Comparison of the repair of DNA damage induced by the benzene metabolites hydroquinone and p-benzoquinone: a role for hydroquinone in benzene genotoxicity

Margaret Gaskell1, Keith I.E. McLuckie and Peter B. Farmer

Cancer Biomarkers and Prevention Group, The Biocentre, University Road, Leicester LE1 7RH, UK

1 To whom correspondence should be addressed. Tel: +44 116 223 1835; Fax: +44 116 223 1840; Email: mg24{at}le.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human population is continually exposed to benzene due to its presence in complex environmental mixtures and exposure has been linked to a range of haematotoxic effects, including an increased risk of leukaemia. Several hypotheses have been postulated on how benzene exerts its toxic and carcinogenic effects, one idea being that following metabolism to more reactive species it can react with DNA to form adducts which subsequently give rise to mutations. Previously, we have demonstrated the formation of four major DNA adducts from the reaction of DNA with the benzene metabolites hydroquinone (HQ) and p-benzoquinone (p-BQ) and the mutagenicity of these adducts when analysed using the supF forward mutation assay after replication in a human kidney cell line. This study demonstrates a potential role in the carcinogenicity of benzene for the DNA adducts formed on 2'-deoxyguanosine 3'-monophosphate. As a continuation of this work, benzene metabolite-treated plasmid pSP189 containing the supF reporter gene was transfected into human nucleotide excision repair (NER)-proficient and NER-deficient (xeroderma pigmentosum, complementation group A) fibroblast cells to determine the method of adduct repair. For all metabolite treatments in both cell lines the majority of mutations were single base substitutions occurring at GC base pairs, predominantly GC->TA transversions and GC->AT transitions. Comparison of mutation frequencies showed a similarity for the HQ treatment for the two cell lines, whereas for the treatments involving p-BQ, an overall higher mutation frequency was observed in the NER-deficient cells compared with the NER-proficient cells. Mutation spectra were significantly different following treatment with HQ in the two cell lines (P = 0.0004). No difference was observed for the control, p-BQ or the combined treatment. The results suggest the involvement of a different repair mechanism for HQ-induced DNA damage and further highlights the potential different roles for the two benzene metabolites in benzene mutagenicity.

Abbreviations: AP, apurinic or apyrimidinic; BER, base excision repair; p-BQ, p-benzoquinone; FCS, fetal calf serum; HQ, hydroquinone; IPTG, isopropyl-ß-D-thiogalactopyranoside; NER, nucleotide excision repair; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Benzene, although a stable aromatic hydrocarbon, has been linked to a number of chronic effects observed in the haematopoietic system of exposed individuals due to its metabolism in the liver and bone marrow to more reactive species (1). These chronic effects include leucopenia, thrombocytopenia, pancytopenia, anaemia, myelodysplastic syndrome and acute myelogenous leukaemia (2). Due to the presence of benzene in our environment from such pollutants as cigarette smoke and automobile exhaust fumes and its use in a number of industries, the mechanism of benzene toxicity has been under investigation for a number of years.

Benzene is metabolized in the liver to a number of metabolites, which include benzene oxide, phenol and several hydroxylated compounds [hydroquinone (HQ) and catechol] (3). These metabolites, especially HQ, are thought to be transported to the bone marrow, the site of benzene toxicity, where they can be further metabolized through the action of peroxidases to the more reactive species p-benzoquinone (p-BQ). The benzene metabolites p-BQ and HQ have been shown to cause a number of effects both in vivo and in vitro, which include the formation of both DNA adducts and mutations (419).

Benzene and several of its metabolites have been investigated using a number of mutation assays, which include both simple short-term in vitro gene mutation assays in bacteria and mammalian cells (1316) and in vivo assays in transgenic animals and humans (1719). In vitro benzene and its metabolites have been shown to be weakly or non-mutagenic, whereas tests using whole systems have demonstrated benzene mutagenicity in a number of mouse tissues, which include both the lung and spleen (17,18) and blood from occupationally exposed individuals (19).

