Several complementary analytical methods, including 32
and quantitative MS/MS,28
have been used to establish the presence of CEdG in human DNA. Initial measurements of adduct levels in human tissue using isotope dilution mass spectrometry range from 1 to 10 CEdG adducts/107
dGs, suggesting it is a relatively abundant adduct in vivo.28
These values are similar to those measured for the oxidative stress marker 8-hydroxy-2′-deoxyguanosine in urine and tissue.38
The miscoding properties of CEdG have been investigated using synthetic oligonucleotides and replicative bacterial polymerases to measure the steady-state kinetics of dNTP incorporation opposite this adduct.24,25
These experiments revealed the preferential pairing of purines opposite CEdG in a polymerase-independent manner, suggesting a preferred geometry of base pairing likely to favor transversions in vivo. These data support a role for CEdG in the mutations resulting from MG treatment of bacteria and mammalian cells, reported to be predominantly G transversions.5,39
Reaction of dG mononucleoside with MG resulted in the formation of the three previously reported adducts (). At neutral pH, cyclic adduct 2
was formed rapidly, although it seemed to partially degrade over a period of 70 h (see the Supporting Information). The attempted isolation of 2
in our hands by HPLC resulted in the recovery of only dG. Frischmann et al. have shown that 2
is not noticeably formed during the reaction of MG with dG under conditions where unreacted MG is scavenged in situ using o
These observations suggest that cyclic adduct 2
, formed from the direct condensation of MG with N1 and N2 of guanine, is likely formed as a kinetic product in equilibrium with MG and dG. We also found that addition of alkaline phosphate buffer to the MG/dG reaction mixture resulted in the rapid and quantitative conversion of 2
to the more thermodynamically stable CEdG (see the Supporting Information). A plausible mechanism for this transformation is shown in . Consistent with these observations, the analogous cyclic adduct of glyoxal with dG has also been reported to be unstable.40,41
At pH >7, we additionally observed the rapid formation of bis-MG adduct 3
(see the Supporting Information).
Figure 6 Proposed mechanism for formation of (R,S)-CEdG 1 via the cyclic adduct 2 arising from the equilibrium reaction of dG with MG. Modified from ref 20.
LC-ESI-MS/MS analyses of hydrolyzed and dephosphorylated nucleoside DNA-AGEs resulting from 1 h reactions of MG with plasmid DNA revealed that only (R,S
)-CEdG was formed in a dose-dependent manner. No evidence of bis-MG adduct 3 in plasmid DNA was obtained. Because the bis-MG adduct of the dG monomer was stable for >40 h at pH 9.0 (see the Supporting Information), the failure to observe this product in plasmid DNA was not likely due to inherent chemical instability, but rather an insignificant rate of formation in double-stranded DNA. It is possible that trace amounts of 2
may have contributed to the observed mutation spectrum. However, the exhaustive removal of unreacted MG from plasmid reaction mixtures as well as the slightly alkaline conditions used for transfection would tend to drive the conversion of 2
. We considered the possibility that the alkaline phosphatase protocol used to prepare DNA for MS/MS analyses may have resulted in underestimation of the amount of 2
in plasmid DNA;40
however, the use of an alternative acidic procedure42
did not reveal the presence of additional 2
. Taken together, these observations indicate that the dose-dependent mutagenic effects observed were due to (R,S
)-CEdG. Moreover, measurements of the intracellular pH of a variety of human cell lines have revealed that the nuclear compartment is slightly alkaline, ranging from pH 7.5 to 8.0.43
This would suggest that alkali labile 2
is not likely to persist in the nucleus in vivo, even under conditions that would favor its initial formation.
