The formation and biological significance of N7-guanine adducts

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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.

Introduction

DNA alkylation or adduct formation occurs at nucleophilic sites in DNA. Of these nucleophilic sites, the N7-position of guanine is the most reactive [1]. A PubMed search on “N7-guanine adducts” resulted in over 300 publications with 9 out of 10 focusing on basic characterization of chemical or biochemical properties of N7-guanine adducts alone or in DNA. In addition, N7-guanine adducts are classified as non-promutagenic since they are chemically unstable and the N7-position does not participate in Watson Crick base pairing [2]. Ever since identification of the first N7-guanine adduct [1], several hundred studies on DNA adducts have been reported and reviewed from different perspectives [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. Consequently, many studies sought to establish the relationship between DNA adduct formation and other biological endpoints (mutations, DNA strand breaks, etc.). Technical limitations, however, did not permit integration into large molecular epidemiological studies during this era of lesion characterization. Despite superior sensitivity of the 32P-postlabeling assay, insufficient chemical specificity made it impossible to identify the chemical source of damage, and chemical depurination of N7-guanine adducts during sample preparation was a major concern. Almost all studies started with in vitro proof of concept experiments demonstrating covalent binding of the compounds of interest or their metabolites to DNA. Surprisingly, the identification and development of sensitive analytical methods remain a primary focus of many DNA adduct studies, even 50+ years later.

N7-Guanine adducts appear to be good biomarkers of internal exposure because of their higher abundance compared to other DNA alkylations. Questions arise, however, regarding the biological significance for 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.

Miller and Miller pioneered the field of chemical carcinogens and were the first to demonstrate covalent binding of chemical carcinogens to macromolecules in vivo [17], [18]. The first evidence for binding of chemical carcinogens or their metabolites to nucleic acid was reported by Wheeler and Skipper [19]. It quickly became apparent that carcinogens comprise a diverse group of chemicals. Some of them were from endogenous sources or natural products, while others arise from synthetic products of modern human life. These chemicals are able to react with nucleophilic sites (electron rich, S, N, and O), in DNA and proteins. Subsequent in vitro and in vivo studies quickly demonstrated that under physiological conditions (pH 7.4, 37 °C), alkylation of DNA primarily occurred at the N7-position of guanine (Table 1) [20]. The distribution of methylation and ethylation adducts in DNA was studied in in vitro reactions, in bacterial or mammalian cell cultures, and in several tissues of mice and rats (reviewed by Beranek [8]). Overall, it confirmed the notion that the relative distribution of alkylation in DNA is similar in vivo and in vitro [21]. However, as technology advanced and allowed examination of lower exposures distinct differences in adduct distribution were established (see Section 2). Binding was shown to mainly occur via monomolecular (SN1, e.g., nitrogen mustards) or bimolecular nucleophilic (SN2, e.g., sulfonyl esters) substitutions [22], [23], [24]. SN2 reactions are heavily dependent on steric accessibility while SN1 reactions generally follow first-order kinetics. In DNA, the ring nitrogens and the exocyclic oxygens are the preferred sites for alkylation. Although the N7-position is the major site of alkylation, the electrophilic species formed by the N-nitroso compounds for example, following SN1 kinetics, will have a greater preference for reaction at the exocyclic oxygens than will the alkanesulfonates, which are limited to SN2 reactions. The larger the alkyl group is, the stronger will be its preference for reaction at the O6-position of guanine. Hence, N-ethyl-N-nitrosourea (ENU) binds more efficiently to the O6-position than does N-methyl-N-nitrosourea (MNU) (Table 1) [8], [25], [26]. The important difference in alkylation agents undergoing SN1 or SN2 reactions is that agents capable of SN1 reactions react more frequently at the O6-position of guanine, thus producing more mutagenic O6-guanine adducts, compared to agents that solely react via the SN2 mechanism.