We have previously demonstrated the formation of four major DNA adducts in vitro following the reaction of DNA or individual nucleotides with the benzene metabolite p-BQ (9). These adducts were identified as (3''-hydroxy)-3,N4-benzetheno-2'-deoxycytidine 3'-monophosphate (4), (3''-hydroxy)-1,N6-benzetheno-2'-deoxyadenosine 3'-monophosphate (5), (3''-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate (68) and (3'',4''-dihydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate (9). The 2'-deoxycytidine 3'-monophosphate adduct was the main adduct formed from the direct reaction of DNA with p-BQ, whereas the two 2'-deoxyguanosine 3'-monophosphate adducts were minor products of the reaction. Reaction of DNA with the benzene metabolite HQ induced only one major DNA adduct, identified as (3''-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate (20). We have previously used the supF forward mutation assay to study mutations induced by these DNA adducts and have demonstrated that the major product of the reaction of DNA with HQ, (3''-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate, or HQ itself via a differing mechanism, i.e. oxidative damage, may play a significant role in benzene mutagenicity (20).

To further validate this finding we have extended this work by comparing the mutagenicity of benzene-induced DNA adducts replicated in nucleotide excision repair (NER)-deficient and NER-proficient human cells using the supF forward mutation assay. This work would hopefully determine a difference in the mechanism of repair for the damage induced by the benzene metabolite HQ compared with p-BQ lending more support to a role of HQ in the mutagenicity of benzene.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
p-BQ and HQ were obtained from Fluka (Gillingham, UK) and Sigma (Poole, UK), respectively. 5-Bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) and isopropyl-ß-D-thiogalactopyranoside (IPTG) were purchased from Melford Laboratories Ltd (Ipswich, UK). Dulbecco's modified Eagle's medium and fetal calf serum (FCS) were obtained from Life Technologies (Paisley, UK). Plasmid extraction kits were purchased from Qiagen (Crawley, UK). The restriction enzyme DpnI was obtained from New England Biolabs (Hitchin, UK). [{gamma}-32P]ATP (>185 TBq/mmol, >5000 Ci/mmol) and Templiphi amplification kits were purchased from Amersham (Amersham, UK). T4 polynucleotide kinase (3'-phosphatase free) and Fugene-6 transfection reagent were obtained from Roche (Lewes, UK). Calf spleen phosphodiesterase was purchased from Merck Biosciences Ltd (Nottingham, UK).

Cell lines, shuttle vector plasmid and bacterial strain
Normal human, NER-proficient, SV40-transformed fibroblast cells (GM00637) and SV40-transformed, NER-deficient, xeroderma pigmentosum fibroblast cells, complementation group A (GM04429) were purchased from the NIGMS Human Genetic Cell Repository (Camden, NJ). Cells were grown in Eagle's minimal essential medium supplemented with either 15 or 10% FCS, respectively, at 37°C in 5% CO2 in air.

The plasmid, pSP189, which contains the supF gene (21), and Escherichia coli strain MBM7070, which carries a lacZ amber mutation, were kindly provided by Dr M.Seidman (National Institute of Aging, NIH, Baltimore, MD). pSP189 is generated from pS189 by the inclusion of a randomly generated 8 bp signature sequence 3' of the supF gene (21) providing a possible 48 (65 536) sequence possibilities (22).

Treatment of plasmid with benzene metabolites
Plasmid pSP189 (100 µg) was treated on three separate occasions with 0, 5, 10 or 20 mM HQ or p-BQ or 0, 5, 10 or 20 mM of each metabolite together in 30 mM ammonium formate buffer, pH 7.0 (500 µl), for 15 h at 37°C. The DNA was precipitated by the addition of 0.1 vol of 2 M sodium acetate and an equal volume of ice-cold ethanol. DNA was recovered, washed twice with 70:30 ice-cold ethanol/water (v/v) followed by one wash with ice-cold ethanol. DNA was resuspended in 1.5 mM sodium chloride, 0.15 mM trisodium citrate.

