Endogenous DNA Adducts: Potential and Paradox - ACS Publications

Chem. Res. Toxicol. 1993,6, 771-785

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Endogenous DNA Adducts: Potential and Paradox Lawrence J. Marnett’ and Philip C. Burchamt A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received August 23, 1993

Progressive accumulation of genetic mutations is thought to be central to the process of tumorigenesis and aging (1). Most of the critical genes that are mutated during the development of human cancer can be broadly classified as either protooncogenes or tumor suppressor genes (2). Although their activity can be altered by a broad range of mutational events, in many cases single base-pair substitutions are sufficient to activate protooncogenes or inactivate tumor suppressor genes. The question arises as to what causes the base-pair substitutions that amass in multiple cellular genes during neoplastic transformation and aging? Replication errors, spontaneous deamination and depurination, and DNA damage induced by exposure to exogenous chemicals are all likely possibilities. Reaction of DNA bases with electrophiles, oxidizing agents, or ultraviolet light produces covalently modified bases that are referred to as DNA adducts (3-6) (Figure 1). Depending upon their structure, DNA adducts may (1)block or significantly slow DNA replication, leading to arrested cell division or chromosomal aberrations (7); (2) direct the misincorporation of bases during replication, leading to mutations (8-14); or ( 3 ) undergo hydrolysis, leading to abasic sites and increasing the probability of strand scission. Relating the chemical structure of a DNA adduct to its biological consequences has been aided by advances in oligonucleotidesynthesis and the development of recombinant DNA technology (15-17). As a result, it is now possible to incorporate individual adducts into defined sequences of DNA to evaluate their effects on replication, mutagenesis, and repair (17-19). Such studies provide an understanding of the structural basis of mutagenesis and repair and an estimate of the toxicological potential of DNA adducts. The ability to relate adduct formation to biological activity forms part of the foundation of the evolving field of molecular epidemiology. Most of the work on the chemistry and biology of DNA adducts has focused on adducts derived from exogenous chemicals such as polycyclic hydrocarbons, aromatic amines, mycotoxins, etc. This emphasis reflects the importance of environmental agents on cancer incidence and mortality in the general human population (20). However, there is a growing realization that adducts are also produced from endogenous chemicalsthat arise during normal metabolism, oxidative stress, and chronic inflammation inter alia (21). If DNA adducts from endogenous chemicals possess similar mutagenic potential to those from exogenous chemicals, these adducts may also contribute to the etiology of genetic disease and therein the base line of human cancer. The developments that have fueled the interest in endogenous DNA adducts are primarily the result of

* Corresponding author.

Phone: (615) 343-7329; FAX: (615) 343-

7534.

+ Present address: Department of Clinical and Experimental Pharmacology,University of Adelaide, GPO Box 498, Adelaide, South Australia 5001, Australia.

4 U”ne

t

CANCER

Figure 1. The role of DNA damage in mutation and cancer.

advances in analytical chemistry. Simply stated, it is now possible to scan large regions of DNA for the presence of low levels of adducts (22-26). Although these techniques were originally developed for the detection and quantitation of adducts from exogenous agents, they are being applied increasingly to the detection of adducts from endogenous agents. Concomitant with advances in analytical chemistry has been the identification of genetic loci that enhance the sensitivity of cells to “spontaneous” mutation (27,281. Some of these loci code for DNA repair enzymes that remove specific types of damage (28). The presence of these enzymes in cells and their role in the modulation of spontaneous mutation imply there is continuous production of DNA adducts from agents generated during normal or aberrant metabolism. The present article will survey what is known about several classes of endogenous adducts from a structural, analytical, and biological perspective. The adducts considered include oxidized bases, alkylated bases, exocyclic base adducts, and a series of unidentified putative adducts called I-compounds. From this survey, it is hoped that readers will appreciate the challenges and implications of the detection and identification of low levels of adducts in mammalian tissues. Furthermore, it is hoped they will recognize the opportunities for important and creative research that exist at this interface of chemistry, toxicology, and molecular biology.

Oxidized Bases Generation of oxidized bases by reaction of DNA with intracellular oxidants is quantitatively the most important class of base modification occurring in mammalian cells. Estimates of the total number of oxidized adducts formed on a daily basis range from lo4to 106 per cell, which rivals the rate of spontaneous depurination of DNA (21). The nature and origin of the oxidants responsible for these lesionsare not known with certainty, but leakage of reactive oxygen species during oxidations in the mitochondria and the endoplasmic reticulum are likely sources (29). Most in vitro studies of DNA oxidation have concentrated on the genotoxicity of species produced by metal-catalyzed decomposition products of H202 such as the hydroxyl

