Chem. Res. Toxicol. 1999, 12, 501-507
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Mutagenicity of Site-Specifically Located in Chinese Hamster Ovary Cell Chromosomal DNA
1,N2-Ethenoguanine
Susumu Akasaka† and F. Peter Guengerich* Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received December 8, 1998
The adduct 1,N2-etheno()-guanine (Gua) can be formed in DNA from exogenous or endogenous bifunctional electrophiles. Previous work with site-specifically modified oligonucleotides has shown all three possible base substitutions at the site of this residue in bacterial cells and in primer extension assays using purified polymerases (with the purified polymerases also showing deletions). A 10-mer was synthesized containing 1,N2--Gua at a specific position and ligated into a modified pCNheIA vector, which was used to insert the modified sequence into the chromosomes of AA8 (wild-type) and UV5 (nucleotide excision repair-deficient) Chinese hamster ovary cells. Transformants were selected by antibiotic resistance; DNA was amplified by polymerase chain reaction, and resistance to the restriction endonuclease NheI was used to estimate mutation frequency. In the AA8 cells, the apparent mutation frequency was elevated >10-fold due to the presence of 1,N2--Gua (to 4.6%). In UV5 cells, the mutation frequency was even higher (7.8%), but the estimate of the frequency in the control system (vector and unmodified sequence only) was 4.5%. Sequence analysis of 21 clones derived from the mutant fraction yielded five that correspond to base pair mutations directly at the 1,N2--Gua site. The remainder of the mutants differed from those generated from the unmodified oligonucleotide and included deletions, rearrangements, double mutants, and base pair substitutions at sites nearby but not at the 1,N2--Gua site.
Introduction Many chemical carcinogens elicit their effects through genotoxic mechanisms, and a relationship between mutagenesis and carcinogenesis has been recognized for many years (1). Even though genotoxicity may not be a complete explanation, it is generally accepted to be the primary basis of the initiation phase of chemical carcinogenesis (2). The number of modifications of DNA that have now been characterized is considerable (3, 4). One group of “exocyclic” DNA-carcinogen adducts is the etheno family, which is named so because of the addition of five-membered rings (with two extra carbons added to purines and pyrimidines) (5). Thus, the group consists of a series of tricyclic ring systems formed from Ade and Gua, bicyclic ring systems formed from Cyt, and their derivatives (6). Such compounds are formed from the reaction of DNA with bifunctional electrophiles. We have characterized mechanisms of formation of these etheno adducts from substituted oxiranes (7, 8), the oxidation products of vinyl monomers. Of interest is the finding that the major etheno adducts can all be formed not only from vinyl halides (9) but also from contaminants of drinking water and from lipid peroxidation products (10-12). The levels arising from the latter * To whom correspondence should be addressed: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Medical Research Building I, 23rd and Pierce Avenues, Nashville, TN 37232-0146. Telephone: (615) 322-2261. Fax: (615) 322-3141. E-mail:
[email protected]. † Present address: Osaka Prefectural Institute of Public Health, Osaka 537, Japan.
source appear to be influenced by diet, in the absence of known exogenous carcinogens (10, 13). The genotoxicity of the major etheno derivatives has been studied in various systems. Since bifunctional electrophilic precursors of these lesions react with Ade, Cyt, and Gua, most of the work has utilized systems in which etheno adducts have been synthesized as nucleosides and incorporated into oligonucleotides by chemical synthesis. 1,N6-Etheno()1-Ade is mutagenic in bacterial cells (14). 3,N4--Cyt is also mutagenic in both bacteria and mammalian cells, with considerable variations reported (14-16). N2,3--dGTP has been incorporated into a vector using enzymatic procedures and used to produce mutants in bacteria (17).
1,N2--Gua has been synthetically incorporated into oligonucleotides and shown to cause polymerase blockage, mispairing, and deletions in vitro (18). In Escherichia coli, 1 Abbreviations: , etheno; CHO, Chinese hamster ovary; R-MEM, alpha minimal essential medium (Eagle’s); PCR, polymerase chain reaction. See the list of standard abbreviations of this journal (January 1999 issue) for the abbreviations for nucleic acid bases and nucleosides.
