Carcinogenesis

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (31) Request Permissions Google Scholar Articles by Arlt, V. M. Articles by Schmeiser, H. H. Search for Related Content PubMed PubMed Citation Articles by Arlt, V. M. Articles by Schmeiser, H. H. Social Bookmarking          What's this?

Carcinogenesis, Vol. 21, No. 2, 235-242, February 2000
© 2000 Oxford University Press

Carcinogenesis

Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene

Volker M. Arlt, Manfred Wiessler and Heinz H. Schmeiser¹

Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

	Abstract

Top Abstract Indroduction Materials and methods Results Discussion References

 
The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA–AAI and dA–AAII) and guanine adducts (dG–AAI and dG–AAII) by the ³²P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC–AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI–DNA and AAII–DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the ³²P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by ³²P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.

Abbreviations: AA, aristolochic acid; AAI, aristolochic acid I (8-methoxy-6-nitrophen-anthro[3,4-d]-1,3-dioxolo-5-carboxylic acid); AAII, aristolochic acid II (6-nitrophen-anthro[3,4-d]-1,3-dioxolo-5-carboxylic acid); dA–AAI, 7-(deoxyadenosin-N⁶-yl)aristolactam I; dA–AAII, 7-(deoxyadenosin-N⁶-yl)aristolactam II; dG–AAI, 7-(deoxyguanosin-N²-yl)aristolactam I; dG–AAII, 7-(deoxyguanosin-N²-yl)aristolactam II.

	Indroduction

Top Abstract Indroduction Materials and methods Results Discussion References

 
The plant extract aristolochic acid (AA) is a potent carcinogen (1–3) and a genotoxic mutagen (4,5). Both major components of the natural extract, aristolochic acid I (AAI) and aristolochic acid II (AAII), are activated by reduction of the nitro group and react with DNA at the exocyclic amino groups of adenine and guanine (Figure 1); this binding occurs at the C7 position of the corresponding aristolactams, the major metabolites of AA (6,7). These DNA adducts appear to trigger carcinogenesis, however, the relative contributions of individual adducts in mutagenesis and carcinogenesis remain unclear. In rodents many chemical carcinogens activate the ras protooncogene by a single point mutation, resulting in the alteration of amino acid residue 12, 13 or 61 (8,9). Likewise, AA-initiated carcinogenesis is associated with a distinct molecular characteristic, i.e. activation of the H-ras gene by an ATTA transversion mutation in codon 61 (CAA). This transversion mutation occurs exclusively (100%) at the first adenine of codon 61 in all tumors of the forestomach and ear duct of rats treated with AAI (10). Although activated AAI and AAII can potentially adduct all purine residues in codons 12, 13 and 61 and, furthermore, mutations in these codons of the H-ras gene can initiate tumorigenesis, all AAI-induced squamous cell carcinomas carry a specific mutation at adenine in codon 61 but not in codon 12 or 13. Since changes of adenine residues at other positions than codon 61 in the ras gene result in amino acid changes that are phenotypically silent, only adenine mutations in codon 61 result in a gain of oncogenic function providing premalignant and malignant cells with a clonal growth and survival advantage (11) and therefore might explain the selectivity for codon 61 mutations. This selectivity of AAI for mutations at adenine residues is consistent with extensive formation of the adenine adduct 7-(deoxyadenosin-N⁶-yl)aristolactam I (dA–AAI) in the target organ, forestomach, in rats (12). Moreover, an apparently lifelong persistence of the dA–AAI adduct in forestomach DNA was found whereas the guanine adduct 7-(deoxyguanosin-N²-yl)aristolactam I (dG–AAI) was removed continuously from the same DNA (13). One important question raised by these results is why almost all AA-induced tumors contain activating mutations at the first adenine of codon 61 and not at the second adenine, although an ATTA transversion mutation at this position also exerts transformation efficiency (14).

View larger version (17K): [in this window] [in a new window]   Fig. 1. DNA adduct formation by AAI and AAII in vivo or in vitro. 1 R = OCH3, AAI; R = H, AAII; the four major AA adducts formed are 2 R = OCH3, dA–AAI; R = H, dA–AAII; 3 R = OCH3, dG–AAI; R = H, dG–AAII.

 
In order to unravel this question, we have used a polymerase arrest assay to detect AAI–DNA and AAII–DNA adducts at the nucleotide sequence level. Recently we reported the synthesis of oligonucleotides containing the major DNA adducts formed by AA in vivo located at a defined site. These site-specifically adducted oligonucleotides were used as templates in primed DNA replication reactions with T7 DNA polymerase (Sequenase) and human polymerase (15,16). Regardless of the type of AA–DNA adduct examined, DNA synthesis was blocked predominantly at the nucleotide 3' to each adduct by both polymerases. Although some subtle differences among the individual AA–DNA adducts were observed these studies demonstrated that all four bulky AA–purine adducts principally lead to arrest of Sequenase 3' to the lesions and bypass was an infrequent event. This phenomenon prompted us to use Sequenase in a polymerase arrest assay as a tool to detect AA–DNA adduct location in a given sequence.