QS93-228~/93/27QS-O77l~Q4.QQlQ 0 1993 American Chemical Society

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46). Targeted G T transversions predicted by these experiments are also the major mutation detected following in vivo replication of bacteriophage vectors containing single 8-oxo-dGuoresidues (47,481. This mutation is also seen in mutant bacteria that are deficient in the repair of dThdg EoxodGuo 5-HMdUrd this lesion, indicating 8-oxo-dGuo is generated by endogenous oxidants in vivo (49, 50). A second well-studied DNA oxidation product is 5,6dihydroxy-5,6-dihydrothymine (thymidine glycol,dThdg). This lesion functions as a sequence-dependent replication block in vitro (51,521. In vivo studies of the replication of randomly modified viral templates imply dThdg is very weakly mutagenic (53). Moreover, mutant bacteria that are unable to repair this lesion by base excision pathways are not markedly more sensitive to mutagenesis by HzOz 5-OHdCyd SOHdUrd dUrdg or ionizing radiation although it is possiblethat other repair pathways participate in the removal of dThdg under these Figure 2. Structures of oxidized bases. conditions (54). A careful study of the mutagenicity of a sole dThdg residue in a single-stranded bacteriophage M13 radical; HzOz itself does not react extensively with DNA genome revealed the induction of a low frequency of T (30-32). Other putative endogenous mediators of DNA C transitions during replication (55). oxidation include excited oxygen species or intermediates formed during the fragmentation of oxidized lipids such A less-studied adduct that is of likely significance for as alkoxy1and peroxyl radicals (33,34). A role for steadyendogenous mutagenesis is the thymine oxidation product state DNA damage by endogenous oxidants has been 5-(hydroxymethyl)-2’-deoxyuridine(5-HMdUrd). In some proposed for cancer and normal aging (35-38). studies 5-HMdUrd has been reported to be produced in amounts equal to that of 8-oxo-dGuo (56). Since 5-HMThe body of experimental evidence documenting the dUrd is only distinguished from thymine by the presence mutagenicity of reactive oxygen species in prokaryotic and of an OH group at the non-base-pairing C-5 methyl eukaryotic cells is quite extensive ( 3 s 4 2 ) . For example, position, this lesion is thought to be only weakly mutagenic. mutant Escherichia coli lacking a component of their However, physical studies have shown 5-HMdUrd assumes antioxidant defense system are more sensitive to oxidative an unusual puckered conformation in both the crystal and mutagenesis than wild-type cells. Transfection of these solvent states, due to unexpected hydrogen bonding mutants with plasmids encoding superoxide dismutase between the C-6 of thymine with the ring oxygen of the reduces their sensitivity to wild-type levels (43). That deoxyribose moiety (57). This unusual structure suggests the mutagenicity of reactive oxygen species is mediated 5-HMdUrd may miscode during DNA replication. Such by direct DNA damage rather than epigenetic effects has a likelihood is further suggested by the finding that been established by the repeated finding that oxidized viral DNA molecules yield elevated mutation frequencies 5-HmdUrd itself and alsoits hydroperoxy precursor induce a high incidence of mutations in Salmonella typhimurium upon replication in both bacterial and mammalian cells (58,591. These considerations may warrant more careful (44,45). The exact nature of the adducts responsible for investigation of the mutagenicity of this lesion. these mutations is not always known. A plethora of base modifications occur during DNA oxidation, and it is Little is known of the mutagenicity in either prokaryotic currently beyond our ability to assign each mutation to a or eukaryotic organisms of the more complex ring-opened specific oxidation product. Indeed, the mutagenic propor ring-contracted pyrimidine oxidation products, or of erties of just a few of the dozens of known oxidized DNA the imidazole ring-opened purines, all of which can be bases are known with any acceptable degree of certainty. formed during DNA oxidation (37). Thus, study of the The most mutagenic lesion induced in DNA by reactive mutagenic potential of judiciously selected DNA oxidation oxygen species may be 7,8-dihydro-8-0~0-2’-deoxygua- products employing site-specific methodologies should be nosine (8-oxo-dGuo)l (Figure 2). In vitro studies of the a major future research goal. The technically-demanding fidelity of DNA synthesis opposite 8-oxo-dGuohave shown nature of such studies would be more than offset by the that the major error is insertion of dAdo, although this is valuable information they would yield on the mutagenicity strongly influenced by the DNA polymerase employed ( 11 , of the dozens of known products of DNA oxidation, whose miscoding potential is currently unknown. ‘Abbreviations: 8-oxo-dGuo, 7,8-dihydrc-8-0~0-2’-deoxyguanosine; Numerous analytical procedures have proven useful for 8-oxo-Gua, 7,8-dihydro-8-oxoguanine;dThdg, 5,6-dihydroxy-5,6-dihythe quantitation of the various products of DNA oxidation. 5-HMdUrd, drothymidme; Thyg, 5,6-dihydroxy-5,6-dihydrothymine; 5-(hydroxymethyl)-2’-deoxyuridine; HPLC-EC, high-performanceliquid Most commonly, these have employed HPLC with a variety chromatography with electroch emical detection; NICI GC/MS, negative of detection methods. For example, a somewhat laborious ion chemical ionization gas chromatography/massspectrometry;ELISA, and insensitive UV-HPLC procedure enabled quantitation enzyme-linked immunosorbent assay, OB-Me-dGuo, OB-methyl-2’-deoxyguanosine; OB-Me-Gua, OB-methylguanine; 7-Me-dGuo, 7-methyl-2’of urinary dThdg (601,whereas a 3H-postlabeling method deoxyguanosine; 7-Me-Gua,7-methylguanine; 3-Me-dAdo, 3-methyl-2‘using HPLC with radioactivity detection enabled simuldeoxyadenosine; 3-Me-Ade, 3-methyladenine; 04-Me-dThd, 04-methylthymidine; 04-Me-Thy, W-methylthymine; cdCyd, 3,W-etheno-2’taneous measurement of 8-oxo-dGuo, 5-HMdUrd, and deoxycytidine; rCyt, 3,Wethenocytosine; cdAdo, Ifl-etheno-2’-deoxy(61). The latter method enabled Frenkel and codThdg adenosine; eAde, 1p-ethenoadenine; cdGuo, 2JVs-etheno-2’-deoxyguaworkers to make the important discovery that the levels nosine; cGua, 2JVs-ethenoguanine; &OH-CMa-PdGuo, 8-hydroxy-6methyl-l,W-propano-2’-deoxyguanosine; 8-OH-PdGuo,8-hydroxy-l JVZof these adducts are elevated in mouse skin DNA during propano-2’-deoxyguanoeine;MlGuo, 3-j3-~-2’-deoxyribofuranotreatment with phorbol ester tumor promoters, a finding sylpyrimido[ 1,2-a]purin-lO(3IO-one;M’Gua, pyrimido[1,2-aIpurin-10(BM-one;04-Et-dThd, P-ethylthymidine; P450, cytochrome P450. that may revise the long-held belief that tumor promotion

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does not involve DNA damage (62-64). Work from several laboratories has shown that the most useful application of HPLC technology to the study of DNA oxidation is in combination with electrochemical detection (HPLC-EC). 8-Oxo-dGuo is especially suited to analysis via this means, and as little as 5-50 fmol can be quantitated in enzyme hydrolysates from 40-100 pg of DNA (65). HPLC-EC has thus been employed to detect increases in 8-oxo-dGuo levels in tissue DNA following treatment of animals with an ever-growing list of diverse prooxidants, including secondary nitroalkanes, diethylstilbestrol, peroxisome proliferators, ionizing radiation, and mycotoxins (66-70). The high sensitivity and selectivity of the HPLC-EC assay especially suits it for the quantitation of the low levels of 8-oxo-dGuo that are formed by endogenousDNA oxidants. For example, 8-oxodGuo was shown to be present at a level of 8 adducts per 106 normal nucleotides in the nuclear DNA of untreated rat livers, and levels in the mitochondrial genome were 16-fold higher, presumably reflecting this organelle’s role in cellular oxygen metabolism (71). More recently, Ames and co-workerssuccessfullyapplied HPLC-EC to the study of oxidation products from dCyd residues in DNA (72). The levels of three oxidized dCyd derivatives in human leukocyte DNA, namely, 5-hydroxy-2’-deoxycytidine,5-hydroxy-2‘-deoxyuridine,and 5,6-dihydroxy-5,6-dihydro-2’deoxyuridine (10 f 5 adducts per lo7 nucleotides, 7 f 6 adducts per lo7 nucleotides, and 20 f 15 adducts per lo7 nucleotides, respectively), were very similar to the levels of 8-oxo-dGuo in these DNA samples (12 f 7 adducts per 107 nucleotides). The mutagenic consequences of the formation of these oxidized dCyd residues in DNA or the pathways involved in the repair of these lesions are poorly understood. The most powerful procedures for the unequivocal chemical characterization and quantitation of DNA oxidation products employ GC/MS with selected ion monitoring (73). These methods have been applied to the analysis of DNA following in vitro exposure to strong oxidants such as ionizing radiation or transition metals plus H202 for simultaneous identification and quantitation of a variety of purine and pyrimidine oxidation products (30, 31, 74-77). Refinement of these methods permits detection of low levels of oxidized bases in DNA from human tissues (78). For example, use of deuterated internal standards enabled Djuric and associates to quantitate trimethylsilylated 5-HMdUrd derivatives via GC/MS in 2-pg DNA samples with a detection limit of 3 adducts per 100 000 thymines (79). This method was then used to quantitate 5-HMdUrd in blood cell DNA from women at risk for breast cancer and showed that adduct levels were decreased from 9.3 f 1.9 adducts per lo4 thymines in a nonintervention control group to only 3.0 f 0.6 adducts per lo4 thymine residues in subjects that changed to a low-fat diet (79). These intriguing findings raise the possibility that oxidative DNA damage may provide a mechanism to explain the epidemiological association between dietary fat consumption and cancer risk. GC/MS analysis of DNA from several human breast samples revealed that oxidized DNA bases are present in tumor tissue at levels approximately 10-fold higher than in surrounding normal tissue (80, 81). A potential complication of the analysis of endogenous levels of oxidized DNA bases is the fact that oxidation of DNA occurs during some of the procedures used for DNA