32P-postlabelling analysis
An aliquot of plasmid DNA (5 µg) from each treatment group was analysed by 32P-postlabelling to allow quantitation of DNA adducts, using the method described previously (9). In brief, samples were analysed using the nuclease P1 enrichment method described by Reddy and Randerath (23). Radiolabelling was by 5'-phosphorylation using [{gamma}-32P]ATP (62.5 µCi) and T4 polynucleotide kinase (6.25 U). Sample analysis was carried out using HPLC with on-line radiochemical detection (9).

Transfection and transformation
Subconfluent cells were transfected with control and treated plasmid (10 µg/9 cm diameter culture plate) in the presence of Fugene-6 transfection reagent (20 µl). Plasmid was recovered 48 h later using a Qiagen mini plasmid extraction kit. Recovered plasmid underwent DpnI digestion (2 U) to remove any unreplicated DNA. Aliquots of recovered DNA were used to transform electrocompetent E.coli (MBM7070) using a Gene Pulser apparatus (2.5 kV, 25 µF, 200 {Omega}) (Bio-Rad, Hercules, CA). Transformed MBM7070 cells were plated onto LB agar plates (525 cm2) containing ampicillin (100 µg/ml), X-gal (75 µg/ml) and IPTG (25 µg/ml). Mutant colonies, which were either white or pale blue when grown on X-gal-containing medium, were purified by restreaking on similar LB agar plates (wild-type colonies were blue).

Sequencing
Plasmid DNA from white or pale blue colonies (mutants) underwent Templiphi amplification and sequencing was carried out using the primer 5'-GGCGACACGGAAATGTTGAA-3' (Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, UK). Sequencing was carried out using an Applied Biosystems Model 377 DNA sequencer using the BigDye version 1.0 sequencing chemistry. Any mutants with identical signature sequences (siblings) were excluded from the analysis. Mutation spectra were analysed by Poisson distribution to determine mutation hot-spots (when the number of mutations at a particular position was ≥4-fold that expected, these positions were considered to be hot-spots for mutation) and mutation spectra were compared using the Cariello Hyperg program where P ≤ 0.05 indicates a significant difference (24).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
32P-postlabeling analysis
Plasmid DNA treated with the benzene metabolite HQ or p-BQ or both metabolites together have previously been assessed by 32P-postlabelling coupled to HPLC (20). Results from this work demonstrated the formation of four main adducts when DNA was treated with p-BQ, with and without HQ. The main adduct formed during this reaction was identified as (3''-hydroxy)-3,N4-benzetheno-2'-deoxycytidine 3'-monophosphate, followed by (3''-hydroxy)-1,N6-benzetheno-2'-deoxyadenosine 3'-monophosphate. The minor products of this reaction were identified as the deoxyguanosine monophosphate adducts (3''-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate and (3'',4''-dihydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate (9). Reaction of the plasmid with HQ alone resulted in only one major adduct, identified as (3''-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate.

Mutation frequency in the supF gene
The mutation frequency for each treatment, which was calculated following transfection of the treated plasmid into either an NER-deficient (GM04429) or NER-proficient (GM00637) cell line, as shown in Table I, reflects the trend observed with the overall adduct number as determined by 32P-postlabelling. HQ treatment in both cell lines gave a mutation frequency and adduct number that did not increase with dose, whereas for the p-BQ treatment and the combined metabolite treatment (HQ + p-BQ) mutation frequency and adduct number both increased in a dose-dependent manner.