In the present paper we describe the modification of a plasmid containing the first two exons of the mouse c-H-ras gene by AAI and AAII. These modified templates were used in a polymerase arrest assay to monitor the distribution of specific AA–DNA adducts throughout exon 2 of the gene. We also investigated the relationship between DNA adduct distribution, structure and the previously reported mutation hot spot in codon 61 of this gene.

	Materials and methods

Top Abstract Indroduction Materials and methods Results Discussion References

 
Chemicals, plasmid and primers
Pure AAI and AAII as sodium salts were kindly provided by Fa. Madaus (Köln, Germany). T4 polynucleotide kinase, T7 DNA polymerase (Sequenase) v.2.0, ultrapure grade dNTPs and Sephadex G-25 columns were purchased from Amersham Pharmacia (Freiburg, Germany). [-³²P]ATP (sp. act. 7000 Ci/mmol) was obtained from ICN Biomedicals.

The plasmid pNPR, a pUC8 derivative containing an insert of exon 1 and 2 of the mouse c-H-ras gene at the PstI site (17), was kindly provided by K. Brown (Beatson Institute for Cancer Research, Glasgow, UK). Six 20mer primers, (A) 5'-GTTTTGCAGGACTCCTACCG-3', (B) 5'-AGACACAGCAGGTCAAGAAG-3' and (C) 5'-GGGCTTCCTCTGTGTATTTG-3' representing the sequences 310–329, 375–394 and 435–454 on the coding strand and (D) 5'-TCTTCTTGACCTGCTGTGTC-3', (E) 5'-ATACACAGAGGAAGCCCTCC-3' and (F) 5'-GTGGCTCACCTGTACTGATG-3' representing the sequences 376–395, 432–451 and 487–506 on the non-coding strand of the plasmid pNPR in exon 2, were used in this study.

Treatment of pNPR with AAI and AAII
Purified pNPR (50 µg) was treated in vitro with 0.12 and 1.2 mM AAI or AAII in 250 µl 50 mM potassium phosphate buffer, pH 5.8, in the presence of 5 mg zinc dust for chemical activation. The incubations and DNA isolation were performed as reported previously (18). Solvent control DNA was treated either with or without 5 mg zinc without carcinogen. The DNA was resuspended in 150 µl TE buffer, pH 8.0. Adducted and control DNA was used for the ³²P-post-labeling analysis and the polymerase arrest studies.

Modification of poly(dA), poly(dC), poly(dT) and poly(dG)^·poly(dC) with AAI and AAII
Aliquots of 50 µg of each polymer were treated in vitro with 1.2 mM AAI or AAII and analyzed as described above.

³²P-post-labeling analysis
DNA adducts in modified pNPR and modified polymer were determined by the standard ³²P-post-labeling procedure (19) with minor modifications. Briefly 1 µg of DNA was digested and labeled using 50 µCi of [-³²P]ATP (sp. act. 7000 Ci/mmol). Subsequently samples were chromatographed on PEI–cellulose thin layer sheets (Macherey and Nagel, Düren, Germany) and analyzed using an Instant Imager (Canberra Co.) as previously described (18).

End-labeling primers
Each primer (20 pmol) was end-labeled with T4 polynucleotide kinase with 50 µCi of [-³²P]ATP (sp. act. 7000 Ci/mmol) in the presence of 40 pmol of cold ATP according to the manufacturer's instructions. Labeled primers were purified on a Sephadex G-25 column, ethanol precipitated, washed with 70% ethanol and resuspended in 10 µl TE buffer, pH 8.0. The incorporation of ³²P-labeled ATP was measured by Cerenkov counting with a Packard Tri-Carb 2000 CA liquid scintillation counter.

Polymerase arrest assay and phosphorimager analysis
Treated pNPR (2 µg) was denatured in the presence of an approximately equimolar amount of ³²P-end-labeled primer with 2 µl of 1 N NaOH in a total volume of 11 µl. After 10 min at 37°C, 2 µl 1 N HCl for neutralization and 2 µl of plasmid reaction buffer (1 M Tris–HCl, pH 7.5, 100 mM MgCl₂, 250 mM NaCl) was added. This mixture was placed at 37°C for 10 min. To the annealed primer–template mixture was added 1 µl 0.1 M dithiothreitol and Sequenase v.2.0 (T7 DNA polymerase), diluted 1:8 in enzyme dilution buffer (10 mM Tris–HCl, pH 7.5, 5 mM 2-mercaptoethanol) to obtain 3.25 U in 2 µl. An aliqout of 4 µl was then transferred to a tube prewarmed to 37°C, containing 2.5 µl of 160 µM deoxynucleotide triphosphates. This mixture was incubated at 45°C for 5 min. To terminate the reaction 4 µl stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF) was added. For electrophoresis the samples were heated to 80°C for 5 min and 4 µl were applied to an 8% denaturating polyacrylamide gel which was dried before exposure to X-ray film for 15–20 h.