10.1021/tx980259j CCC: $18.00 © 1999 American Chemical Society Published on Web 05/14/1999
502 Chem. Res. Toxicol., Vol. 12, No. 6, 1999
Figure 1. Capillary gel electrophoresis of 5′-ATGCTA[1,N2-Gua]CAT-3′.
analysis of base pair substitutions resulting from 1,N2-Gua indicated that A, C, G, and T could all be inserted at the site opposite the lesion (19). Further, the distribution of misincorporations observed from purified polymerases (in vitro) differed considerably among the enzymes used and from the in vivo bacterial pattern (18). Because of the presence of 1,N2--Gua in DNA due to a variety of sources and the positive but variable genotoxic events observed in our various studies (18, 19), characterization of events in mammalian cells is of interest, particularly in light of some of the differences from bacterial systems reported by Moriya et al. (16). A number of mammalian systems have been employed for DNA adduct site-specific mutagenesis (20-22). Most involve extrachromosomal replication. Essigmann and his associates have developed and utilized a system involving genomic integration of the modified plasmid vector (containing the adduct) and analysis of the product (23, 24). In this system, O6-alkylGua and O4-alkylThy adducts yielded only the expected respective G f A and T f C transitions, and these were attenuated in the presence of the appropriate methyl transferase systems. Although this system is more complex than the shuttlevector approaches and has a higher background, it has a potential advantage in that the modes of replication must be identical to the situation normally encountered in cells (with the caveat that the cells are in culture). We utilized this intrachromosomal system to study events in the Chinese hamster ovary (CHO) cell genome resulting from the presence of 1,N2--Gua.
Experimental Procedures Oligonucleotide Synthesis. The unmodified 10-mer (with deoxy sugars) 5′-ATGCTAGCAT-3′ was prepared by Midland Certified Reagent Co. (Midland, TX). 1,N2--dGuo phosphoramidite was prepared as previously described (18) and used to prepare 5′-ATGCTA[1,N2--Gua]CAT, using an Expedite Nucleic Acid Synthesis System (Millipore Corp., Bedford, MA) and 4-tertbutylphenoxyacetyl-protected reagents (PerSeptive Biosystems, Framingham, MA). Both oligonucleotides were purified by HPLC and subsequent polyacrylamide gel electrophoresis (18, 19). The purity of the oligonucleotides was established by capillary gel electrophoresis (Figure 1) (see refs 18 and 19 for details of the conditions). The identity of the oligonucleotides was further established by enzymatic digestion/HPLC/UV detection of the 1,N2--Gua-containing oligonucleotide (18) (results not presented).
Akasaka and Guengerich Construction of pCNheIA (Figure 2). Plasmid pCXmnI, which contains the Neor gene (23, 24), was kindly provided by J. M. Essigmann (Massachusetts Institute of Technology, Cambridge, MA). pCNheIA was constructed by inserting a double-stranded 10-mer oligonucleotide (5′-ATGCTAGCAT-3′ plus complement), which includes the 6 bp NheI site, into the XmnI site. Plasmid DNAs were isolated from transformed E. coli DH5R cells with a Qiagen Maxi Kit (Qiagen, Studio City, CA) and purified by CsCl-ethidium bromide equilibrium ultracentrifugation. Preparation of pCNheIA Containing 1,N2-E-Gua. The gapped heteroduplex was prepared following the method of Altshuler et al. (24) with minor changes. XmnI-digested pCXmnI and HindIII-digested pCNheIA were mixed at a ratio of 1:1 in 10 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl and 1.0 mM EDTA (Figure 2). The mixture was incubated at 95 °C for 8 min, cooled to 65 °C in a water bath over the course of 25 min, and incubated for an additional 10 min at 65 °C. The resultant gapped circular DNA and linear DNA were separated by repetitive gel electrophoresis (twice, 1.0% low-melting point agarose). The gapped duplex was extracted from the agarose gel with β-agarase I (New England BioLabs, Beverly, MA). Single-stranded 10-mer oligonucleotides containing Gua or 1,N2--Gua at the seventh base position (G7) were phosphorylated at the 5′ end with T4 polynucleotide kinase and ATP. The gapped duplex and a 100-fold molar excess of each 10-mer oligonucleotide were incubated at 37 °C for 10 min and cooled to room temperature. Ligation was performed by incubation with T4 DNA ligase for 2 h at 16 °C. Another aliquot of ligase was added and incubation carried out overnight. An aliquot of the product was digested with NheI to measure the extent of ligation. The digested mixtures were separated by electrophoresis (1.0% agarose gel) and stained with ethidium bromide. The amounts of linear and open circular DNA were estimated with a Nucleovision transillumination imaging system (Nucleotech, San Carlos, CA) and the associated (GelExpert) software. Transfection of pCNheIA into CHO Cells. CHO cell lines AA8 and UV5 were purchased from American Type Culture Collection (Rockville, MD). UV5 is a derivative of AA8 and is hypersensitive to