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Table I. Adduct number and mutation frequency observed after treatment of plasmid pSP189 with varying doses of HQ, p-BQ and the two metabolites together with transfection into repair-proficient (GM00637) and repair-deficient (GM04429) cells and subsequent transformation into E.coli, MBM7070

 
The spontaneous mutation frequencies observed were 12.92 x 10–4 and 1.65 x 10–4 for the NER-proficient and NER-deficient cells, respectively, as determined by screening of the untreated plasmid (Table I). The mutation frequency for the HQ treatments was between 3.5 and 7 times higher in the NER-proficient cells compared with the NER-deficient cells. No significant difference was observed for either the p-BQ or combined treatments when comparing the two cell lines. However, when the data were normalized for the spontaneous mutation frequency a similar mutation frequency for the HQ treatment was observed, whereas for the treatments involving p-BQ an overall higher mutation frequency was observed in the NER-deficient cells compared with the NER-proficient cells, as shown in Table I. For the treatments involving p-BQ, mutation frequencies in the NER-deficient cells ranged from ~4 to 9 and 4 to 15 times greater than that observed in the NER-proficient cells for p-BQ and both p-BQ + HQ together, respectively. The difference observed for the HQ treatment was not as great, ranging only from 1.2 to 2.2 times that observed in the repair-proficient cells.

Mutation types found in the supF gene
Mutant colonies as defined by their white or pale blue colour were selected for sequencing of the supF gene to allow both the type and location of the mutation to be determined. Only colonies from the highest dose for each treatment were selected for sequencing. The majority of mutations for the three treatments and for both cell lines were base substitutions, as shown in Table II, and of these single base substitutions predominated. For the NER-proficient cell line GC->AT transition single base mutations predominated, followed by GC->TA transversions for the control and all treatment groups. A similar result was observed for the NER-deficient cell line, except for the combined treatment, which resulted in GC->TA transversions predominating over GC->AT transitions, as shown in Table III.


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Table II. Mutation types observed in the supF gene following treatment of pSP189 with 20 mM HQ, 20 mM p-BQ or 20 mM of both metabolites together after replication in NER-deficient XPA fibroblasts (GM04429) and NER-proficient human fibroblasts (GM00637)

 

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Table III. Types of single and tandem base substitution mutations observed in the supF gene after treatment of pSP189 with 20 mM HQ, 20 mM p-BQ or 20 mM of both metabolites together replicated in NER-deficient XPA fibroblasts and NER-proficient human fibroblasts

 
A similar level of multiple base substitution mutations was observed for the control and all treatment groups for the NER-proficient cells. This type of mutation accounted for 14, 16, 18 and 16% of all mutation types for the control, HQ, p-BQ and combined treatments, respectively (Table II). The predominant mutation was different in each case, with AT->CG transversions, GC->AT transitions and GC->TA transversions predominating for the HQ, p-BQ and combined treatments, respectively, as shown in Table IV. For the control group AT->CG transversions and GC->AT transitions predominated. For the NER-deficient cell line fewer multiple mutations were observed compared with the NER-proficient cell line. Multiple mutations accounted for 0, 1.5, 10 and 7% for the control, HQ, p-BQ and combined treatments, respectively. Frameshift mutations accounted for 45, 21, 4 and 3% of the total mutations observed in the control, HQ, p-BQ and combined treatments, respectively, for the NER-proficient cell line. A similar level was observed in the NER-deficient cell line, with this type of mutation accounting for 46.5, 23.5, 8 and 5%, respectively.


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Table IV. Types of multiple base substitution mutations observed in the supF gene after treatment of pSP189 with 20 mM HQ, 20 mM p-BQ or 20 mM of both metabolites together replicated in NER-deficient XPA fibroblasts and NER-proficient human fibroblasts.

 
Mutation spectra
The mutation spectra, which include single, tandem and multiple base substitutions for the metabolite treatments in both cell lines, can be seen in Figures 1 and 2. For both cell lines a number of hot-spots for mutation, as determined by Poisson distribution, were observed for the single and tandem mutations (Figure 1) that in all but one case occurred at GC base pairs. The exception to this was for the combined treatment in the NER-deficient cells, where a hot-spot for mutation was observed, at an AT base pair (position 139). In the NER-proficient cells hot-spots for mutation occurred at positions 127, 133 and 150 for the control sample compared with no observed hot-spots for the NER-deficient cell line. Treatment of the plasmid with HQ only resulted in mutation hot-spots at positions 108, 109, 123, 129, 133 and 175, with the strongest hot-spot at position 133, following replication in NER-proficient cells, whereas in the NER-deficient cells mutation hot-spots occurred at positions 108, 129, 139 and 155 for this treatment. Base substitutions at these hot-spots accounted for 53 and 31% of the total base substitutions detected for the NER-proficient and NER-deficient cell lines, respectively.