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digestion and analysis. Although the yields of oxidized bases produced during sample workup are low, trace amounts are significant because the level of sensitivity provided by modern chemical analysis is 1adduct per 108 base pairs or greater. Thus, great care must be taken to avoid artifactual generation of oxidized bases during sample preparation. Precautions include the use of antioxidants in buffers and the avoidance of hydrolytic processes that employ formic acid (78, 82). Understanding of the repair of DNA oxidation products is limited to just a few adducts. For example, bacteria possess an elaborate tripartite system of protection against 8-oxo-dGuo, involving a DNA glycosylase, a mismatch repair activity, and even a nucleotide triphosphatase that removes 8-oxodeoxyguanosine triphosphate from the nucleotide pool (83,84). Eukaryotic mechanisms of repair for this lesion are incompletely characterized, but the fact that high levels of both 8-oxo-dGuo and 7,8-dihydro-8oxoguanine (8-oxo-Gua)are excreted in mammalian urine strongly implies that base and nucleotide excision-repair operate on this lesion in animal tissues (82,85). Indeed, urinary excretion of 8-oxo-dGuoappears to be a very useful biomarker for DNA damage by endogenous oxidants since it correlates strongly with basal 0 2 consumption in three species (85). Urinary levels of 8-oxo-dGua are less meaningful due to the confounding influence of dietary nucleic acids (82). The usefulness of 8-oxo-dGuo as a biomarker for oxidative DNA damage in humans was underlined in a recent Danish study that revealed an influence of smoking, gender, and body mass on urinary 8-oxo-dGuo levels (86). In 30 smokers, the mean 8-oxodGuo excretion was 320 f 99 pmol/kg/24 h compared to 213 f84 pmol/kg/24 h in 53 nonsmokers (86). This finding concurs with reports of increased 8-oxo-dGuo levels in the leukocyte DNA of smokers compared to nonsmokers and also with the demonstrated ability of tobacco combustion products to generate oxidized DNA bases in vitro (87-89). The mediators of the DNA oxidation associated with inhalation of tobacco combustion products could well be oxygen-centered radicals, given that the gas phase from a single puff of a cigarette contains about 10’5 radicals (90). Unraveling the contribution DNA oxidation makes to the overall carcinogenic risk associated with tobacco usage is likely to be an active area of future investigation. Removal of the oxidized thymidine residue dThdg from both prokaryotic and eukaryotic genomes also proceeds via either base or nucleotide excision-repair (91-94).Both (thythe free base 5,6-dihydroxy-5,6-dihydrothymidine mine glycol, Thyg) and deoxynucleoside dThdg resulting from the repair of this lesion are present in urine and are useful markers for endogenous exposure to oxidants, although the lengthy HPLC assay for Thyg and dThdg renders themless useful than 8-oxo-dGuo(36,951. Urinary levels of Thyg and dThdg in mice, rats, monkeys, and humans correlate strongly with metabolic rate, confirming the contention that oxidative damage to DNA is a consequence of normal aerobic metabolism (96).Background urinary concentrations of Thyg and dThdg in humans were 390 pmol/kg/24 h and 100 pmol/kg/24 h, respectively (60). These values have been used to provide an estimate of 1000oxidative hits to thymine residues per day for each of the body’s 6 X 1013cells (97). Given the likely mutagenicity of this lesion, dThdg may make an important contribution to the background rate of spon-


s

FH3

y

R

CH3

2

’

7-Me-dGuo

J-Me-dAdo

d-Me-dGU3

d-MedThd

Figure 3. Structures of alkylated bases.

taneous mutagenesis in the event DNA replication occurs prior to adduct removal. The finding of high levels of oxidized DNA bases in human and animal urine provides strong support for the contention that mammalian genomes are continually barraged with endogenous oxidants. At present, specific knowledge regarding the extent of intracellular formation, repair, and mutagenicity is limited to just a few of the dozens of oxidative base modifications that can be induced in DNA. The scope for future work on the role of other DNA oxidation products in spontaneous mutagenesis is enormous, as is that for unraveling the precise mechanisms of their formation in vivo. For the time being, the hypothesis that steady-state oxidative damage to DNA plays a role in carcinogenesis and aging is attractive but incompletely tested.

Alkylated Bases DNA adducts from alkylating agents are the best studied of all DNA base modifications, due mainly to the fact that they are formed upon treatment with certain classical carcinogens such as the nitrosoureas and nitrosamines. However, DNA alkylation appears to be common even in cells that have not been treated with alkylating agents because DNA repair enzymes capable of excising N- and 0-methylated bases are present in virtually all cell types. Alkylated bases can arise from exposure of DNA to both endogenous and exogenous agents (98,99).Candidates for endogenous DNA-methylating agents include quaternary amines such as betaine and choline, and the trialkylsulfonium agent S-adenosylmethionine (100, 101). The multiple nucleophilic oxygensand nitrogens present in DNA vary in their reactivity toward alkylating agents, with N-7 and N-3 of guanine and the N-3 of adenine showing highest reactivity toward S~2-typealkylating agents (Figure 3). Less discriminating SN1-type compounds react more extensively with exocyclicbase oxygens and also with oxygens in the phosphodiester backbone (102,103).Methylation products at N-7, N-3, and 0-6 of guanine and N-3 of adenine have been detected in DNA following in vitro incubations with the endogenous methyl donor S-adenosylmethionine (101, 104). Efforts to detect alkylated DNA adducts in biological samples have exploited a wide range of contemporary analytical tools. For example, HPLC with electrochemical detection has been used for the estimation of base-line 7-methylguanine (7-Me-Gua)levels and also those induced by N-methyl-N-nitrosourea (105,106).Mass spectrometry