bulky-adduct mutagens. Cells [1-2 × 105/60 mm dish, in 4 mL of alpha minimal essential medium (Eagle’s) (R-MEM) growth medium, with 10% fetal calf serum, v/v] were incubated for 18-24 h to give 40-60% confluence. Control pCNheIA (constructed with the normal 10-mer oligonucleotide) or pCNheIA containing 1,N2--Gua was digested with EcoRI and transfected into AA8 or UV5 CHO cells using the liposome formulation Lipofectin (Life Technologies, Gaitherburg, MD) following the supplier’s instructions; 1 µg of DNA (in 100 µL of OPTI-MEMI medium) and 5 µL of Lipofectin (in 100 µL of OPTIMEMI medium) were combined and incubated at room temperature for 10-15 min. Serum-free R-MEM (1.8 mL) was added to the tube containing the Lipofectin-DNA complex, and the complex was overlayed onto the cells. The cells were incubated for 8 h at 37 °C in a CO2 incubator; the DNA containing medium was replaced with 4 mL of growth medium (10% fetal calf serum and R-MEM), and the cells were incubated at 37 °C in a CO2 incubator for an additional 48 h. The transfected cells (in 60 mm dishes) were divided into three 100 mm dishes containing 10 mL of selective medium (R-MEM medium with 10% fetal calf serum and 800 µg of G418 sulfate per milliliter). Cells were cultured for 3 weeks, with changes of the selective medium every 3 days to select cells with pCNheIA integrated in the chromosome. The colonies (in the 100 mm dishes) were washed with a phosphate-buffered saline solution and counted without staining. Cells were gathered (using a scrubber) for subsequent extraction of chromosomal DNA. Polymerase Chain Reaction (PCR) Recovery of DNA. Chromosomal DNA of the neomycin-selected cells was isolated with a QIAamp Blood Kit (Qiagen, Chatsworth, CA). Extracted DNA samples from three 100 mm dishes (which had been divided from an original 60 mm dish) were mixed as one
1,N2--Guanine Mutations
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Figure 2. Strategy for mutagenesis experiments. independent transfectant. A 285 bp DNA fragment of pCNheIA integrated in the chromosome was recovered by PCR following the method of Altshuler et al. (24) with minor changes. The primers were P1a (5′-GCGGCGACGATAGTCATGCC-3′) and P3a (5′-CACACCTCCCCCTGAACCTG-3′). Vent DNA polymerase (New England Biolabs) was used insted of Taq polymerase. PCR products were separated by gel electrophoresis (1.8% low-melting point agarose) using 0.5× TBE buffer [45 mM
Tris-borate (pH 8.3) with 1.0 mM EDTA] and isolated from the gel with β-agarase I. The deletion frequency was estimated by semiquantitative PCR (Figure 2), using two pairs of primers, neo1 (5′-CCGCTCAGAAGAACTCGTCAAGAA-3′) and neo2 (5′-TCCTGCCGAGAAAGTATCCATCAT-3′), which give a NeoR gene 476 bp fragment, and primers P1a and P3a (vide supra).
504 Chem. Res. Toxicol., Vol. 12, No. 6, 1999 Table 1. Transfection Results with the Site-Specifically Adducted pCNheIA Vector number of colonies (µg of pCNheIA)-1 cell line G7 site in vector AA8 UV5 AA8 UV5
Gua Gua 1,N2--Gua 1,N2--Gua
experiment 1
experiment 2
446 ( 24 424 ( 48 411 ( 60 339 ( 112
555 ( 36 374 ( 34 486 ( 38 283 ( 28
Estimation of the Mutation Frequency. The purified 285 bp fragment (2 µg) was digested with the restriction enzyme NheI. If a mutation occurs in the NheI site, the 285 bp fragment cannot be digested by the restriction enzyme (Figure 2). The reaction was started by adding 5 units of the enzyme, with incubation at 37 °C for 10 h, and the addition of an additional 5 units of NheI every 2.5 h (i.e., total of 20 units). The reaction mixture was extracted with phenol and phenol/CHCl3/isoamyl alcohol (25:24:1, v/v/v) and then ethanol precipitated. The digested fragment (194 bp) and undigested fragment (285 bp) were separated by gel electrophoresis (1.8% agarose) and blotted to Hybond-N nylon membranes (Amersham, Des Plaines, IL) with a PosiBlot system (Stratagene, La Jolla, CA). The membrane was hybridized with the 32P-labeled P3a 20-mer oligonucleotide. The amounts of undigested and digested PCR products were quantified by PhosphorImager analysis (model 400E, Molecular Dynamics, Sunnyvale, CA). Cloning of Mutated DNA Fragments. NheI-treated PCR products were ligated into the SmaI site of cloning vector pUC19 and introduced into E. coli DH5R cells. The transformed cells with inserted pUC19 were selected by colony hybridization under stringent conditions. Probe 200A recognizes the region 23 bp 5′-upstream of the 10-mer inserted site (5′-CTTATCATGTCTGGATCCGAACCAT-3′). Probe NheIA is a 25-mer including the 10-mer insert (5′-CGAACCATGCTAGCATCTTCGTCGA3′). Probe 200A-positive and probe NheIA-negative colonies were selected as mutants of the target site. The DNA from selected colonies was isolated and digested with NheI or XmnI. The DNA that was not digested by NheI or XmnI was purified for sequencing. Sequence analysis of the 285 bp fragments was performed using a ABI BigDye Terminator Cycle Sequencing kit and ABI Prism 310 Genetic Analyzer (Perkin-Elmer/Applied Biosystems, Foster City, CA).