DNA sequencing of untreated pNPR was carried out as described above, except that the extension used a dideoxynucleotide termination mix consisting of 80 µM each deoxyribonucleotide and 8 µM appropiate dideoxynucleotide in 50 mM NaCl.

The intensity of each band was measured with a phosphorimager (Storm 860) from Molecular Dynamics using ImageQuant software (Molecular Dynamics Inc.). For each track the area under each peak was expressed as a percentage of the total area under all peaks.

	Results

Top Abstract Indroduction Materials and methods Results Discussion References

 
Adduct formation by AAI and AAII in plasmid pNPR
Plasmid pNPR, containing an insert of exons 1 and 2 of the mouse c-H-ras gene, was modified with AAI and AAII by chemical reduction with zinc. Specific AAI–DNA and AAII–DNA adducts were determined by the standard procedure of the ³²P-post-labeling method. As shown in Figure 2, the major DNA adducts were identified as reported previously (18) as dA–AAI (spot 1), dG–AAI (spot 2), 7-(deoxyadenosin-N⁶-yl)aristolactam II (dA–AAII) (spot 3) and 7-(deoxyguanosin-N²-yl)aristolactam II (dG–AAII) (spot 4). As reported before (18), dA–AAII (spot 3) is formed from AAII as well as from AAI by reductive activation systems and in vivo through a demethoxylation reaction of AAI. Quantitative analysis obtained by the ³²P-post-labeling procedure (18), shown in Table I, revealed for both concentrations used that the extent of DNA modification by AAII was higher than by AAI. Total adduct levels induced with the high dose (1.2 mM) reached 1 adduct/200 nucleotides for AAI and 1 adduct/100 nucleotides for AAII, resulting in an average number of adducts per fragment of the ras gene (60–70 nt) analyzed by polymerase arrest of 0.4 and 0.7, respectively. Reductively activated AAI strongly favored guanine adduct formation in a ratio of ~4:1, whereas in reactions with reductively activated AAII adenine adducts were preferentially formed in a ratio of ~2:1. Besides the identified dA–AAII and dG–AAII adducts an additional spot 5 was found in AAII-modified pNPR DNA. This prompted us to investigate the nature of this unknown adduct spot. To this end, poly(dA), poly(dG)^·poly(dC), poly(dT) and poly(dC) were reacted with AAI and AAII under the same conditions used for modification of the plasmid. ³²P-post-labeling analyses, shown in Figure 3, revealed that spot 5 was chromatographically indistinguishable from an adduct spot found in incubations with poly(dC) and AAII (Figure 3F) and therefore was assigned as dC–AAII. Similarly, poly(dC) modified by AAI also exhibited a single adduct spot, although at a considerably lower level (assigned as dC–AAI). Co-chromatographic analysis revealed that the two cytosine-derived adducts were different (data not shown). Besides the major AA–DNA adducts identified previously, some minor adduct spots were found, especially in digests of poly(dA). The dC–AAII adduct was also found in double-stranded poly(dG)^·poly(dC) modified by AAII, whereas in analogous incubations with AAI the dC–AAI spot was not detectable. No adduct formation was observed in digests of poly(dT) modified by AAI and AAII (data not shown).

View larger version (77K): [in this window] [in a new window]   Fig. 2. Autoradiographic profiles of AA–DNA adducts obtained from plasmid pNPR modified with 1.2 mM AAI (A) and AAII (B) by activation with zinc at pH 5.8. The standard method of 32P-post-labeling was used. Origins, in the bottom left corner, were cut off before exposure. Screen enhanced autoradiography was at room temperature for 30–60 min. Chromatographic conditions: D1, 1 M sodium phosphate, pH 6.8; D3, 3.5 M lithium formate, 8.5 M urea, pH 4.0; D4, 0.8 M LiCl, 0.5 M Tris–HCl, 8.5 M urea, pH 9.0; D5, 1.7 M NaH2PO4, pH 6.0. Spot 1, dA–AAI; spot 2, dG–AAI; spot 3, dA–AAII; spot 4, dG–AAII; spot 5, dC–AAII.