Marnett and Burcham

has also enabled unambiguous identification of 7-Me-Gua and 3-methyladenine (3-Me-Ade) in complex biological mixtures (107-11 1). Other approaches to the quantitation of N-methylpurines employed enzyme-linked immunosorbent assay (ELISA) techniques either as stand-alone procedures (112-114) or, more successfully,in combination with HPLC (115). Inclusion of a prior chromatographic purification step also greatly assists determination of 7-methyl-2’-deoxyguanosine (7-Me-dGuo) by 32P-postlabeling (116, 117 ) . Interestingly, use of anion-exchange chromatography in conjunction with 32P-postlabeling reveals a smoking-related increase in 7-Me-dGuo levels in lymphocyte DNA in humans, yielding mean values of 14 adducts per lo7 nucleotides in nonsmokers compared to 24 adducts per lo7 nucleotides in smokers (117). Sensitive analytical methods are available for detection of 06-methyl-2’-deoxyguanosine(06-Me-dGuo) in DNA from anumber of sources. Anovel competitive DNA repair assay employingEscherichia coli alkyltransferase enabled quantitation of 06-Me-dGuo in lymphocyte DNA from cancer patients following treatment with procarbazine (118). Good specificity and sensitivity for 06-Me-dGuo in human lung DNA were achieved through the use of a combined HPLC-32P-postlabeling procedure (119, 120). Mean 06-Me-dGuolevels obtained during triplicate measurements in peripheral lung tissue DNA from individuals with known occupational and smoking histories ranged from 1 f 1adducts per lo7dGuo residues to 52 f 3 adducts per lo7 dGuo residues (120). The range of procedures currently available for the measurement of 04-alkyl-dThd adducts is much more limited. An immuno-slot-blot procedure enabled quantitation of 04-ethylthymidine (04-Et-dThd) in bacterial DNA following treatment with N-ethyl-N-nitrosourea (121). Also, evidence suggesting ethylation of the 0-4 of dThd may be important during human carcinogenesis was obtained using HPLC-ELISA (122). Whether 04-MedThd levels are also elevated in human malignancies has yet to be determined. In light of the pronounced mutagenicity of 04-Me-dThd,more work needs to be directed toward the quantitation of this adduct in human-derived materials. The various DNA base methylation products differ markedly in their ability to induce mutations during DNA replication. Methylation at N-7 of guanine is usually the major outcome following exposure of DNA to methylating agents, but this site does not participate in base-pairing so 7-Me-dGuois usually considered only weakly mutagenic if at all (123). However, 7-Me-dGuo can undergo basecatalyzed ring-opening of the imidazole ring to form a 5-formamide-4,6-pyrimidine derivative, a lesion that has been found to be a block to DNA replication (124-126). Next to N-7 of dGuo, N-3 of dAdo is perhaps the second most nucleophilic center in the various DNA bases. Methylation at this site has profound consequences. Indeed, S-methyl-2’-deoxyadenosine(3-Me-dAdo) seems largely responsible for the toxicity associated with exposure to cytotoxic methylating agents (127). Bacteria deficient in the repair of this lesion are exquisitely sensitive to killing by monofunctional alkylating agents, whereas transfection with plasmids expressing repair enzymes restores normal sensitivity to such agents (128-130). As with 7-Me-dGuo, the mutagenicity of 3-Me-dAdo has not been investigated in site-specificmutagenesis experiments in either bacterial or mammalian cells. Nevertheless, 3-Me-dAdo appears


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to be an SOS-dependent premutagenic lesion in repairdeficient bacteria (131). It is more difficult to assess whether 3-Me-dAdo is cytotoxic and mutagenic in mammalian systems because cell lines unable to repair this lesion have yet to be identified. Furthermore, studies on the effect of overexpression of repair enzymes selective for 3-Me-dAdo on the mutagenicity of methylating agents in mammalian cells have yielded conflictipg results (132, 133). Clearly, more work needs to be done on the mutagenicity of these N-methylpurines in both bacterial and mammalian cells. Final judgment of the true miscoding potential of such adducts as 3-Me-dAdo or ringopened 7-Me-dGuo awaits performance of site-specific mutagenesis experiments employing well-defined DNA templates. Less ambiguity surrounds the mutagenicity of O-methyl adducts generated during the reaction of methylating agents with thymidine and deoxyguanosine. Numerous site-specificmutagenesis experiments employingsynthetic oligonucleotides or viral genomes containing 06-Me-dGuo A have consistently found the adduct induces G transitions during DNA replication both in vitro (134,135) and also in bacterial and mammalian cells (136-140).The G A transition is the major mutation that is induced in animals by alkylating agents (123). The biological properties of 06-Me-dGuo have also been assessed by studying the mutagenicity of alkylating agents in cells with differing repair capacity for this lesion. For example, bacterial mutants unable to repair 06-Me-dGuo are sensitive to mutagenesis by N-methyl-N’-nitro-N-nitrosoguanidine (141).Evidence implicating 06-Me-dGuo as a major premutagenic lesion in mammalian cells comes from the finding that alkylating agents are less mutagenic in cells that overexpress repair enzymes selective for this adduct (142-144).06-Me-dGuo has also been shown to be important during carcinogenesis in a number of model systems (145,146).However, most intriguing are recent findings that bacterial and yeast cells deficient in the repair of this lesion exhibit an elevated mutation frequency in the absence of chemical treatment. This suggests that endogenous methylating agents contribute to spontaneous mutagenesis via the generation of 06-Me-dGuo(28,141). Collectively,these findings indicate 06-Me-dGuois a good candidate as a mediator of spontaneous mutagenesis, in the event it is replicated prior to repair. Similar studies employing site-specifically positioned 04-Me-dThdresidues have shown this adduct to be very mutagenic both in vitro and in vivo (135).In virtually all instances, the predominant mutation results from incorporation of dGuo opposite the adduct during DNA synthesis (147-151).Side-by-sideanalysis of the bacterial mutagenicity of 06-Me-dGuo and 04-Me-dThd when positioned in homologous sequences revealed 04-Me-dThd was much more mutagenic than 06-Me-dGuo,apparently because the latter was more efficiently repaired by bacterial enzymes (152). N-Methylpurines suffer a number of fates upon formation in cellular DNA. First, the presence of a methyl group on N-7 of dGuo or N-3 of dAdo labilizes the glycosyl bond, so a proportion of these adducts undergo spontaneous depurination (153). The resultant apurinic sites are likely to be mutagenic if they are replicated prior to repair (154),and abasic sites may mediate part of the mutagenicity of cytotoxic alkylating agents in vivo (155). Indeed, recent evidence indicates endogenously generated