Results Transformation and PCR. In vitro studies with oligonucleotides containing 1,N2--Gua indicated strong polymerase blockage (18, 19) and UV induction of the SOS response was needed for mutagenesis in bacterial cells (19). Toxicity to the CHO cells is not directly observed in the system used here, because stable integration precedes replication. The transformation efficiency was somewhat reduced by the presence of 1,N2--Gua in the vector, but not sufficiently to interfere with the analysis (Table 1). The ratios of PCR products between the Neor site and the target oligmer were determined, as a measure of deletion frequency (Figure 2). The presence of 1,N2--Gua led to a slight decrease. The ratio (target/Neor) with Gua in the G7 postion (of the oligmer ATGCTAG*CAT) was 0.97 ( 0.04 in AA8 cells and 0.93 ( 0.03 in UV5 cells. With 1,N2--Gua at the site, the ratio was 0.82 ( 0.02 in AA8 cells and 0.88 ( 0.05 in UV5 cells. Mutation Frequency. The mutation frequency in this system is calculated from the fraction of recovered DNA that is not cleaved by the restriction enzyme NheI, which cleaves only wild-type DNA (Figure 2). As pointed out before, with a self-complementary site this value is 1/2 the actual frequency (23). In wild-type CHO cells (AA8),
Akasaka and Guengerich
the mutant fraction was increased from 0.4 to 4.6% when 1,N2--Gua was incorporated (Table 2). The control level is excellent when compared to literature values for a similar system (0.2-2.5%) (23, 24), and the increase of >10-fold indicates that mutants should all be the result of events induced by 1,N2--Gua. Under these conditions, the increase in cell count is considerable (doubling time of ∼12 h), so the possibility that only the integrated vector is contributing to the mutant fraction is unlikely. UV-sensitive cells (UV5) were also used. These cells are deficient in nucleotide excision repair because they lack the helicase (ERCC2) of transcription factor TFIIH that opens the duplex at the lesion site before dual incisions on the damaged strand by ERCC4/ERCC1 and EERC5. This activity is essential in nucleotide excision repair, and UV5 cells are highly defective in bulky adduct removal (25). We were unsuccessful in further lowering the mutant fraction in the UV5 cells transformed with the control vector. This difficulty appears not to be attributable to reagent conditions (e.g., PCR and NheI), which were the same as those used with the AA8 cells.2 Sequence Analysis. DNA that was resistant to NheI digestion was used for cloning and sequencing analysis of a 285 bp element surrounding the target region of the vector (Table 2). The parent oligonucleotide that was used was the self-complementary 5′-ATGCTAG*CAT-3′, which, with G* being 1,N2--Gua, would be expected to yield base pair substitutions at this position (denoted G7). In the AA8 cells transfected with the control vector, very few clones were obtained and many of these were false positives, suggesting that the mutation frequency that has been reported (0.4%) is probably an overestimate. Sequence analysis showed a G f A transition at the G7 position. With the UV5 cells, three clones were analyzed and all showed a G f A transition at the G3 site. (This is a self-complementary sequence, and any changes could be interpreted as occurring on the complementary strand; i.e., a change reported as G7 f A could be a reflection of a C4 f T transition.) The sequences of the mutants derived from the cells transformed with the 1,N2--Gua-containing vector were varied and unusual. A total of eight clones from the AA8 cells were analyzed, only two of which contained the sequence found in the control vector. The remainder contained base pair mutations at other sites, one double (base pair) mutation, and two rearrangements at the fourth and ninth positions of the 10-mer vector site were deleted. Of the 13 clones derived from the UV5 cells, two corresponded to the sequences seen in the AA8 (but not UV5) cells into which the unmodified vector had been moved. The remaining 11 clones were all different and contained one deletion, five rearrangements, and seven base pair substitutions at sites removed from the 1,N2-Gua. The independence of four of the clones can be questioned. Clone 3110 is a double mutant, but the C4 f G (or G7 f C) transversion is considered to be independent 2 As pointed out, the mutation frequency was not dramatically enhanced in UV5 cells compared to that in AA8 cells. Deficiency in ERCC2 (UV5 cells) has been shown not to influence sensitivity of AA8 cells to methylation damage (26) or that induced by cross-linking events (27, 28); although the degree of accumulation of cross-links was somewhat higher than in AA8 cells, UV5 cells showed almost the same survival curve as AA8. One possibility is that the adduct 1,N2--Gua is not efficiently recognized as a substrate for nucleotide excision repair in CHO cells, although uvrA deletion clearly enhanced the mutation frequency in E. coli cells (19).
