The formation and biological significance of N7-guanine adducts

Abstract

DNA alkylation or adduct formation occurs at nucleophilic sites in DNA, mainly the N7-position of guanine. Ever since identification of the first N7-guanine adduct, several hundred studies on DNA adducts have been reported. Major issues addressed include the relationships between N7-guanine adducts and exposure, mutagenesis, and other biological endpoints. It became quickly apparent that N7-guanine adducts are frequently formed, but may have minimal biological relevance, since they are chemically unstable and do not participate in Watson Crick base pairing. However, N7-guanine adducts have been shown to be excellent biomarkers for internal exposure to direct acting and metabolically activated carcinogens. Questions arise, however, regarding the biological significance of N7-guanine adducts that are readily formed, do not persist, and are not likely to be mutagenic. Thus, we set out to review the current literature to evaluate their formation and the mechanistic evidence for the involvement of N7-guanine adducts in mutagenesis or other biological processes. It was concluded that there is insufficient evidence that N7-guanine adducts can be used beyond confirmation of exposure to the target tissue and demonstration of the molecular dose. There is little to no evidence that N7-guanine adducts or their depurination product, apurinic sites, are the cause of mutations in cells and tissues, since increases in AP sites have not been shown unless toxicity is extant. However, more research is needed to define the extent of chemical depurination versus removal by DNA repair proteins. Interestingly, N7-guanine adducts are clearly present as endogenous background adducts and the endogenous background amounts appear to increase with age. Furthermore, the N7-guanine adducts have been shown to convert to ring opened lesions (FAPy), which are much more persistent and have higher mutagenic potency. Studies in humans are limited in sample size and differences between controls and study groups are small. Future investigations should involve human studies with larger numbers of individuals and analysis should include the corresponding ring opened FAPy derivatives.

Figure 1

Guanine and adenine methylation by MNU relative to N7-Me-Gua formation (100%).

Figure 2

Formation of N7-guanine adducts form diazonium ion metabolites of NNK. NNK is metabolized by P450–catalyzed α-hydroxylation, producing hydroxymethyl NNK (2) and α-methylenehydroxy NNK (3), which spontaneously decompose to their corresponding diazonium ions (4,5), and keto aldehyde and formaldehyde, respectively. The highly reactive diazonium ions (4,5) subsequently form DNA adducts including N7-guanine adducts [82].

Figure 3

Example of olefin metabolism and formation of N7-guanine adducts illustrated on BD. BD is metabolized by P450s to several epoxides that form DNA adducts including N7-guanine adducts. Shown are N7-HB-Gua, bisN7-Gua-BD-diol and THB-Gua as representative N7-guanine adducts formed from EB, DEB and EB-diol, respectively.

Figure 4

Comparison of the dose-responses for formation of N7-hydroxyalkyl-Gua adducts following repeated exposure to the olefins ethylene (A) and propylene (C), and their epoxy metabolites EO (B) and PO (C), respectively. Formation of N7-guanine adducts is much more efficient for the epoxide metabolite compared to the parent olefin. Data are from rat lung after 20 days inhalation exposures to olefins and their epoxides [33, 127, 133, 149].

Figure 5

Determination of the number of N7-Me-Gua adducts present with an imbalance in BER. NAD(P)H values for CHO AA8 cells were plotted against the corresponding cumulative dose (the product of mM MMS and exposure duration). The X value corresponding to the intersection of the resulting linear regression line and y = 100 (i.e., 100% NAD(P)H relative to controls) was determined to be the start of an imbalance in BER. In the case of AA8 cells, exposure to 1.96 mM MMS for 1 hr initiated the imbalance of BER (unpublished results from Pachkowski et al).

Figure 6

Formation of endogenous and exogenous N7-Me-Gua in AHH-1 cells exposed to [¹³C₂] MMS for 24 h (Modified from Swenberg et al [27]). Exogenous N7-Me-Gua adducts were distinguished form endogenous N7-Me-Gua base on mass differences due to stable isotope labeled [¹³C₂] MMS used for cell treatment. Adduct amounts were compared to mutation frequency in HPRT gene as reported by Doak et al. [283].