 

View this table: [in this window] [in a new window]   Table I. Quantitative analysis of DNA adducts formed by AAI and AAII in plasmid pNPR

 

View larger version (85K): [in this window] [in a new window]   Fig. 3. Autoradiographic profiles of AA–DNA adducts obtained after incubation of poly(dA), poly(dG)·poly(dC) and poly(dC) with 1.2 mM AAI and AAII with activation with zinc. The standard method of 32P-post-labeling was used. Origins, in the bottom left corner, were cut off before exposure. Screen enhanced autoradiography was at room temperature from 30–180 min. Chromatography was performed as described in Figure 2. RALs were expressed as adducts/103 nucleotides given in parentheses. (A) poly(dA)/AAI: spot 1, dA–AAI (0.46 ± 0.11); spot 3, dA–AAII (0.2 ± 0.01); spots 7 (0.12 ± 0.03) and 8 (0.16 ± 0.02), unknown. (B) Poly(dA)/AAII: spot 3, dA–AAII (3.2 ± 0.1). (C) Poly(dG)·poly(dC)/AAI: spot 2, dG–AAI (0.31 ± 0.08). (D) Poly(dG)·poly(dC)/AAII: spot 4, dG–AAII (1.7 ± 0.3); spot 5, dC–AAII (0.51 ± 0.6); spot 9 (0.15 ± 0.02), unknown. (E) Poly(dC)/AAI: spot 6, dC–AAI (0.55 ± 0.18), structure not known. (F) Poly(dC)/AAII: spot 5, dC–AAII (2.9 ± 1.4), structure not known.

 
Polymerase arrest spectra
In the polymerase arrest assay six primers were used to investigate the adduct distribution of AAI and AAII within exon 2 of the mouse H-ras gene. Figure 4 shows the results of a representative polymerase arrest assay with the DNA polymerase Sequenase and modified pNPR DNA as template. Dideoxy sequencing products were also loaded on the gel to determine the site in the ras sequence at which the DNA polymerase had arrested. The control lane containing pNPR DNA incubated with zinc or solvent alone caused only very weak premature polymerase arrests in some sequences. Similarly, clean control lanes were seen with each primer used. In contrast, in lanes containing DNA treated with AAI and AAII numerous sites of polymerase arrest were apparent, indicating that an AA–DNA adduct was present at or near this arrest site. The bands varied in intensity, indicating that some sites in the sequence were more readily adducted than others. As can be seen, both AAI and AAII induced an increase in the intensity of polymerase arrest bands with dose, however, arrest sites were qualitatively similar. Comparison of the arrest sites found in the adducted templates with those seen in the sequencing lanes showed that there was a resemblance between the polymerase arrest caused by the AA adducts and the ddTTP and ddCTP lanes. However, the arrest bands produced by the adducted templates appeared to be 1 nt shorter, suggesting that DNA polymerase is arrested at the nucleotide 3' to an adenine or guanine adduct in most cases. These findings are consistent with results published before that AAI and AAII adducts were mainly formed at adenine and guanine bases (6,7) and that Sequenase was terminated predominantly one base prior to these purine adduct sites in site-specifically adducted oligonucleotides (15). Some arrest bands in the AAII-modified template were found at the nucleotide 3' to a cytosine residue, indicating the presence of cytosine modifications and thereby confirming the ³²P-post-labeling results shown above.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Autoradiograph of a representative sequencing gel showing the arrest sites of polymerase Sequenase in a region of exon 2 of the mouse H-ras gene. Plasmid pNPR was chemically modified with 1.2 (lane 1) and 0.12 mM (lane 2) AAI and 1.2 (lane 4) and 0.12 mM (lane 5) AAII. The control lane (lane 3) contains plasmid DNA treated with zinc in the absence of carcinogen. The lanes C, T, A and G correspond to the dideoxy sequencing termination mix used to determine the exon 2 DNA sequence. The sequence shown corresponds to nucleotides 340–400 on the coding strand of exon 2 (17).

 
Figure 5 illustrates the DNA binding affinity determined by polymerase arrest for both strands in exon 2 of the mouse H-ras gene, assuming that the DNA polymerase was totally blocked at the nucleotide 3' to each adduct. The data shown are those for the highest dose in each case. Detailed examination of Figure 5 indicates that the patterns of arrest sites are different for AAI and AAII. However, some obvious similarities were present. For example, polymerase arrest sites were absent for both AAs in the pyrimidine-rich sequences 451–453 on the upper strand and 389–396 on the lower strand. In addition, there was a triplet of adenines on the lower strand at 451–453 where almost no polymerase arrest was detectable. For all the arrest bands, the average values of the relative band intensity are given in Table II. For AAI the majority of polymerase arrest sites corresponded to adenine bases with an average value of 1.35, whereas for AAII the average values corresponding to adenine, guanine and cytosine adducts were similar.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Relative band intensities (RIs) of polymerase arrest in both strands of exon 2 of the mouse H-ras gene in plasmid pNPR chemically modified with 1.2 mM AAI (A) and AAII (B). Arrest bands were quantified in a phosphorimager; for each track the area under each peak was expressed as a percentage of the total area under all peaks. The background from control plasmid DNA treated with zinc in the absence of carcinogen was subtracted. The relative intensities from the separate phosphorimager analyses in one strand were normalized to each other. The relative values given are the mean of at least two separate experiments within a maximal error range of 15%. Total AA–DNA adduct levels in the modified plasmids were 5.2 for AAI and 9.9 for AAII per 103 nucleotides as determined by 32P-post-labeling. The vertical line represents the starting point of the polymerase arrest spectrum on each strand; dotted lines represent the average RI; values above the dashed line represent arrest hot spots in the sequence defined as sites in which the band intensity is 4-fold higher than the average band intensity. The numbering system used is from Brown et al. (17).