1,N2--Guanine Mutations
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Table 2. Mutations Induced by 1,N2-E-Gua-Containing pCNheIA Inserted into the CHO Cell Genome plasmid G*
host background
mutation rate with Nhe I (%)
G Gua 1,N2--Gua
AA8 (wild-type) AA8
0.4 4.6
Gua 1,N2--Gua
UV5 UV5
4.5 7.8
clone number
sequence at target site
change
1222 3110 3107 3108 3207 3302 3303 3307 3217 2101, 2204, 2404 4107 4101 4104 4212 4213 4216 4305 4311 4313 4401 4405 4406 4412
ATGCTAGCATa ATGCTAACAT TTGGTAGCAT ATGCTAACAT ATGCTAGC- - ATG- - - - - - ATGCTAACAT ATTCTAGCAT ATGTTAGCAT ATACTAGCAT ATACTAGCAT (x3) ATGCTAGAAT ATGTTAGCAT ATGCTAACAT ATGCAA- - - ATGCTAGC- ATGCTAG- - ATG- - - ATGCTAGC- ATGCTAGCAATGCTAACAT ATGATAGCAT ATTCTAGCAT ATGCTATCAT
G7 f A C4 f G, A1 f T G7 f A rearrangement rearrangement G7 f A G3 f T C4 f T G3 f A G3 f A C8 f A C4 f T G7 f A T5 f A, four-base deletion rearrangement rearrangement rearrangement rearrangement rearrangement G7 f A C4 f A G3 f T G7 f T
a The site underlined is the position to be substituted with 1,N2--Gua. In all other cases, the underlined sites are the changes found in the mutants.
Figure 3. Possible equilibria of 1,N2--Gua derivatives.
because it is unique in this set (3100 series, Table 2). However, the C4 f T transition in clones 3307 and 4101 and the C4 f A tranversion in clone 4405 are not unique in the same transformants. For instance, the possibility exists that the C4 change occurred opposite 1,N2--Gua on one strand and also opposite 1,N2--Gua on the other; these mutations might have occurred in the same cell but on distinct pCNHeIA fragments.