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N-methylpurines contribute to the spontaneous mutation rate in Saccharomyces cerevisiae via the intermediacy of abasic sites (28). The second fate of N-methylpurines in DNA is removal by enzyme-catalyzed repair (156).Two glycosylases are responsible for base excision-repair in bacteria (127,157). MammalianN-methylpurine glycosylasesremove a variety of methylated bases from DNA, including 3-Me-dAdo and 7-Me-dGuo (92,158,159). These enzymes release free N-methylpurines, which eventually are excreted into the urine. This raises the possibility that urinary levels of such adducts may serve as biomarkers for exposure to methylating agents (160). Interesting results were obtained in this respect through the use of GC/MS to quantitate urinary 7-Me-Gua and 3-Me-Ade in a Columbian population at risk for gastric cancer (107,161). A significant increase in the mean values of urinary 7-MeGua was seen in smokers (8.1 f 3.6 mg/subject/day, n = 40) compared to nonsmokers 16.5 f 2.2 mg/subject/day, n = 116,P = 0.002 in t-test (161)l.An elevation in urinary 3-Me- Ade excretion was also associated with tobacco smoking in this population 17-05f 5.7 pg/subject/day in nonsmokers (excluding 3 outliers) versus 9.4 f 5.2 pg/ subject/day in smokers, P = 0.025 (161)l.Unfortunately, high background urinary levels of 7-Me-Gua and 3-MeAde limit their usefulness as biomarkers for exposure to either exogenous or endogenous alkylating agents (110, 124). Confounding sources of urinary N-methylpurines that frustrate efforts to associate their excretion with particular lifestyle factors include their widespread contamination of foodstuffs and the release of 7-Me-Gua during tRNA turnover (114,160,162). Multiple pathways also appear to participate in the repair of 06-Me-dGuo in cellular DNA. Bacteria possess two distinct methyltransferases that effect the error-free removal of methyl groups from 06-Me-dGuo and a nucleotide excision pathway that removes this lesion (92, 121, 141, 163). Mammalian repair of 06-Me-dGuo is effected by an 06-alkylguanine alkyltransferase that possesses a cysteine alkyl acceptor residue at the active site (164).Base excision-repair pathways are not thought to be involved in the removal of 06-Me-dGuofrom bacterial genomes, and the same probably holds true for mammalian cells. Thus, it is not overly surprising that Farmer and associates were unable to detect 06-Me-dGuo in human urine via GC/MS (110).If nucleotide excision participates in the removal of 06-Me-dGuo from mammalian DNA as is the case in bacteria, it may be more fruitful to search for the intact 06-adducted deoxynucleoside in human urine. 04-Me-dThdcan also suffer a number of fates within cellular DNA. In E. coli, 04-Me-dThdmay be repaired by either methyl transfer or nucleotide excision pathways (92,121,165). Uncertainty surrounds the question of how efficiently 04-Me-dThd is repaired in mammalian cells. For example, 04-Me-dThd was found to be very persistent in hepatic DNA following treatment of rats with dimethylhydrazine, presumably due to poor repair (166).Neither rat nor human 06-alkylguanine alkyltransferases were found capable of repairing 04-Me-dThd in randomlymodified DNA, although more recent studies using welldefined oligodeoxynucleotide substrates suggested the human methyltransferase can repair this lesion (167-169). Thus, mammalian cells may be able to repair 04-Me-dThd via methyl transfer, although somewhat less effectively

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peroxidation. tdAdo and tdCyd were detected in a human liver and a human colon sample at a level of approximately 1 adduct per lo8 bases. Both DNA samples were from cancer patients, and their exposure to antitumor agents was uncertain. Swenberg and associates have recently applied immunoaffinity chromatography with a2P-postcdGw labeling to the analysis of tdAdo and edCyd in human livera3 The level of tdAdo in 10 different liver samples 0 OH 0 OH 0 averages 7 f 4 adducts per lo8 bases, and the level of tdCyd averages 2.8 f 0.9 adducts per lo7 basesa3 Nath and Chung combined HPLC purification with 32Ppostlabeling to quantitate 8-OH-6-Me-PdGuoin liver DNA R H R from rodents and humans (179). Surprisingly high levels MldGw 8-OH-6-Me-PdGuo &OH-PdGUo of the adduct, ranging from 2 to 6 per lo7 guanines were present in samples from mouse, rat, and humans.* FurFigure 4. Structures of exocyclic base adducts. thermore, Nath and Chung isolated the spot that coeluted than they repair Oe-Me-dGuo. If nucleotide excision repair with the 3’,5’-bisphosphate of 8-OH-6-Me-PdGuo during is found to be involved in the removal of this lesion from postlabeling and analyzed it by reversed-phase HPLC and mammalian genomes, it would be interesting to know anion-exchange HPLC. The recovered adduct cochrowhether O*-Me-dThd is present in mammalian urine. matographed with an authentic standard in both chroAlthough most of the interest that has been shown in matography systems. In addition to 8-0H-6-Me-PdGu0, the acrolein-derived adduct %hydroxy-1JV2-propano-2’alkylated DNA bases arose because of their relevance to cytotoxic alkylating agent-induced cancer, their presence deoxyguanosine (8-OH-PdGuo) was detected at comparable levels. in DNA from unexposed animal and human tissues indicates that they are also formed by endogenous Fedtke et al. developed a negative ion chemical ionalkylating agents. Although the true miscoding potential ization (NICI) GC/MS method for determination of cdGuo of certain N-methylpurines has yet to be definitively residues in DNA that involves converting the base to a determined, the mutagenicity of O-alkyl derivatives of pentafluorobenzyl derivative (23). Quantitation was acdGuo and dThd is very well established. The finding that complished by selected ion monitoring with an internal prokaryotic and simple eukaryotic cells that are deficient standard of the base IP,3-ethenoguanine (tGua) labeled in the repair of O-alkylated bases exhibit elevated mutation with four 13C’s. The limit of detection was 6 adducts per frequencies in the untreated control state strongly suggests lo8 base pairs. Fedtke et al. applied this method to the endogenous methylating agents contribute to spontaneous analysis of tdGuo in several tissues of Sprague-Dawley mutagenesis by generating these lesions (28,141). If rats exposed to vinyl chloride (180).Although substantial similar events occur in mammalian cells, N - and especially amounts of the adduct were detected in liver, lung, and O-alkylated bases could make an important contribution kidney, detectable amounts were present in the liver DNA to spontaneous mutagenesis and carcinogenesis. of animals that were not exposed to vinyl chloride. The level of tdGuo in rat liver was 4 adducts per lo7base pairs. Significant amounts of tdAdo, cdCyd, and edGuo are Cyclic Nucleic Acid Adducts detectable in the DNA of all rodent tissues examined.3 Difunctional electrophiles react with exocyclic and ring Our laboratory has recently developed a GUMS method nitrogens to form cyclic nucleic acid adducts, examples of for quantitation of MldGuo (181).Pyrimido[l,2-a]purinwhich are depicted in Figure 4 (170). 1JP-Etheno-2‘10(3H)-one (M1Gua) is isolated from DNA hydrolysates deoxyadenosine(edAdo)and 3f14-etheno-2‘-deoxycytidine and converted to a pentafluorobenzyl derivative that is (edCyd) are produced by several different compounds, analyzed by NICI GC/MS. Quantitation is by selected including vinyl carbamate epoxide, vinyl chloride epoxide, ion monitoring using an internal standard of MlGua and chloroacetaldehydeand as a result of lipid peroxidation isotopically labeled with four 13C’sand one 15N. The limit (171-174).IP,3-Etheno-2’-deoxyguanosine (tdGuo) is also of sensitivity of the assay is approximately 2 adducts per produced from vinyl chloride epoxide and chloroacetallo8 base pairs starting with 300 pg of DNA. Preliminary dehyde (173). 8-Hydroxy-6-methyl-l,W-propano-2’- results indicate that MlGua is present in the DNA of deoxyguanosine (&OH-&Me-PdGuo) is a product of the normal Sprague-Dawley rats at levels ranging from 2 to reaction of crotonaldehyde with deoxyguanosine, and 3-p9 per lo7 base pairs. The identity of the MlGua was D-2’-deoxyribofuranosylpyrimido[1,2-alpurin-lO(3H)confirmed by recording the mass spectrum of material to one (MldGuo)2is produced by reaction of malondialdehyde which no internal standard had been added. The spectrum with deoxyguanosine (175-1 78). was identical to that of an authentic standard of the Barbin et al. recently combined immunoaffinity chropentafluorobenzyl derivative of M1Gua. matography with 32P-postlabelingfor the analysis of tdAdo tdGuo, tdCyd, and to a lesser extent tdAdo have been and edCyd in normal rat liver (174).The limit of detection shown to induce base-pair substitutions in site-specific of the assay was 5 adducts per 1Olo bases, and both adducts mutagenesis experiments (182-185). Likewise, 1,Wwere detected in the DNA from livers of Sprague-Dawley propano-2’-deoxyguanosine,which is a model for both rats ( I 1per loQbases) (174).The authors speculated that 8-OH-6-Me-PdGuo and MIGua, has been shown to induce these adducts may have arisen as a consequence of lipid base-pair substitutions and frameshifts in site-specific mutagenesis experiments (186,187).The frequencies of