 

View this table: [in this window] [in a new window]   Table II. Average intensity of polymerase arrest in exon 2 of mouse H-ras

 
In exon 2 of the H-ras gene 20 arrest hot spots, defined as sites in which the band intensity was 4-fold higher than for average band intensity in the arrest spectra, were observed for AAI. For AAII a total of 10 arrest hot spots were found. It is clear that codon 61 does not represent an arrest hot spot for either activated AAI or activated AAII. However, both AAs formed metabolites that produced polymerase arrest at the two adenines of codon 61 in the coding strand, but the arrest bands at this codon are weak and below average intensity.

Effect of flanking bases on polymerase arrest
In order to determine the sequence effect on the intensity of polymerase arrest the relative intensities of each arrest site were summed and averaged for all four nucleotides on the 5'- and/or 3'-side of the presumed adduct site. The results of the dinucleotide analysis shown in Table III suggest that for AAI as well as for AAII the arrest band is most intense when pyrimidines flank the adenine 3' to which the arrest occurred. For arrests associated with guanine adducts of AAI, T > G > A > C is the preferred order of the base on the 5'- and 3'-side. For AAII the identity of the 5'- and 3'-nucleotide influence arrest intensity to a lesser extent. Similarly, the 16 trinucleotide combinations were analyzed (Table IV). The most intense arrest bands corresponding with adenine adducts of AAI and AAII were seen in the sequences CA*C, TA*C and CA*T, whereas in most sequences with flanking purines arrest bands were weak. Substantial arrest intensity was also observed in the six CA*A sequences harbored in exon 2, although the first adenine of codon 61 showed only a very weak response. For guanine adducts optimal sequences for polymerase arrest were AG*T and TG*G for AAI and CG*A and TG*G for AAII.

View this table: [in this window] [in a new window]   Table III. Sequence dependence of polymerase arrest by nearest 5'- or 3'-neighbor base to the presumed adduct site

 

View this table: [in this window] [in a new window]   Table IV. Sequence dependence of polymerase arrest by nearest 5'- and 3'-neighbor base to the presumed adduct site

 

	Discussion

Top Abstract Indroduction Materials and methods Results Discussion References

 
In this study a polymerase arrest assay was used to determine the distribution of DNA adducts formed by the two main components of the carcinogenic plant extract AA throughout exon 2 of the mouse H-ras gene. The arrest of DNA polymerase by the presence of bulky adducts in a defined sequence has been used by others to examine the sites preferentially modified by reactive carcinogen metabolites (20–25). Since reactive metabolites of AAI and AAII are not available, activation by zinc, the most efficient in vitro activation system for both AAs (18), was used to induce an average modification level of 0.4–0.7 adducts in the 60–70 nt DNA fragment analyzed by polymerase arrest (see Table I). Since the average number of adducts per DNA fragment is less than one, a saturation effect produced by the first adduct encountered by polymerase and thereby preventing detection of a second adduct downstream is unlikely. Because such high adduct levels have been found necessary for the polymerase arrest assay used (21), activation by rat liver microsomes, the most appropriate in vitro system mimicking target tissue activation, could not be used (18). When the adducted DNA was analyzed by the ³²P-post-labeling method an as yet unknown adduct was detected in AAII-modified pNPR. Results obtained from experiments with single-stranded homo-polydeoxyribonucleotides and the polynucleotide duplex poly(dG)^·poly(dC) revealed that this minor adduct is derived from cytosine. Among the various in vitro systems known to activate AAII only chemical reduction by zinc resulted in formation of the dC–AAII adduct spot, which is in agreement with our former study where, however, only the major AA adducts were analyzed (18). ³²P-post-labeling analyses of the other four known AA–DNA adducts were consistent with results reported earlier except that adduct levels of the dA–AAII adduct found in incubations with AAI were much higher. These purine adducts of AAI and AAII are the major adducts identified in rodents and man (12,26,27).

Using the polymerase arrest assay, we showed that AAI–DNA and AAII–DNA adducts are detectable in a gene sequence as primer extension products on a sequencing gel. The relative absence of arrest sites in control DNA and the increase in band intensities with increasing AA adduct levels are consistent with previous reports on polymerase arrest assays with other carcinogen adducts (21–24).