Discussion The major finding of this work was that 1,N2--Gua appears to be mutagenic in mammalian cells (at least AA8 CHO cells), inducing base pair mutations. In the AA8 cells, the mutation frequency was >10-fold higher than the control value. The case for genotoxicity of 1,N2-Gua in mammalian cells is also supported by the distribution of mutant sequences and comparison with the mutants derived from the control sequence. If we restrict the analysis of the results to the base pair substitutions seen at the site of the inserted 1,N2--Gua, the apparent preference is for a G f A transition (Table 2). This was the most prominent change observed in uvrA- E. coli cells (19). Comparisons can be made with 1,N2--Gua-generated events seen in other systems. Work with purified exonuclease-deficient polymerases yielded all four possible insertions opposite the lesion with the pattern varying with different enzymes (18). With the particular sequence that was analyzed, some one- and two-base deletion products were detected (18). In E. coli cells, progeny
corresponding to all four possible base pair substitutions were detected (19). However, it should be emphasized that the hybridization system used in the bacterial work would not have detected any deletions, rearrangements, or base pair substitutions at sites other than those that were assayed. Also, in work with purified polymerases, one would not expect to detect large deletions and other complex events with short oligonucleotides. The pCNheI system used here is apparently the only intrachromosomal site-specific mutagenesis system used to date. In the work with O6-alkylGua and O4-alkylThy lesions, only the expected base pair transitions were observed upon sequence analysis (23, 24). O6-AlkylGua lesions are known to block polymerases (29) but not as strongly as 1,N2--Gua (18, 19). Further, O6-alkylGua lesions yield predominantly only dTTP incorporation with all the polymerases that were tested (23, 30, 31), so similar forces appear to drive the substitution in all systems. Complex mutations may also occur at sites other than those directly opposite this modified lesion, but some caveats are in order. Little work has been done to address mechanisms related to such events with any chemical lesions. A potential problem is that the lack of complete ligation of the vector could introduce mutations.3 The 3 To minimize the chance of such a contribution, we purified the constructed vector after ligation. One useful control used in the work on O-alkyl adducts reported by Essigmann and his associates was treatment of vector with an alkyl transferase to show that mutants can be abolished (23, 24). Unfortunately, no such repair control was available for 1,N2--Gua.
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potential for introduction of artifacts into the plasmid during handling cannot be dismissed, although the control plasmid was processed at the same time and in the same way. As an example of a complex mutation, the simple modification of Gua by O6-methylation has been reported to induce intrachromosomal homologous recombination in several human cell lines (32). Although a specific DNA adduct has not been implicated, about onehalf of the genetic changes resulting from treatment of TK cells with butadiene diepoxide are rearrangements (31, 34). This behavior is not unusual for potential crosslinking agents (20), and one possibility is that 1,N2-Gua can rearrange to a ring-opened form, N1-(2-oxoethyl)Gua (18), that could bind to an exocyclic amine on a neighboring DNA strand, in a manner analogous to that demonstrated for the six-membered ring homologue (35) (Figure 3).4 In summary, evidence is presented that 1,N2--Gua is mutagenic in mammalian cells. Because of the very different natures of the systems, direct comparison of the frequencies in mammalian and bacterial cells is not possible. Finally, all of the DNA lesions formed by the vinyl chloride oxidation product 2-chlorooxirane appear to miscode or cause mutations in some system. The list includes 1,N6--Ade (14), 3,N4--Cyt (14-16), N2,3--Gua (17), and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2a]purine and 1,N2--Gua (refs 18 and 19 and this report). Thus, it may not be appropriate to focus on one of these lesions as being the major contributor to cancer, in the absence of further information about repair rates, effects on specific genes, etc. Another point to consider is that the amount of modification at the Gua N7 atom is 1 order of magnitude greater than the apparent sum of the etheno adducts (9) and may contribute to the mutagenic load by increasing the size of the pool of abasic sites.
Acknowledgment. This work was supported in part by U.S. Public Health Service Grants R35 CA44353 and P30 ES00267.
Akasaka and Guengerich
(8)
(9)
(10)
(11)
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(13)
(14)
(15)
(16)
(17)
(18)
References (1) Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D. (1973) Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U.S.A. 70, 2281-2285. (2) Brusick, D. (1989) Genetic toxicology. In Principles and Methods of Toxicology (Hayes, A. W., Ed.) 2nd ed., pp 407-434, Raven Press, New York. (3) Searle, C. E. (1984) Chemical Carcinogens, Vol. 1 and 2, American Chemical Society, Washington, DC. (4) Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerba¨ck, D., and Bartsch, H. (1994) DNA Adducts: Identification and Biological Significance, IARC Scientific Publications, Lyon, France. (5) Singer, B., and Bartsch, H. (1986) The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis, IARC Scientific Publications, Lyon, France. (6) Leonard, N. J. (1993) Etheno-bridged nucleotides in enzyme reactions and protein binding. Chemtracts: Biochem. Mol. Biol. 4, 251-284. (7) Guengerich, F. P., and Raney, V. M. (1992) Formation of etheno adducts of adenosine and cytidine from 1-halooxiranes. Evidence 4 Although no evidence for actual cross-linking is available at this point, 1,N2--Guo does undergo a selective exchange of the H-7 atom at neutral and basic pH (8), and the equilibria shown in Figure 3 are consistent with this result. On the other hand, treatment of 1,N2-dGuo with NaBH4 in CH3OH/H2O did not yield new products, which does not support facile ring opening under such conditions. Further studies on the cross-linking potential of 1,N2--Gua have not been carried out.
(19)
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