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Fig. 1. Mutation spectra showing single and tandem base substitutions induced in the supF gene in control (A), 20 mM HQ (B), 20 mM p-BQ (C) or 20 mM HQ + 20 mM p-BQ (D) modified pSP189 plasmid replicated in NER-proficient (GM00637) and NER-deficient (XPA) human fibroblast cells. The 5'->3' sequence of the transcribed strand of the wild-type supF tRNA gene is shown for each treatment (21). Letters underneath the wild-type sequence indicate the position and type of point mutations induced in the repair-proficient cells whereas those letters above the wild-type sequence indicate the position and type of point mutation induced in repair-deficient cells. Tandem mutations (mutations which occur at two adjacent bases) are underlined. Mutational hot-spots are indicated by {blacksquare}.

 


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Fig. 2. Mutation spectra showing multiple base substitutions induced in the supF gene in control (A), 20 mM HQ (B), 20 mM p-BQ (C) or 20 mM HQ + 20 mM p-BQ (D) modified pSP189 plasmid replicated in NER-proficient (GM00637) and NER-deficient (XPA) human fibroblast cells. The 5'->3' sequence of the transcribed strand of the wild-type supF tRNA gene is shown for each treatment (21). Letters underneath the wild-type sequence indicate the position and type of point mutations induced in the repair-proficient cells whereas those letters above the wild-type sequence indicate the position and type of point mutation induced in repair-deficient cells. Tandem mutations (mutations which occur at two adjacent bases) are underlined. Mutational hot-spots are indicated by {blacksquare}.

 
Plasmid treatment with the benzene metabolite p-BQ induced mutation hot-spots at different positions than those observed for the HQ-treated plasmid after transfection into NER-proficient cells. These hot-spots occurred at positions 115, 122 and 155. Only one hotspot was observed for this treatment in NER-deficient cells, and this occurred at position 155, a hot-spot previously observed for HQ-treated plasmid transfected into this cell line. The combined treatment gave three mutation hotspots at positions 115, 122 and 123 in the NER-proficient cells compared with two at positions 132 and 139 in NER-deficient cells. For the NER-proficient cells 77, 92 and 94% of the base substitutions detected at all the mutation hot-spots in the single and tandem spectra were for either an adenine or thymine base compared with 94, 75 and 50% of mutations reported for the NER-deficient cells following treatment with HQ, p-BQ or the combined treatment, respectively.

The multiple base substitutions shown in Figure 2 again give a number of mutation hot-spots for replication of a benzene metabolite-treated plasmid following transfection into NER-proficient cells, whereas only one hot-spot was observed for the NER-deficient cell line. The single mutation hot-spot for the NER-deficient cell line occurred in the p-BQ treatment, at position 108, and each base substitution observed at this site was a GC->AT transition. In the NER-proficient cells two hot-spots were observed in the HQ-treated plasmid at positions 133 and 145, whereas in the p-BQ-treated plasmid five hot-spots were observed, three of which were clustered at the 5'-end of the supF gene sequence at positions 102, 103 and 105 and two at positions 145 and 154. Mutations at these hot-spots accounted for 31 and 49% of the total mutations for the HQ and p-BQ treatments, respectively. The predominant base substitution at these hot-spots for both the HQ and p-BQ treatments was an AT->CG transversion.