T h e abbreviation MlGua refers to the presence of one molecule of malondialdehyde and one molecule of guanine in this adduct. It was proposed because malondialdehyde polymerizes and ita polymers also form adducts to nucleic acid bases.

3J. Swenberg, personal communication. *F.-L.Chung, personal communication.

Chem. Res. Toxicol., Vol. 6, No. 6,1993 777


mutations induced in plasmid-based mutagenesis assays by some of these adducts are comparable to or greater than those induced by large aromatic adducts derived from polycyclic hydrocarbons and aromatic amines (188-190). Relatively little information is available on the rate of repair of cyclic adducts from mammalian DNA. Swenberg et al. determined the levels of edGuo, edCyd, and edAdo in liver DNA of preweanling rats exposed to 600 ppm vinyl chloride for 5 days (4h/day) (191).EdGuo was removed very slowly (t1p 30 day) whereas edCyd and EdAdo did not appear to be repaired. These observations suggest that some cyclic adducts are repaired very slowly or not at all. A similar conclusion was reached by Vaca et al., who studied the removal of MldGuo from mouse liver followinga single intraperitoneal dose of malondialdehyde (192). The t l p for adduct disappearance was approximately 12.5 days. Despite the apparently long half-lives of these cyclic adducts in rodent liver, several groups have described the removal of etheno bases from DNA by glycosylases, including one from human placenta (193-195). The existence of such glycosylases implies that etheno adducts are repaired by a base excision-repair mechanism. If the excised bases appear unchanged in urine, it would provide the basis for a noninvasive method for estimation of adduct formation and repair. The levels of etheno adducts in rodent or human urine are unknown, but the base derived from MlGua has been reported to be present in human urine (196). However, a subsequent analysis of human and rat urine by thermospray LC/MS indicated that the levels of MlGua were below the limit of detection of the assay (0.5 pmol/mL of urine) (197). Thus, the presence of MlGua in human urine should be considered unconfirmed. The limited data available at present indicate that several different cyclic nucleic acid adducts are present in the hepatic DNA of normal rats and humans at levels ranging from 1 per 109 to 1 per lo6 base pairs. 8-OH-6Me-PdGuo and MldGuo probably derive from crotonaldehyde and malondialdehyde, respectively. The source of the etheno adducts is uncertain because there are several pathways for their formation. Although recent work suggests that etheno adducts can arise from reaction of DNA with unspecified products of lipid peroxidation (174), the levels of etheno adducts are not elevated in animals fed choline-deficient dietsa3 Previous studies suggest choline deficient diets increase oxidative stress in rodent liver (198). CdGuo and edCyd appear to be highly mutagenic, and model studies suggest 8-OH-6-Me-PdGuo and MldGuo are also highly mutagenic. The etheno adducts and MldGuo appear to be removed slowly from liver DNA and are persistent. Thus, cyclic nucleic acid adducts are produced endogenously, are efficient premutagenic lesions, and are repaired slowly. As such, they are ideal candidates for endogenous mediators of mutagenesis and carcinogenesis.

N

N

N

X

I

32P-Postlabeling is a sensitive technique developed by Randerath and colleagues for the detection and quantitation of DNA adducts (Figure 5) (22).Samples of DNA are treated with micrococcal nuclease and spleen phosphodiesterase to produce deoxynucleoside 3’-monophosphates. The 5’-hydroxyl group is then phosphorylated with [y32PlATPby the action of polynucleotide kinase.

N

N

N

1. Micrococcal Nuclease1

J

phosphodiesterase P-ATPIPNK

SJIIWI

2.

-

I-Compounds

N

2D-TLC Remove unmodifed dN’s

P

PO

1

PD-TLC

Resolve modified dXb

Figure 5. Outline of procedures for 32P-postlabeling.

This produces a mixture of deoxynucleoside 3’,5’-bisphosphates derived from normal and adducted bases. The 3’,5’-bisphosphates of normal deoxynucleosides are removed by enzymatic hydrolysis, solvent extraction, or chromatography, and then the 3‘,5‘-bisphosphates of adducted deoxynucleosides are resolved by multidimensional thin-layer chromatography on poly(ethy1eneimine)cellulose plates. Labeled compounds are detected by autoradiography and quantitated by Cerenkov counting of the 32P. Postlabeling will detect a wide range of DNA adducts so long as their deoxynucleoside 3‘-monophosphates are phosphorylated by polynucleotide kinase and their deoxynucleoside 3’,5’-bisphosphates are resolved from unmodified ones by the workup procedures. The sensitivity of the original version of 32P-postlabelingenabled adduct detection at a level of 1adduct per 108normal nucleotides (22).Subsequent refinements of the technique increased the sensitivity to 1 adduct per 1O1O normal nucleotides (199,200).32P-Postlabeling has been used to explore a wide range of problems related to adduction and repair of DNA by exogenous chemicals but suffers from the drawback that it does not provide any information about the structures of the adducts isolated. Experienced investigators have circumvented this problem by synthesizing standards of deoxynucleoside 3’,5’-bisphosphate adducts of known structure for chromatographic comparison. The development of the high-sensitivity version of 32Ppostlabeling enabled Randerath to detect putative adducts in DNA samples from animals that were not deliberately exposed to exogenous carcinogens (201).His group found that multiple adduct spots are detectable in DNA digests from liver and kidney of untreated rodents (201).Fewer numbers of spots are detectable in DNA digests from lung and heart. 32Pincorporation into adduct spots is not observed if DNA is omitted from the assay, if RNA is substituted for DNA, if nucleases are omitted, or if polynucleotide kinase is omitted (201).These requirements suggest the spots detected on TLC are derived from DNA adducts. Randerath terms the compounds respon-


