Increase, Oxidative DNA damage leads to Increase, Mutations

Mutagenicity of oxidative DNA lesions has been extensively studied; incorrect base insertion opposite unrepaired oxidative DNA lesions during replication is a well-established event.

For example, 8-oxo-dG and FaPydG, the two most prominent oxidative DNA lesions, are able to form base pairs with dATP, giving rise to G:C→T:A transversions with subsequent DNA synthesis (Gehrke et al., 2013; Freudenthal et al., 2013; Markkanen, 2017). Replicative DNA polymerases such as DNA polymerase α, δ, and ε (pol α, δ, ε) have a poor ability to extend the DNA strand past 8-oxo-dG:dCTP base pairs and may cause replication to stall or incorrectly insert dATP opposite 8-oxo-dG (Hashimoto et al., 2004; Markkanen et al., 2012). In stalled replication forks, repair polymerases may be recruited to perform translesion DNA synthesis (TLS). Human Y-family DNA polymerases (Rev 1, pol κ, ι, and η) are DNA repair polymerases mainly involved in TLS for stalled replication forks. However, TLS is not free of error and its accuracy differs for each repair polymerase. For example, it is known that pol κ and η perform TLS across 8-oxo-dG and often insert dATP opposite the lesion, generating G:C→T:A transversions. The error-prone nature of bypassing unrepaired oxidative lesions has been described in many previous studies and reviews (Greenberg, 2012; Maddukuri et al., 2014; Taggart et al., 2014; Sha et al., 2017).

In vitro studies

• Concentration-dependent increase in oxidative lesions observed in TK6 human lymphoblastoid cells exposed to KBrO₃ and glucose oxidase (GOx; enzyme that produces H₂O₂) for 1 hour; increase in mutation frequency measured in TK assay after 14 days (3 days in non-selective medium and 11 days in selective medium) following 1 hour exposure corresponded with the concentration-response observed in oxidative lesions (Platel et al., 2011).
  • NOGEL could be determined in TK assay (KBrO3: 1.75 mM; bleomycin: 0.6µM; GOx: 1.17x10-5 units/mL) but not in the Fpg-modified comet assays (First statistically significant concentrations: KBrO3: 1 mM; bleomycin: 0.5µM; GOx: 1.08x10-5 units/mL)
    • These results indicate that statistically significant increases in oxidative lesions (measured in Fpg comet assay) occur at lower concentrations of the above three stressors than mutations measured by the Tk gene mutation assay at a later time point (after 14-day recovery)
    • Demonstrates concentration concordance in oxidative DNA lesions and mutation

In vivo studies

• Klungland et al. (1999) measured and compared the level of 8-oxodG in the liver of OGG1-null Big Blue mice and Ogg1+/+ Blue Blue mice at 13-15 weeks of age. (Klungland et al., 1999).
  • The amount of 8-oxodG in the OGG1-null mice was 1.7-fold higher than in wild-type mice at the time of measurement.
  • Spontaneous mutation frequencies in the liver of OGG1 null (Ogg1 -/-) Big Blue mice and wild type (Ogg1 +/+) Big Blue mice were measured at 10 and 20 weeks of age:
• At 10 weeks, mutation frequency increased by 2- to 3-fold in OGG1 -/- mice compared to the wild type mice. No further increase was observed at 20 weeks.
• Of the 16 base substitutions detected in Ogg1 -/- mutant plaques analyzed by sequencing, 10 indicated G→T transversions.
• This study demonstrates that increased levels of oxidative DNA damage in the null mice was concordant with increased incidence of mutations.
• Unfried et al. (2002) measured the level of 8-oxodG and mutations in the omenta of rats exposed to crocidolite for various durations (Unfried et al., 2002).
  • Statistically significant increases in 8-oxodG were observed compared to control after 10 and 20 weeks of exposure.
  • The number of G→T transversions after 4, 12, and 24 weeks of exposure was significantly higher compared to control and G→T transversions were the most prominent base substitution in these samples.
  • This mutation spectrum supports that oxidative DNA lesions were the source of mutations.
• Five-week-old male gpt delta mice were given drinking water containing 85 ppm sodium arsenite for 3 weeks and sacrificed 2 weeks after administration was stopped (Takumi et al., 2014).
  • The gpt mutation assay and 8-OHdG quantification was performed using genomic DNA isolated from the liver
  •  Significantly higher levels of 8-OHdG were observed in the arsenite group (1.15/10⁶ dG) compared to the control group (0.86/10⁶ dG)
  •  Elevated mutation frequency was observed in the arsenite group with an average of 1.10 x10^-5, compared to that of the control group (0.71x10^-5 )
  • G:C→T:A made up 46% of the mutations in the arsenite-fed mice
  • These data demonstrating a positive correlation between incidence of oxidative lesions in DNA and elevation in mutation frequency support that these events are associated, and the mutation spectrum further suggest that mutations were the result of oxidative lesions.

The provided empirical evidence examined only the quantities of 8-oxo-dG and related the observed mutations to this oxidative lesion; the level of overall DNA oxidation is inferred from the level of 8-oxo-dG present. It is unclear how other oxidative DNA lesions such as FapyG, FapyA, and thymidine glycol contribute to the mutation spectra and frequencies.