When comparing the mutation spectra obtained for the single and tandem base substitutions using the Cariello Hyperg program (24) a significant difference (P = 0.03) was observed between the mutation spectra obtained for the HQ treatment and that obtained for the p-BQ treatment in the NER-proficient cell line. Comparison of the spectra achieved for the same treatment in the different cell lines showed a significant difference between those spectra obtained for the HQ treatment (P = 0.0004). Spontaneous control spectra were shown not to be significantly different between the cell lines. No significant difference was observed for any of the other spectra, irrespective of cell line or treatment.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using the supF forward mutation assay we have previously studied the mutagenicity of benzene–DNA adducts following replication in a human kidney cell line (Ad293 cells) (20). This work demonstrated a potential difference between the mechanism of mutagenicity induced by the benzene metabolites HQ and p-BQ, which may suggest a role for the benzene–DNA adducts formed with 2'-deoxyguanosine 3'-monophosphate or a different mechanism of mutagenicity entirely, which may involve oxidative damage with subsequent strand breaks (20). The work described here details the replication of a benzene metabolite-modified plasmid in two cell lines, one which is NER-proficient (GM00637) and one which is NER-deficient (GM04429).

The background mutation frequency in NER-deficient cells was 1.65 x 10–4 colonies, compared with 5 x 10–5 colonies as reported by Seidman et al. (25). This group also reported a much higher spontaneous mutation frequency for the NER-proficient cell line, 7 x 10–4 colonies (25), which is comparable to the level observed here of 12.92 x 10–4 colonies. The higher spontaneous mutation frequency in the NER-proficient cell line is probably due to individual differences between the two cell lines. To allow for comparison of the mutation frequency data between the cell lines the data were normalized for the spontaneous mutation frequency. After normalization a similar mutation frequency for the HQ treatment was observed for each cell line. In contrast, for both treatments involving p-BQ a higher mutation frequency was observed in the NER-deficient cells compared with NER-proficient cell lines. These results suggest a differing mechanism for the repair of HQ-induced damage compared with the bulky DNA damage induced by p-BQ.

Transfection of a benzene metabolite-modified plasmid into both cell lines gave a predominance of single base substitutions in each case. In all treatments the majority of these mutations occurred at GC base pairs, with GC->TA transversions and GC->AT transitions predominating. Again, as observed previously for transfection of a treated plasmid into Ad293 cells (20), the majority of base substitutions for each treatment group were for either an adenine or thymine base. This result suggests application of the ‘A rule’ (reviewed in 26).

Hot-spots of mutation for the single and tandem base substitutions occurred at positions 108, 109, 123, 129, 133 and 175 for HQ-treated plasmid transfected into repair-proficient cells compared with 108, 129, 139 and 155 for the repair-deficient cells. Comparison of these two spectra using the Cariello Hyperg program (24) demonstrated a significant difference (P = 0.0004). Transfection of p-BQ-treated plasmid into NER-proficient cells resulted in hot-spots at positions 115, 122 and 155, whereas for NER-deficient cells only one hot-spot was recorded, again at position 155. These results were similar to the findings of Nakayama et al. (16), who reported mutation hot-spots in similar regions of the supF gene. For the combined treatment different hot-spots were observed for each cell type, positions 115, 122 and 123 and positions 132 and 139 for the repair-proficient and repair-deficient cells, respectively. Two of the hot-spots observed for p-BQ and the combined treatment in repair-proficient cells were common to both treatments; these were located at positions 115 and 122. One hot-spot observed in the NER-deficient cells (combined treatment) was at position 132, a thymine base. A hot-spot at a thymine base was not detected in either NER-proficient or Ad293 cells (20) for single or tandem base substitutions.

A common pattern with regard to sequence context was not observed for the hot-spots present in the spectra recorded for the single and tandem base substitutions. In the NER-proficient cells HQ treatment, which resulted in only one detectable adduct located on 2'-deoxyguanosine 3'-monophosphate, resulted in three hot-spots located at multiple cytidine sites, suggesting that the mutations may have originated on the non-transcribed strand. One hot-spot occurred within three adjacent guanine bases and the remaining two were flanked either by adenine or thymine bases. A similar finding was observed for the same treatment following transfection into NER-deficient cells. For the hot-spots detected for the repair-proficient cells, two out of three and three out of three hot-spots were observed in sites where two or more guanine bases were present for the p-BQ and combined treatments, respectively. For the repair-deficient cells (combined treatment) each hot-spot was flanked by either cytidine or thymine bases.