sible for these spots I-compounds because the evidence suggests they arise indigenously. Recently, his group has detected I-spots in DNA from human brain (202). The numbers and levels of I-spots exhibit significant tissue, species, and sex dependence (201,203,204). The patterns in agiven tissue are quite reproducible and appear to reflect genetically determined differences between cell types in the production of endogenous electrophiles. The species and tissue differences seen in the pattern of I-spots are in marked contrast with the comparatively uniform DNA adduct profiles that are seen in different tissues and rodent species following administration of exogenous chemical carcinogenssuch as dibenz[a jlacridine or safrole (205). This reflects the fact that the same reactive derivatives are produced in most tissues from each exogenous chemical albeit at different levels. The levels of I-compounds demonstrate a significant increase with age, an effect that is especially pronounced in rats (206). For example, the total level of liver I-spots in rat liver DNA increases from 8.5 per 109 nucleotides to 83 per 109 nucleotides as the animals age from 1 to 4 months. In 8-10-month-old rats, the levels of liver I-spots are as high as 2 per lo7 dinucleotides. These levels approach those of DNA adducts measured in target organs following acute or chronic intoxication of rats with exogenous carcinogens (207). The levels of I-compounds in the livers and kidneys of different strains of rodents appear positively correlated with median life span (208). Thus, the longer an animal lives, the higher are the levels of its I-compounds. Significant variation in I-compound levels are observed with diet (206,209). The highest levels are observed with natural diets; lower levels are detected with several semipurified diets. Liver levels of I-spots appear positively correlated with protein and carbohydrate content and negatively correlated with fat content (209). Although these findings raise the possibility that I-spots are derived from genotoxic agents present in the diet, the available evidence suggests this is not the case (209). First, the chromatographic behavior of the I-spots is different from that of adducts derived from a number of genotoxic agents including natural carcinogens such as aflatoxin, safrole, nitrosamines, polycyclic hydrocarbons, and protein pyrolysates. Second, similar patterns of I-spots are observed when rats are fed diets that are certified for minimal xenobiotic contamination. Third, substantial tissue and species differences are observed in I-spot patterns in animals fed the same diets. These observations are more consistent with a mechanism in which natural or synthetic diets stimulate the production of endogenous electrophiles within tiwues to differing extents depending upon the genetic programming of the cell. Several different enzyme inducers diminish I-compound formation in rodent liver (204,210). These include 2,3,7,8tetrachlorodibenzo-p-dioxin,peroxisome proliferators, Aroclor 1254, pregnenolone-l6a-~arbonitrile, and phenobarbital. Since most of these compounds are inducers of different isoenzymes of cytochrome P450 (P450), the inverse dependence on enzyme induction implies a role for P450s in the metabolic removal of chemicals leading to I-compounds. Likewise depletion of P450s with agents like CCll increases the level of I-compounds (211). An implicit assumption of carcinogenesis research is that DNA adduction increases the probability of genetic mutations, which may eventually lead to the development

Marnett and Burcham

of cancer. Since I-compounds appear to represent DNA adducts, one might expect a positive correlation between the levels of one or more I-spots and induction of cancer. In fact, a negative correlation is observed. Examination of liver, lung, and kidney DNA from five strains of mice that differ in susceptibility to spontaneous liver and lung tumors reveals that lower levels of I-spots are present in the livers and lungs of strains that are most susceptible to tumors at those sites (201,203). Likewise, feeding rats a choline-deficient diet, which results in liver tumorigenesis, depresses the levels of I-spots in the liver relative to animals fed a choline-sufficient diet (212). The levels of I-spots in uninvolved liver tissue from tumor-bearing rats are lower than the levels in normal liver, and the levels in tumor tissue are drastically reduced relative to uninvolved or normal tissue (212). In contrast, caloric restriction of normal animals, which is associated with a reduction in susceptibility to tumorigenesis, increases the levels of I-spots (213). Finally, determination of the levels of I-spots in the DNA from a series of Morris hepatomas grown in vivo reveals a depression of 7-16-fold relative to normal liver (224). Comparable reductions are observed in minimal-deviation hepatomas, but there is no correlation between reduction of adduct levels and tumor growth rate. Randerath has suggested that reduction in the levels of I-compounds with tumor development may indicate a functional role for these DNA adducts in normal cells. Some support for this is provided by the finding that the levels of I-spots exhibit circadian rhythm, implying that their formation is regulated (215). Levels of several I-spots in mouse liver are lowest in early morning and rise severalfold during the day. The levels of one particular I-spot decreases during the same time period (225). In contrast, the levels of DNA adducts produced from the exogenous carcinogen safrole do not vary throughout the day (215). Likewise, the levels of 5-methyldeoxycytidine in DNA, which is believed to modulate gene expression, do not exhibit diurnal variation (215). On the surface, these findings appear to challenge our assumptions that DNA adducts are harmful to the cell and lead to mutation and cancer. In fact, the compounds detected as I-spots may play a role in regulation of gene expression and proliferation. In this regard, recent findings linking DNA repair to transcriptional control are provocative (216-218). However, it is also possible that decreases in I-spot levels with tumorigenesis reflect increases in P450 activity. For example, some tumors exhibit elevated P450 levels, and Randerath has shown that P450 inducers lead to a decrease in the levels of I-spots (204,210). Thus, the reduction in I-spots associated with tumorigenesis may reflect modulation of mixed-function oxidase activity. It is difficult to speculate on the importance of I-compounds in carcinogenesis without information on their structures. A t present, the available data indicate what they are not: oxidative lesions, 5-methyldeoxycytidine, or previously identified DNA adducts arising from exogenous chemicals (205,219). Although the levels of most I-compounds in DNA are rather low, some of these adducts attain levels approaching 1 per lo7nucleotides. Improvements in mass spectroscopic methods for analysis of trace amounts of low-volatility organic compounds suggest it should be possible to acquire structural information on some of these compounds in the near future. For example, from the DNA isolated from a single rat liver, we have

Chem. Res. Toxicol., Vol. 6, No. 6, 1993 779


recorded a complete mass spectrum of a derivative of the endogenous malondialdehyde-deoxyguanosine adduct, MldGuo (181). Once the structures of the I-spots are solved, retrosynthetic analysis should identify the reactive intermediates that lead to them and suggest possible biosynthetic pathways for their formation. Elucidation of the biosynthetic pathways may identify genetic loci that could be modulated by diet to increase or decrease their levels. Furthermore, chemically synthesized I-compounds could be incorporated into oligodeoxynucleotides to evaluate their effects on DNA replication, mutagenesis, and repair. This would provide the ultimate test of the hypothesis that there are biological consequences to the formation of this class of endogenous DNA adducts.