Evidence that Sequenase is effectively blocked at the base immediately preceding AAI and AAII adducts comes from several sources. Firstly, where activated AA caused strong arrest bands there was a strong resemblance between the polymerase arrest lane and the ddTTP and ddCTP dideoxy sequencing lanes, consistent with published data that both AAI and AAII react extensively at purine residues in DNA (6,12,18,27). Secondly, during examination of the AAII-modified plasmid additional arrest bands associated with the ddGTP dideoxy sequencing lane were found, confirming the presence of an uncharacterized cytosine adduct as determined by ³²P-post-labeling. Thirdly, in primer extension studies with site-specifically mono-adducted oligonucleotides containing purine–AA adducts DNA synthesis by Sequenase was blocked predominantly (80–90%) at the nucleotide 3' to the adduct regardless of the type of adduct examined (15).

Thus, these results suggest that DNA polymerase is effectively arrested one base prior to most AA adducts and that the intensity of arrest bands should, therefore, reflect the degree of adduct formation at that site. However, as discussed by Ross et al. (21), it is generally difficult to extrapolate from the sites of polymerase arrest to the precise site of adduct formation responsible for this arrest and adduct distribution spectra obtained by this technique must not be considered as quantitative.

The arrest spectra in exon 2 of the mouse H-ras gene obtained with activated AAI revealed that polymerase arrest was associated mainly with adenine, i.e. arrest bands were 3' to an adenine in the template rather than guanine, although the ³²P-post-labeling analysis showed that the majority of reactions with plasmid DNA were at guanine residues. Similarly, activated AAII resulted in polymerase arrest sites associated with adenine and guanine to almost the same extent, whereas twice as many adenine adducts as guanine adducts were determined by ³²P-post-labeling. Therefore, it appears that the intensity of arrest bands obtained by polymerase arrest in the ras sequence does not reflect the overall AA–DNA adduct distribution in the plasmid. However, such a quantitative comparison is based on a number of potentially incorrect assumptions, the foremost being that different adducts are labeled with similar efficiencies and that all adducts block the polymerase equally well. Similar results have been observed by Ross et al. (21) in a study with benzo[c]phenanthrene dihydrodiolepoxide compounds, which like the AAs react to a substantial extent at both adenine and guanine residues. Since most polymerase arrest studies have used reactive carcinogen metabolites that react predominantly with one base forming a single major adduct, such observations were not possible, instead Thrall et al. (28) reported a dose-dependent arrest of Sequenase through benzo[a]pyrene diolepoxide-induced adducts in a run of guanines. Moreover, despite the structural similarities among the AAI–DNA and AAII–DNA adducts the action of Sequenase on the individual AA–DNA adducts might be different, leading to differential arrest efficiencies. This hypothesis is supported by a study reported previously (15) showing that although all four purine AA adducts provide severe blocks to DNA replication, adenine adducts arrest Sequenase more effectively than guanine adducts. A suitable explanation for this observation is provided by the different adduct structures, namely the stronger rigidity due to the imino structure of the N⁶-adenine adducts compared with the amino structure of the N²-guanine adducts. Furthermore, the aristolactam moiety of guanine adducts is located in the minor groove, whereas the aristolactam moiety of adenine adducts is situated in the major groove of the B-DNA strand (15). Therefore, it seems likely that structural characteristics of these bulky DNA adducts can account for differences in the response by polymerase.

Amongst the adenines and guanines in exon 2 of the H-ras gene there was an uneven distribution of polymerase arrest, indicating that some sites in the sequence were more readily adducted than others and therefore AA adduct formation is not random but sequence specific. Results in Tables III and IV suggest that neighbor bases affect the efficiency of DNA binding of AAI and AAII depending on the adduct structure. 5'-Nearest neighbor pyrimidine bases display the greatest effect on AA–DNA binding. In particular, adenine binding is favored by flanking pyrimidines rather than by purines, which can be tentatively attributed to steric effects arising from the presence of bulkier purines flanking the reacting adenine residues. The binding affinity in trinucleotide binding is consistent in most cases with the dinucleotide analysis. However, there are some notable exceptions; for example, binding to guanine residues in the sequence AG*T, TG*A and GG*G differs strongly for AAI and AAII.

Our results show that both adenines in codon 61 are arrest sites for polymerase and therefore AA DNA-binding sites, suggesting that the mutations observed in AA-treated rodents may originate from adduct formation in this codon. It would appear, however, that the mutational hot spot is not dictated by initial DNA adduct formation by the chemical mutagen. Such a lack of direct correlation between the sites of polymerase arrest and the sites of mutation hot spots has been seen in other studies (21–24). It seems likely that the non-random action of DNA repair processes on adducted DNA could account for differences between polymerase arrest and mutational spectra. On the other hand, mutations presumably arise when the endogenous cellular DNA polymerase is not completely arrested by an adduct and it consequently places an incorrect base across from the adduct site. These observations demonstrate the importance of determining DNA damage at the sequence level in order to understand its biological consequences.