Tandem base substitutions have been demonstrated to be products of pyrimidine dimers or intrastrand cross-links and in particular a tandem mutation occurring at a CC base pair has been identified as a potential marker for oxidative damage during carcinogenesis (27). All the tandem mutations occurring in NER-proficient cells transfected with HQ-treated plasmid occurred at CC base pairs. As observed for the mutations induced in Ad293 cells, a high percentage of the mutations induced by treatment with HQ alone were frameshifts, which accounted for 21 and 23.5% of the total for NER-proficient and NER-deficient cells, respectively.

A number of multiple mutations were detected for all treatments for both cell lines, with a higher percentage occurring in the repair-proficient cells compared with the repair-deficient cells. A similar observation was made by Seidman et al. (25) and Courtemanche and Anderson (28) after passage of a UV-irradiated or aflatoxin B1-treated shuttle vector into NER-deficient cells. They concluded that multiple mutations occur during NER by a gap-filling error-prone polymerase (25) and the secondary mutation is thought to occur 3' to the original lesion during DNA replication (28). A number of hot-spots were recorded for the individual treatments with HQ and p-BQ in the repair-proficient cells. As observed in the Ad293 cells, the majority of base substitutions at these hot-spots were for a guanine base, suggesting that as the ‘A rule’ does not apply to this situation, a different polymerase or repair mechanism is involved.

The major multiple base substitution mutation in the NER-proficient cell line, although different for each treatment, predominated at GC base pairs except when HQ was involved alone. The major mutation in this case was at AT base pairs.

Work done by Singer et al. has demonstrated the role of apurinic or apyrimidinic (AP) endonucleases, without the need for glycosylases, in the repair of the benzene–DNA adducts (3''-hydroxy)-3,N4-benzetheno-2'-deoxycytidine 3'-monophosphate, (3''-hydroxy)-1,N6-benzetheno-2'-deoxyadenosine 3'-monophosphate and (3''-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate (2931). These enzymes are utilized in an unusual mode of action which involves the enzyme directly incising the oligonucleotide 5' to the adduct without the generation of an AP site. The adduct is left as a ‘dangling base’ on the 5'-terminus (29), and it is this localized effect on the DNA sequence, i.e. sequence distortion, rather than the adduct itself which allows recognition by the repair enzyme (32). Previously obtained results with the deoxyadenosine adduct demonstrated the importance of sequence context with regard to enzymatic action (30). This work demonstrates the role of a base excision repair (BER)-like process in the repair of benzene–DNA adducts, which does not fit with our findings following p-BQ treatment. However, the work carried out by Singer et al. was carried out in vitro using a short sequence of double-stranded DNA and therefore may not reflect the repair processes in vivo, when the complexity of the DNA structure, position of the adduct (major groove, minor groove, sequence context, etc.) and the cellular environment may play a role in the recognition process of repair enzymes.

The work described here suggests that the damage induced by the benzene metabolite HQ, whether it be due to the formation of bulky DNA adducts or a different mechanism such as oxidative damage, is repaired by a BER process, whereas the bulky DNA damage induced by p-BQ is repaired by NER. Further work would be required using a BER-deficient cell line to corroborate this hypothesis.

Although the work described here confirms our earlier findings, i.e. that HQ induces mutagenicity by a differing mechanism to p-BQ (20), it is still not clear whether the mutagenicity is brought about by the minor DNA adduct, (3''-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine 3'-monophosphate, or whether it is due to a differing mechanism, such as oxidative damage. Due to the number of adducts induced by the benzene metabolite p-BQ, compared with only one adduct induced by HQ, it may be of interest to study the mutagenicity of the individual adducts using a site-selective mutation assay. This work, particularly when carried out in repair-proficient and repair-deficient cells would further clarify whether the mutations induced are due to benzene–DNA adducts or to a differing mechanism.


    Acknowledgments
 
This work was supported by the Medical Research Council, UK (grant reference G0100873).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 3, 2004; revised December 13, 2004; accepted December 15, 2004.





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