The availability of an ensemble of shuttle vector plasmids containing precisely defined levels of DNA damage made it possible to examine the effects of CEdG adduct density on mutation induction and the repair response. Moreover, this strategy obviated the potential issue of sequence context-dependent effects that can arise using single-adduct substituted plasmids, because DNA damage was randomly distributed throughout the marker gene. Transfection of pSP189 vectors bearing defined CEdG levels into fibroblasts differing solely in the ability to conduct NER revealed dramatic differences in induced mutation frequencies that indicated that NER is the primary, if not exclusive, pathway for repair of this adduct in DNA. It is striking that even though other DNA repair mechanisms were presumably active in the XP-G deficient cells, no attenuation of mutation frequency was observed with increasing adduct levels.
In the absence of alternative mechanisms for its removal, CEdG is predicted to accumulate to genotoxic levels in individuals with somatic or inherited defects in NER. This might be particularly important in patients with poor glucose control as a result of metabolic disease who display elevated levels of MG.10,11,44,45
Recent reports linking adiposity and diabetes with impairment of NER and DNA repair in general46,47
lend credence to a potential role for CEdG in contributing to somatic mutagenesis in these individuals. The accumulation of CEdG in patients compromised in their ability to repair it suggests a molecular mechanism that may explain in part the increased incidence of certain cancers associated with diabetes.48,49
Comparison of mutation spectra induced by high and low CEdG adduct levels in human fibroblasts differing in NER capacity indicated a secondary, indirect mutagenic pathway induced by this adduct. A >2-fold increase in the transition frequency was observed in XP-G+ cells at high adduct levels, a change ascribed solely to the sudden appearance of A · T → G · C base substitutions (). In contrast, in NER deficient XP-G cells, the induced pattern of base substitutions and deletions was unchanged relative to background following transfection of plasmids with elevated levels of CEdG, in spite of a >18-fold increase in the overall mutation frequency. Thus, the changes in the base substitution pattern observed in the XP-G+ cells at high adduct densities appeared to require a functional NER apparatus.
It initially appeared puzzling that the sole changes in the base substitution pattern induced by high CEdG adduct levels occurred at A · T rather than G · C base pairs. It was formally possible that this mutation arose as a result of replication past an adenine adduct(s) produced during reaction with high concentrations of MG. Frischmann et al. have described the formation of carboxyethyl-2′-deoxyadenosine (CEdA) in DNA in low yield following prolonged (1 week) reaction with a 20-fold excess of MG.20
However the fact that A · T → G · C transitions were not a significant feature of the mutation spectra when identically substituted plasmids were transfected into repair deficient XP-G cells suggests that this base substitution did not arise from replication past adenine adducts.
We considered the possibility that recruitment of an error-prone Y-family polymerase with poor fidelity opposite T or A could lead to the observed result. Evidence of the involvement of Y-family polymerases in the gap filling step of NER has recently been presented.50
However, most Y-family polymerases characterized to date induce mispairs at relatively low frequencies opposite undamaged DNA.51,52
An important exception to this general trend is pol ι
, which exhibits a 10-fold preference for the incorporation of dGTP over dATP opposite T in template DNA.53-55
Recruitment of pol ι
during “repair crisis” induced by high levels of CEdG could result in the observed increase in the level of A · T → G · C mutations as a result of this mispairing event during the gap-filling step of NER. The fact that significant increases in this level of base substitution only occurred in XP-G+
but not XP-G fibroblasts could imply a potential role for XP-G in the recruitment of pol ι
Although CEdG is only modestly mutagenic in human cells possessing a fully functional NER pathway, it can induce mutations at high frequencies when this repair mechanism is ablated. It has become clear in recent years that substantial interindividual variability in the efficiency of NER exists, with the various xeroderma pigmentosum phenotypes representing one extreme of the range. It has been suggested that these NER polymorphisms may be biomarkers for cancer susceptibility.56,57
Because it appears that NER may be the primary, if not exclusive, mechanism for repair of CEdG in human cells, any loss of efficiency in NER cannot be offset by other repair pathways. Thus, the presence of NER polymorphisms that limit the efficiency of NER would tend to render these individuals susceptible to CEdG-induced genetic instability and possibly increase their risk for cancer.