Mutagenesis by acrolein-derived propanodeoxyguanosine adducts in human cells

Acrolein, which is widely spread in the environment and is produced by lipid peroxidation in cells, reacts with DNA to form two exocyclic 1,N2-propanodeoxyguanosine (PdG) adducts. To establish their relative contribution to the acrolein mutagenicity, the genotoxic properties of alpha-OH-PdG and gamma-OH-PdG together with their model DNA adduct, PdG, were studied in human cells. DNA adducts were incorporated site-specifically into a SV40/BK virus origin-based shuttle vector and replicated in xeroderma pigmentosum complementation group A (XPA) cells. Analysis of progeny plasmid revealed that alpha-OH-PdG and PdG strongly block DNA synthesis and that both adducts induced base substitutions with G --> T transversions predominating. Primer extension studies, catalyzed by the 3'-->5' exonuclease-deficient Klenow fragment of Escherichia coli pol I, revealed limited extension from the 3' primer termini opposite these two adducts. In contrast, gamma-OH-PdG did not strongly block DNA synthesis or miscode in XPA cells. Primer extension from a dC terminus opposite gamma-OH-PdG was much more efficient than that opposite alpha-OH-PdG or PdG. These results indicate that the minor alpha-OH-PdG adduct is more genotoxic than the major gamma-OH-PdG. Furthermore, experiments using a HeLa whole cell extract indicate that all three DNA adducts are not efficiently removed from DNA by base excision repair.     

The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA-topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

Crotonaldehyde is a representative alpha,beta-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA-Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA-Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA-Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde.     

Mutagenic properties of estrogen quinone-derived DNA adducts in simian kidney cells

DNA damage caused by catechol estrogens has been shown to play an etiologic role in tumor formation. Catechol estrogens are reactive to DNA and form several DNA adducts via their quinone forms. To explore the mutagenic properties of 2-hydroxyestrogen-derived DNA adducts in mammalian cells, N(2)-(2-hydroxyestrogen-6-yl)-2'-deoxyguanosine and N(6)-(2-hydroxyestrogen-6-yl)-2'-deoxyadenosine adducts induced by quinones of 2-hydroxyestrone, 2-hydroxyestradiol, or 2-hydroxyestriol were incorporated site-specifically into the oligodeoxynucleotides ((5)(')TCCTCCTCXCCTCTC, where X is dG, dA, 2-OHE-N(2)-dG, or 2-OHE-N(6)-dA). The modified oligodeoxynucleotides were inserted into single-stranded phagemid vectors followed by transfection into simian kidney (COS-7) cells. Preferential incorporation of dCMP, the correct base, was observed opposite all 2-OHE-N(2)-dG adducts. Only targeted G --> T transversions were detected; the highest mutation frequency (18.2%) was observed opposite the 2-OHE(2)-N(2)-dG adduct, followed by 2-OHE(1)-N(2)-dG (4.4%) and 2-OHE(3)-N(2)-dG (1.3%). When 2-OHE-N(6)-dA adducts were used, preferential incorporation of dTMP, the correct base, was observed. Targeted mutations representing A --> T transversions were detected, accompanied by small numbers of A --> G transitions. The highest mutation frequencies were observed with 2-OHE(1)-N(6)-dA and 2-OHE(3)-N(6)-dA (14.5 and 14.1%, respectively), while 2-OHE(2)-N(6)-dA exhibited a mutation frequency of only 6.0%. No mutations were detected with vectors containing unmodified oligodeoxynucleotides. Thus, 2-OHE quinone-derived DNA adducts are mutagenic, generating primarily G --> T and A --> T mutations in mammalian cells. The mutational frequency varied depending on the nature of the 2-OHE moiety.     

Mutagenic potential of benzo[a]pyrene-derived DNA adducts positioned in codon 273 of the human P53 gene

Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo[a]pyrene. We incorporated a single (+)- or (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells. Oligodeoxyribonucleotides ((5)(')GAGGTGCG(BPDE)TGTTTGT) modified with (+)- or (-)-trans-dG-N(2)-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells. Progeny plasmids were then used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base. We determined the mutational frequencies associated with (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE adduction to be 26.5% and 17.5%, respectively. The predominant mutations generated by both stereoisomers were G --> T transversions, with some G --> A transitions. When the cytosine 5' to dG-N(2)-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged. Thus, the mutational "hot spot" at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE-DNA adducts positioned at this site.     

Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II).

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.     