	Acknowledgments

 
We thank Dr J.Boer for help with the phosphorimager analyses and Dr T.Broschard for helpful comments and critical reading of the manuscript.

	Notes

 
¹ To whom correspondence should be addressedEmail: h.schmeiser{at}dkfz-heidelberg.de

	References

Top Abstract Indroduction Materials and methods Results Discussion References

 

1. Mengs,U., Lang,W. and Poch,J.A. (1982) The carcinogenic action of aristolochic acid in rats. Arch. Toxicol., 51, 107–119.[Web of Science]
2. Mengs,U. (1988) Tumor induction in mice following exposure to aristolochic acid. Arch. Toxicol., 61, 504–505.[Web of Science][Medline]
3. Cosyns,J.-P., Goebbels,R.-M., Liberton,V., Schmeiser,H.H., Bieler,C.A. and Bernard,A.M. (1998) Chinese herbs nephropathy-associated slimming regimen induces tumors in the forestomach but no interstitial nephropathy in rats. Arch. Toxicol., 72, 738–743.[Web of Science][Medline]
4. Robisch,G., Schimmer,O. and Goggelmann,W. (1982) Aristolochic acid is a direct mutagen in Salmonella typhimurium. Mutat. Res., 105, 201–204.[Web of Science][Medline]
5. Schmeiser,H.H., Pool,B.L. and Wiessler,M. (1986) Identification and mutagenicity of metabolites of aristolochic acid formed by rat liver. Carcinogenesis, 7, 59–63.[Abstract/Free Full Text]
6. Pfau,W., Schmeiser,H.H. and Wiessler,M. (1990) Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA. Carcinogenesis, 11, 313–319.[Abstract/Free Full Text]
7. Pfau,W., Schmeiser,H.H. and Wiessler,M. (1991) N⁶-Adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen. Chem. Res. Toxicol., 4, 581–586.[Web of Science][Medline]
8. Balmain,A. and Brown,K. (1988) Oncogene activation in chemical carcinogenesis. Adv. Cancer Res., 51, 147–182.[Web of Science][Medline]
9. Barbacid,M. (1987) Ras genes. Annu. Rev. Biochem., 56, 779–827.[Web of Science][Medline]
10. Schmeiser,H.H., Janssen,J.W., Lyons,J., Scherf,H.R., Pfau,W., Buchmann,A., Bartram,C.R. and Wiessler,M. (1990) Aristolochic acid activates ras genes in rat tumors at deoxyadenosine residues. Cancer Res., 50, 5464–5469.[Abstract/Free Full Text]
11. Hussain,S.P. and Harris,C.C. (1998) Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes. Cancer Res., 58, 4023–4037.[Free Full Text]
12. Stiborova,M., Fernando,R.C., Schmeiser,H.H., Frei,E., Pfau,W. and Wiessler,M. (1994) Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by ³²P-postlabelling analysis using different chromatographic procedures. Carcinogenesis, 15, 1187–1192.[Abstract/Free Full Text]
13. Fernando,R.C., Schmeiser,H.H., Scherf,H.R. and Wiessler,M. (1993) Formation and Persistence of Specific Purine DNA Adducts by ³²P-Postlabelling in Target and Non-target Organs of Rats Treated with Aristolochic Acid I. IARC Scientific Publications no. 124, IARC, Lyon, pp. 167–171.
14. Topal,M.D. (1988) DNA repair, oncogenes and carcinogenesis. Carcinogenesis, 9, 691–696.[Free Full Text]
15. Broschard,T.H., Wiessler,M., von der Lieth,C.W. and Schmeiser,H.H. (1994) Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid. Carcinogenesis, 15, 2331–2340.[Abstract/Free Full Text]
16. Broschard,T.H., Wiessler,M. and Schmeiser,H.H. (1995) Effect of site-specifically located aristolochic acid DNA adducts on in vitro DNA syntheses by human DNA polymerase . Cancer Lett., 98, 47–56.[Web of Science][Medline]
17. Brown,K., Bailleul,B., Ramsden,M., Fee,F., Krumlauf,R. and Balmain,A. (1988) Isolation and characterization of the 5' flanking region of the mouse c-Harvey-ras gene. Mol. Carcinog., 1, 161–170.[Web of Science][Medline]
18. Schmeiser,H.H., Frei,E., Wiessler,M. and Stiborova,M. (1997) Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by ³²P-post-labelling: evidence for reductive activation by peroxidases. Carcinogenesis, 18, 1055–1062.[Abstract/Free Full Text]
19. Gupta,R.C., Reddy,M.V. and Randerath,K. (1982) ³²P-postlabeling analysis of aromatic carcinogen–DNA adducts. Carcinogenesis, 3, 949–955.
20. Reardon,D.B., Bigger,C.A.H., Strandberg,J., Yagi,H., Jerina,D.M. and Dipple,A. (1989) Sequence selectivity in the reaction of optically active hydrocarbon dihydrodiol epoxides with rat H-ras DNA. Chem. Res. Toxicol., 2, 12–14.[Web of Science][Medline]
21. Ross,H.L., Bigger,C.A.H., Yagi,H., Jerina,D.M. and Dipple,A. (1993) Sequence specificity in the interaction of the four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides with the supF gene. Cancer Res., 53, 1273–1277.[Abstract/Free Full Text]
22. Ross,H.L., Lee,H., Harvey,R.G. and Dipple,A. (1994) Comparison of the sites of reaction of three polycyclic aromatic hydrocarbons in the supF gene. Mutat. Res., 306, 91–101.[Web of Science][Medline]
23. Endo,H., Schut,H.A.J. and Snyderwine,E.G. (1995) Distribution of the DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the supF gene as determined by polymerase arrest assay. Mol. Carcinog., 14, 198–204.[Web of Science][Medline]
24. Page,J.P., Ross,H.L., Bigger,C.A.H. and Dipple,A. (1996) Mutagenic specificities and adduct distributions for 7-bromomethylbenz[a]anthracenes. Carcinogenesis, 17, 283–288.[Abstract/Free Full Text]
25. Roy,D. and Abul-Hajj,Y.J. (1997) Estrogen–nucleic acid adducts: guanine is major site for interaction between 3,4-estrone quinone and COIII gene. Carcinogenesis, 18, 1247–1249.[Abstract/Free Full Text]
26. Schmeiser,H.H., Bieler,C.A., Wiessler,M., van Ypersele de Strihou,C. and Cosyns,J.P. (1996) Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy. Cancer Res., 56, 2025–2028.[Abstract/Free Full Text]
27. Bieler,C.A., Stiborova,M., Wiessler,M., Cosyns,J.-P., van Ypersele de Strihou,C. and Schmeiser,H.H. (1997) ³²P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy. Carcinogenesis, 18, 1063–1067.[Abstract/Free Full Text]
28. Thrall,B.D., Mann,D.B., Smerdon, M.J. and Springer,D.L. (1992) DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide. Carcinogenesis, 13, 1529–1534.[Abstract/Free Full Text]