Conclusions The study of the chemistry and biology of endogenous DNA adducts is still in its infancy. Nevertheless, the available information indicates that a multitude of endogenous adducts are present in rodent DNA at levels of 1per 109 base pairs to as high as 1 per 106 base pairs. It is likely that analogous adducts are present in human DNA. Some of these endogenous adducts (oxidized bases, alkylated bases, exocyclic adducts) appear to be efficient premutagenic lesions, which raises questions about their contribution to human cancer. For example, do endogenous adducts provide a background of structurally diverse premutagenic lesions waiting to be fixed by stimulation of cell proliferation? Does stimulation of the formation of endogenous adducts provide a mechanism for the carcinogenic activity of nongenotoxic carcinogens? The contribution of endogenous DNA adducts to mutations and genetic disease relative to other endogenous pathways of mutagenesis is also unknown. Replication errors introduced by DNA polymerases are a major mechanism of spontaneous mutagenesis although mammalian cells use exonucleolyticproofreading and mismatch repair to minimize them (14, 220, 221). In addition, deamination of purines and pyrimidines may contribute to the genesis of transition mutations (222). Although the spontaneous rate of deamination in double-stranded DNA is remarkably low, recent studies have found that deamination is significantly accelerated by reaction of DNA with nitric oxide (223, 224). Nitric oxide is a ubiquitous mediator of signal transduction and a cytotoxic product of inflammatory cells (225). Examination of the mutation spectra of the p53 gene in several human tumors (e.g., colon) reveals a preponderance of C T transitions, which probably result from deamination or replication errors (226). However, p53 mutation spectra in other human tumors are dominated by transversion mutations, which are more commonly associated with replication of DNA adducts from exogenous or endogenous sources (226). The rates of deamination of purines and pyrimidines pale in comparison to the rates of spontaneous depurination of DNA and introduction of strand breaks. Depurination is estimated to occur at a rate of 5801h per cell and strand breaks at a rate of 2300lh per cell (227). These estimates are based on the rates of spontaneous hydrolysis and strand breakage of pure DNA molecules in solution and have not actually been measured in cells. Despite these high rates of spontaneous DNA degradation, abasic sites and strand breaks appear to be efficiently repaired, so their importance as premutagenic lesions is uncertain

-

Table I. Levels of Selected Endogenous and Exogenous DNA Adducts in Human Tissues ~~

adduct levels ( r 107 adduct tissue Lee) 12 f 7 8-oXo-dGuo leukocyte 10f5 5-OH-dCyd leukocytes 7*6 5-OH-dUrd leukocytes leukocytes 20 15 dUrdg leukocytes 230 8-oxo-dAdob 5-HMdUrd white blood cells 2300 t 480 lung 0.25 f 13 OB-Me-dGuo lymphocytes 14 7-Me-dGuo 6 8-OH-6-Me-PdGuo liver 10 8-OH-PdGuo liver 0.7 f 0.4 cdAdo liver tdCyd liver 2.8 f 0.9 lymphocytes 0.3-0.2 P AH-DNA' bladder 2-3 4-ABP-dGL

~

method EC-HPLCO EC-HPLCa EC-HPLQ EC-HPLC" GC/MSc GC/MSd mP-p~~tlabeling' azp-poetlabe1ingi a2P-poetlabelii SP-postlabelingr 82P-poetlabelinP S2P-poetlabeing" a2P-postlabelingj aZP-poetlabelin&

a From ref 72. 7,8-Dihydro-8-0~0-2'-deoxyadenosine.From ref 80. d From ref 78. The levels shown were calculated assuming thymidine comprises 25 % of totaldeoxynucleotides. * From ref 120. The levels shownwere calculated assumingdeoxyguanosine comprises 25% of total deoxynucleotides. f From ref 117.

(228). Considering the extent of DNA damage from spontaneous depurination, strand breaks, deamination, and endogenous adduction, there appears to be a multitude of covalent modifications of DNA occurring constantly in normal cells, but the biological consequences are unknown. Perhaps it will eventually be possible to determine the total inventory of DNA damage in an individual cell type and relate the occurrence of particular lesions to genetic mutations that arise in that cell. Although this is an attractive and worthwhile goal, we are currently a long way from achieving it. The presence of endogenous adducts in cellular DNA at levels approaching 1 per lo6 base pairs also raises provocative questions about the significance of the formation of adducts from exogenouscarcinogens. The latter are usually produced at levels of 1per lo7 base pairs or lower in target tissues (26, 207). Comparable levels are formed in human DNA during chronic exposure to very low doses of carcinogens (Table I) (26,229-234). Since exogenous and endogenous adducts appear comparable in efficiency as premutagenic lesions, one might question the biological consequences of increasing the total adduct burden in a cell by 1-10% following exposure to an exogenous chemical. Perhaps differential rates of DNA repair alter the relative biological potencies of exogenous and endogenous adducts. Therefore, studies of the rates and pathways of repair of endogenous adducts should be accorded a high priority. A critical deficiency in our knowledge has to do with the relationship of endogenous adduct levels to the development of cancer. A priori, one might expect that endogenous adducts increase in concentration in association with chemical, dietary, or physiological modifications that lead to cancer. If so, quantitation of their levels might provide an estimate of the relative risk of developingcertain cancers and could be useful for comparison to the levels of adducts from exogenouschemicals. However, the only information linking endogenous adduct formation to risk of cancer relates to levels of I-compounds. Surprisingly, an inverse association appears to exist; adduct levels decrease with susceptibility to tumorigenesis. These studies challenge our concepts of the role of DNA modification in carcinogenesis by suggesting that some endogenous adducts may actually play a role in normal cells in the of control gene expression and perhaps proliferation. However, this


speculation can only be tested by the identification of individual I-compounds and evaluation of their biological function. Finally, it is important to reiterate the contribution that chemistry has made to our awareness of the potential importance of endogenous DNA adducts. This is a field that has evolved from the activities of several research groups pushing the envelope of analytical chemistry as applied to DNA adduct detection and identification. As in many cases, our technological capabilities currently exceed our understanding of the biological significance of the results. However, this gap will close quickly as toxicologistsand molecular biologists apply their expertise to evaluating the genotoxic potential of this class of adducts. In the meantime, the detection, identification, and quantitation of endogenous DNA adducts represents an exciting challenge to investigators at the frontiers of structural and analytical chemistry, mechanistic toxicology, and molecular epidemiology.

Acknowledgment. Work in the principal investigator’s laboratory on endogenous DNA adducts is supported by a research grant from the National Institutes of Health (CA47479). We are grateful to FungLung Chung, Helmut Bartsch, and James Swenberg for sharing data in advance of publication, to Richard Setlow for a helpful discussion, and to F. Peter Guengerich and Kurt Randerath for critical readings.

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