Protein-template-directed synthesis across an acrolein-derived DNA adduct by yeast Rev1 DNA polymerase

Acrolein is generated as the end product of lipid peroxidation and is also a ubiquitous environmental pollutant. Its reaction with the N2 of guanine leads to a cyclic gamma-HOPdG adduct that presents a block to normal replication. We show here that yeast Rev1 incorporates the correct nucleotide C opposite a permanently ring-closed form of gamma-HOPdG (PdG) with nearly the same efficiency as opposite an undamaged G. The structural basis of this action lies in the eviction of the PdG adduct from the Rev1 active site, and the pairing of incoming dCTP with a "surrogate" arginine residue. We also show that yeast Polzeta can carry out the subsequent extension reaction. Together, our studies reveal how the exocyclic PdG adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Rev1 and Polzeta provides for accurate and efficient synthesis through this potentially carcinogenic DNA lesion.     

Mutation induction and killing of Escherichia coli by DNA adducts and crosslinks: a photobiological study with 8-methoxypsoralen.

Low doses of 350 nm radiation (NUV) in the presence of 8-methoxypsoralen (8-MOP) induce predominantly mono-adducts in bacterial DNA. Further exposure to NUV in the absence of 8-MOP converts a proportion of these mono-adducts to interstrand cross-links. Using this approach the relative effects of adducts and cross-links on bacteria with different repair capacities was studied. Escherichia coli WP100 uvrA recA, believed to be totally deficient in the ability to repair 8-MOP plus NUV damage to DNA, was inactivated on average by a single photon event occurring with a quantum efficiency of about 0.03. We conclude that the inactivating lesion is probably a single mono-adduct. E. coli WP2 uvrA, deficient in excision endonuclease activity, may be inactivated by a very small number of cross-links, probably one. These conclusions are consistent with present knowledge of the repair capabilities of these bacteria. Conversion of mono-adducts to cross-links in WP2 uvrA (which occurs with a quantum efficiency of around 0.3) greatly increases lethality but results in a reduction of the induced mutation frequency presumably because cross-links are (almost) invariably lethal. In the repair-proficient strain WP2 both adducts and cross-links can be repaired but the latter are more likely than the former to lead to either death or mutation.     

Mutagenic potential of anti-herpes agents

A number of anti-herpes agents which are either licensed for clinical use (acyclovir) or subject of clinical studies (bromovinyldeoxyuridine, fluoroiodoaracytidine, dihydroxypropoxymethylguanine) or under preclinical investigation (i.e., fluoroiodoarauridine), fluoromethylarauridine, dihydroxybutylguanine, bromovinyldeoxycytidine, bromovinylarauridine and carbocyclic bromovinyldeoxyuridine) were evaluated for their ability to induce sister chromatid exchange (SCE), an indicator of mutagenesis. SCE was scored on metaphase chromosomes of human lymphocytes which had been exposed to 5-bromo-2'-deoxyuridine and varying concentrations of the test compounds. The antiviral assays were based on the inhibition of the cytopathogenicity of herpes simplex virus for human diploid fibroblasts. Most compounds, i.e. acyclovir, bromovinyldeoxyuridine or carbocyclic bromovinyldeoxyuridine, did either not induce SCE or only so at concentrations far above their minimum antiviral concentrations. However, fluoroiodoaracytidine and dihydroxypropoxymethylguanine were found to affect the SCE rate at a concentration (greater than or equal to 4.5 micrograms/ml) that is readily achievable in blood following intravenous injection.     

Mutagenic bypass of the butadiene-derived 2'-deoxyuridine adducts by polymerases eta and zeta

Butadiene is a ubiquitous environmental chemical carcinogen that when activated to its monoepoxide intermediate can react with the N3 position of cytosine, resulting in two stereoisomeric adducted bases that rapidly deaminate to N3 2′-deoxyuridine lesions. We have previously shown that replication of DNAs containing these adducts through mammalian cells resulted in 97% mutagenicity, predominantly C to T transitions. Since replicative DNA polymerases were blocked by these lesions in vitro, translesional polymerases were assessed for their ability to bypass these adducts. While polymerases ι, κ and ζ were significantly blocked one nucleotide prior to the lesion, pol η incorporated nucleotides opposite the adducts with a preference for insertion of a G or A. Following polymerase dissociation and reassociation, pol η was also able to extend primers with mispaired termini opposite the lesions, with extensions from the A and T mismatched primer termini being the most efficient. Pol ζ was also able to extend primers containing all mismatched nucleotides opposite the lesions, with the most efficient extension occurring off of the A mismatched primer.     

Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms

Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells.     

Structure-dependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.     

Determination by fluorescence quenching of the environment of DNA crosslinks made by malondialdehyde

DNA crosslinks made by malondialdehyde are fluorescent. The fluorescence is quenched by collision or intercalation. A 3.3-fold higher concentration of the external (collision) quencher KI was required to cause 50% quenching of the fluorescence of the interstrand-DNA crosslinks than to cause 50% quenching of the fluorescence of the model compounds Val2MDA and the malondialdehyde-crosslinked heterodimer of GMP and CMP. Thus, the crosslinked nucleotide dimers in the DNA were shown to be 70% shielded from the solvent. Similarly, DNA-protein crosslinks made by malondialdehyde were shown to be 55% shielded. The internal (intercalation) quencher Ag+ enhanced the fluorescence of the DNA crosslinks at concentrations below 0.3 mM; higher concentrations quenched the fluorescence. Concentrations of Ag+ below 10 mM did not affect the fluorescence of the model compounds. The calculated dissociation constant for Ag+ was much less at pH 5 than at pH 7 or 9. The observed binding of Ag+ and its pH dependence suggest that pi-stacking of adjacent bases strengthens the binding of Ag+ to the crosslinks. These results indicate that the crosslinks are in the interior of the DNA, so they may not easily be recognized by a repair system.     

Formation of DNA adducts by formaldehyde-activated mitoxantrone.

Recent studies with the anthracycline Adriamycin have demonstrated its activation by formaldehyde and subsequent binding to DNA in vitro. Since formaldehyde levels are known to be higher in cells of myeloid origin and the structurally related drug mitoxantrone is most effective against cancers of myeloid origin, this indicates a possible role of formaldehyde in the activation of mitoxantrone. In vitro studies revealed that the activation of mitoxantrone by formaldehyde leads to the formation of drug-DNA adducts. These adducts stabilised DNA such that they functioned as virtual interstrand crosslinks. The interstrand crosslinks were formed in the presence of mitoxantrone and formaldehyde in a time- and concentration-dependent manner. In the absence of formaldehyde no crosslinks were formed, indicating a key role in drug activation and DNA binding. The adducts (virtual crosslinks) were relatively unstable with 50% crosslinks remaining after 10 min at 60 degrees C in 45% formamide. Like Adriamycin, the mitoxantrone-formaldehyde-DNA crosslinks are heat labile and do not display the stability associated with covalent interstrand crosslinks.     

Platinated DNA adducts enhance poisoning of DNA topoisomerase I by camptothecin

Camptothecins constitute a novel class of chemotherapeutics that selectively target DNA topoisomerase I (Top1) by reversibly stabilizing a covalent enzyme-DNA intermediate. This cytotoxic mechanism contrasts with that of platinum drugs, such as cisplatin, which induce inter- and intrastrand DNA adducts. In vitro combination studies using platinum drugs combined with Top1 poisons, such as topotecan, showed a schedule-dependent synergistic activity, with promising results in the clinic. However, whereas the molecular mechanism of these single agents may be relatively well understood, the mode of action of these chemotherapeutic agents in combination necessitates a more complete understanding. Indeed, we recently reported that a functional homologous recombination pathway is required for cisplatin and topotecan synergy yet represses the synergistic toxicity of 1-beta-D-arabinofuranosyl cytidine in combination with topotecan (van Waardenburg, R. C., de Jong, L. A., van Delft, F., van Eijndhoven, M. A., Bohlander, M., Bjornsti, M. A., Brouwer, J., and Schellens, J. H. (2004) Mol. Cancer Ther. 3, 393-402). Here we provide direct evidence for Pt-1,3-d(GTG) poisoning of Top1 in vitro and demonstrate that persistent Pt-DNA adducts correlate with increased covalent Top1-DNA complexes in vivo. This contrasts with a lack of persistent lesions induced by the alkylating agent bis[chloroethyl]nitrosourea, which exhibits only additive activity with topotecan in a range of cell lines. In human IGROV-1 ovarian cancer cells, the synergistic activity of cisplatin with topotecan requires processive DNA polymerization, whereas overexpression of Top1 enhances yeast cell sensitivity to cisplatin. These results indicate that the cytotoxic activity of cisplatin is due, in part, to poisoning of Top1, which is exacerbated in the presence of topotecan.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.