These early binding experiments in DNA, cell culture and animal studies also showed that some carcinogens required metabolic activation to gain their ability to form DNA adducts and to exhibit their mutagenic and carcinogenic effects. Consequently, compounds were classified as “direct-acting” or “metabolically activated” carcinogens. The latter type is also termed a pro-carcinogen. In addition to mono adducts, compounds with multiple reactive groups were shown to have the ability to form protein–protein, DNA–DNA or protein–DNA cross-links [20]. The decades following have produced a better understanding of the relationship between carcinogen exposures, DNA adduct formation, mutagenesis, and carcinogenesis [4], [5], [6], [7], [10], [12], [27]. Various technologies have been applied to animal and human exposure studies for routine analysis of N7-guanine adducts and other DNA adducts. These studies have increased our understanding of formation and persistence of DNA adducts, and their relationship to carcinogenesis. It has become clear that cancer is a complex, multi-step process that varies with types of exposure, site of tumor induction, and species. Understanding the implications of N7-guanine adducts has significantly contributed to identification of the mode of action in chemically induced mutagenesis and carcinogenesis. These findings have subsequently led to a better understanding of the role of DNA adducts in mutagenesis and mechanism-based risk assessment [27].

Compared to many other DNA adducts, N7-guanine adducts are chemically unstable, with half-lives in double-stranded DNA (dsDNA) ranging from as little as 2–150 h. The instability of N7-guanine adducts is created by the formal placement of an additional positive charge on the guanine ring system. In general, larger alkyl groups promote depurination in dsDNA. This has been demonstrated under physiological conditions (pH 7.4, 37 °C), where the half-lives for N7-methyl-guanine (N7-Me-Gua), N7-(2-hydroxy-3-butenyl)-guanine (N7-HB-Gua) and N7-(trihydroxy-benzo[a]pyreneyl) guanine are 150, 50, and 3 h, respectively [28], [29], [30], [31]. In addition, N7-guanine adducts accumulate in DNA with continuous exposure or treatment and usually reach a plateau (steady state) after ∼7–10 days [15], [32], [33], [34]. Steady state is reached when the rate of N7-guanine adducts formed is equal to the rate of adducts lost. In contrast, adducts that are more persistent, such as O4-ethyl-thymidine (O4-Et-Thy), accumulate over a period of 4 weeks [35], and O6-methyl-guanine (O6-Me-Gua) in the brain continue to accumulate over 6 weeks of dosing [36]. The formal placement of an additional positive charge on the guanine ring system also promotes further reactions that have been reviewed by Gates et al. [37]. Relative to guanine, N7-Me-Gua depurinates 106 times more rapidly at pH7, 37 °C [37]. Reactions characteristic for N7-guanine adducts are: (i) loss of C8 proton, (ii) depurination, (iii) ring opening to yield 5-N-alkyl-2,6-diamino-4-hydroxyformamidopyrimidine (alkyl-FAPy), (iv) hydrolysis of the N7-alkyl bond, and (v) rearrangement to C8 adducts. For details of the chemical reactivity of N7-guanine adducts, the reader is referred to the thorough review by Gates [37].

During the past 50 years many technologies have been used for analysis of N7-guanine adducts. These technologies have been applied to rodent and human exposure studies for routine analysis of N7-guanine adducts and other DNA adducts. In the earliest studies, radiolabeled carcinogens were administered to rodents and binding to protein, RNA, and DNA was assessed by scintillation counting of the corresponding cellular fractions [38]. After DNA isolation and hydrolytic treatments, individual DNA adducts could be purified and quantified by basic column chromatography. This approach allowed analysis of one sample per day, with a detection limit of 1 adduct per 106 normal nucleotides (nnt) using ≥5 mg DNA [29], [39]. Longer exposure regimes were laborious and expensive, due to the requirement of radiolabeled carcinogen for these studies. Consequently, most studies employed single exposures [40], [41].

By the 1980's, HPLC with fluorescence detection, radioimmunoassay, or enzyme-linked immuno sorbent assay were commonly used for the analysis of DNA adducts. These approaches significantly increased throughput, reduced cost via elimination of custom radioisotope synthesis, and allowed application to study designs that included multiple exposure protocols. The extended exposure protocols provided information on the steady state concentrations of DNA adducts and demonstrated that what had previously appeared to be minor adducts following single exposures could actually become major adducts if they were poorly repaired and accumulated with extended exposure [42]. These methods, however, had limited sensitivity compared to present day technology and some of the immunoassays were prone to false positive results due to cross-reactivity.

A major break through in methodology occurred in 1982, when Randerath and colleagues developed 32P-postlabeling methods for DNA adducts [43]. The limit of detection for the early 32P-postlabeling assays was 1 adduct per 108 nnt using as little as 1–2 μg DNA [43], [44]. Subsequently, combinations of 32P-postlabeling with HPLC or immunoaffinity permitted larger amounts of DNA to be analyzed and improved the sensitivity by one or more orders of magnitude. The major problems associated with this methodology include the lack of chemical-specific identity and poor reproducibility [45], [46]. The 32P-postlabeling method was most suitable for more stable DNA adducts, such as etheno adducts [47], [48], [49] and N2-guanine adducts derived from polycyclic aromatic hydrocarbons (PAH) [50], and less so for N7-guanine adducts, due to their instability.

More recently, advances in mass spectrometry have lowered the limit of detection for this chemical-specific and quantitative technology, making it the method of choice in contemporary investigations. Earlier studies applied gas chromatography–negative ion chemical ionization-mass spectrometry (GC–MS), after hydrolysis and derivatization, to the analysis of DNA adducts [51], [52], [53], [54], [55], [56]. Presently, however, the vast majority of quantitative analysis of DNA adducts is performed with liquid chromatography tandem mass spectrometry (LC–MS/MS). The application of mass spectrometry for DNA adducts has been recently reviewed by Singh and Farmer [57] and others [15], [16], [58], [59], [60], [61], [62], [63]. Major advances in instrumentation for both mass spectrometry and chromatography have increased the detection limits for DNA adducts up to 100-fold, making it possible to routinely measure 1 adduct per 108 nnt. A major advantage of GC– and LC–MS/MS methods is the utilization of chemically identical stable isotope labeled internal standards for accurate quantitation.

The greatest sensitivity for measuring DNA adducts, however, is achieved with accelerator mass spectrometry (AMS), which can quantitate down to 3 adduct per 1011 nucleosides using 1 μg DNA [64]. While this method is extremely sensitive, it is limited to the following radioisotopes (3H, 14C, 26Al, 41Ca, 10Be, 36Cl, 59Ni, 63Ni, and 129I), of which 14C and 3H are the most commonly used in biomedical research. Therefore, specific chemical syntheses are required to either obtain radioisotope-labeled test compounds or for chemical labeling of compounds of interest (postlabeling, derivatization). Unfortunately, access to AMS is limited worldwide (only 5 instruments as of 2007), mainly due to the expense of the mostly custom-made instruments [65].

Section snippets

Formation of N7-guanine adducts in animal models

Several investigators have successfully utilized N7-guanine adducts as biomarkers to answer important toxicology questions in rodent models. These studies used multi-dose exposure protocols to generate comprehensive dose–response curves. Data from studies in mice and rats are presented in supplemental materials (Table S1 and Table S2). Adduct formation was compared to other biological endpoints such as unscheduled DNA synthesis, mutation frequency, micronucleus, apurinic sites (AP sites), gene

Formation of N7-guanine adducts in human specimens

Despite the ubiquitous nature of N7-guanine adducts, their application as a biomarker of exposure in larger molecular epidemiology studies is not common practice. A review of the literature demonstrated limited numbers of studies using N7-guanine adducts as biomarkers for exposure to environmental or occupational pollutants. Furthermore, reported data are not extensive and mostly contain small numbers of individuals per group.

Similar to the data from animal studies, the presence of N7-Me-Gua

N7-Me-Gua and N7-Et-Gua and mutagenesis in mammalian cells

Early efforts aimed to compare DNA alkylation with mutation frequency (MF) and mutation spectra to identify adducts involved in mutagenesis. Beranek et al. reported a good correlation between DNA methylation (N7-Me-Gua and O6-Me-Gua) and mutation frequency (MF) in the HPRT gene in CHO cells after treatment with MMS or MNU [205]. In contrast, the formation of N7-Et-Gua did not correlate with mutation frequency in HPRT or Na-K-ATPase genes in CHO cells treated with diethylsulfate (DES), EMS, or

Conclusions

After decades of research on N7-guanine adducts in animals and humans, it has become clear that specific N7-guanine adducts are excellent biomarkers for internal exposure when they are determined in tissue DNA. In contrast, N7-guanine adducts that can be formed from endogenous or background sources are less reliable for estimating low external exposures. While the presence of N7-guanine adducts clearly demonstrate exposure to the tissues or cells, subsequent interpretations and conclusions need

Conflicts of interest

None.

Acknowledgements

The authors are thankful to Lynn Pottenger for constructive and productive discussion and editorial assistance. This work was supported in part by NIH grants P42-ES05948, P30-ES10126, R01-ES012689 and the American Chemistry Council.

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