Received June 23, 1999; revised August 25, 1999; accepted October 19, 1999.

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				H. L. Greetham, S. A. Bingham, and P. A. Burns Adduction of Human p53 Gene by Fecal Water: An In vitro Biomarker of Mutagenesis in the Human Large Bowel Cancer Epidemiol. Biomarkers Prev., December 1, 2007; 16(12): 2681 - 2685. [Abstract] [Full Text] [PDF]

				V. M. Arlt, M. Stiborova, J. vom Brocke, M. L. Simoes, G. M. Lord, J. L. Nortier, M. Hollstein, D. H. Phillips, and H. H. Schmeiser Aristolochic acid mutagenesis: molecular clues to the aetiology of Balkan endemic nephropathy-associated urothelial cancer Carcinogenesis, November 1, 2007; 28(11): 2253 - 2261. [Abstract] [Full Text] [PDF]

				V. M. Arlt 3-Nitrobenzanthrone, a potential human cancer hazard in diesel exhaust and urban air pollution: a review of the evidence Mutagenesis, November 1, 2005; 20(6): 399 - 410. [Abstract] [Full Text] [PDF]

				M. Stiborova, E. Frei, B. Sopko, K. Sopkova, V. Markova, M. Lankova, T. Kumstyrova, M. Wiessler, and H. H. Schmeiser Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: evidence for reductive activation by human NAD(P)H:quinone oxidoreductase Carcinogenesis, October 1, 2003; 24(10): 1695 - 1703. [Abstract] [Full Text] [PDF]

				V. M. Arlt, M. Stiborova, and H. H. Schmeiser Aristolochic acid as a probable human cancer hazard in herbal remedies: a review Mutagenesis, July 1, 2002; 17(4): 265 - 277. [Abstract] [Full Text] [PDF]

				V. M. Arlt, H. H. Schmeiser, and G. P. Pfeifer Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR Carcinogenesis, January 1, 2001; 22(1): 133 - 140. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (31) Request Permissions Google Scholar Articles by Arlt, V. M. Articles by Schmeiser, H. H. Search for Related Content PubMed PubMed Citation Articles by Arlt, V. M. Articles by Schmeiser, H. H. Social Bookmarking          What's this?

Online ISSN 1460-2180 - Print ISSN 0143-3334

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics