vivo supports the potential importance of the one-electron oxidations, but the significance of such processes relative to the cytochrome P450-mediated metabolic transformations to diolepoxides has not been determined at this point. The next overhead is another FYI, but it shows that radical mechanisms are messy and difficult to work out. The slide represents the proposed adducts of the DMBA cation radical with dGuo in vitro using I2 as a 1-electron model oxidant:
[OH: pathway for adducts of DMBA cation radical with dGuo]
The oxidation of the PAH dibenzo[a,l]pyrene has also been investigated in vitro and adducts of its electrochemically generated cation radical with dAdo characterized.
[OH: adducts of DB[a,l]P cation radical with dAdo]
The final slide in this series shows the damage generated by reagents that abstract H1′ from the deoxyribose. Of the two compounds shown, the “ene-diyne” is of interest because it is an antineoplastic (i.e., anticancer) drug.
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