Mutational spectrum induced by acetaldehyde in the HPRT gene of human T lymphocytes resembles that in the p53 gene of esophageal cancers
Peri Noori and
Sai-Mei Hou,1
Environmental Medicine Unit, Department of Biosciences, The Karolinska Institute, CNT/NOVUM, S-141 57 Huddinge, Sweden
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Abstract
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As the primary metabolite of alcohol, acetaldehyde (AA) may be responsible for many pathological effects related to consumption of alcohol, such as esophageal cancer. The spectrum of p53 mutations in esophageal tumors is indicative of the involvement of exogenous agents, such as tobacco smoke. There is, however, no experimental proof for the involvement of alcohol as data on mutational spectrum induced by AA in human genes is completely lacking. The aim of this study is to investigate whether AA leaves mutational fingerprint in the HPRT reporter gene in human peripheral T cells. Pre-existing in vivo HPRT mutants were removed from PHA-stimulated T lymphocytes before in vitro treatment with 2.4 mM AA for 24 h. Following cell growth to allow mutation expression, independent 6-thioguanine-resistant mutants were selected from large numbers of subcultures showing a 3-fold induction of mutant frequency on average. A total of 73 induced and 36 spontaneous mutants were found to carry a missense, nonsense, frameshift or splice mutation. Base substitutions were identified in the coding or splicing sequences of 55 induced and 26 control mutants. The induced base changes were mainly G > A transition (40%, G on non-transcribed strand) followed by A > T transversions (14.5%, A on non-transcribed strand). The control mutants had significantly (P = 0.04) less G > A transition (15.4%) and completely lacked A > T transversions. We also identified 5'-AGG-3' or 5'-AAG-3' as potential target sequences for AA-induced G > A transitions. This specific mutational spectrum induced by AA is consistent with the known formation and persistency of N2-ethyl-2'-guanosine adduct and with the predominance of G > A transitions and mutations at A:T base pairs in the p53 gene of esophageal tumors. We conclude that AA may be involved in the pathogenesis of esophageal cancer.
Abbreviations: AA, acetaldehyde; HPRT, hypoxanthine-guanine phosphoribosyl transferase; BPDE, benzo[a]pyrene diolepoxide; XP, xeroderma pigmentosum.
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Introduction
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Acetaldehyde (AA) is an important industrial chemical that is used in the plastic and resin industry for the synthesis of organic chemicals (1). It is present in automotive exhausts and tobacco smoke. In addition, it is a primary product during the metabolic oxidation of ethanol by alcohol dehydrogenase in the liver, to be further oxidized to acetate by aldehyde dehydrogenase. Recent findings (2) suggest that alcohol ingestion increase the proportion of Neisseria in the oral microflora, the bacteria species that has extremely high activity of alcohol dehydrogenase. A great number of epidemiological data have identified chronic alcohol consumption as a significant risk factor for upper alimentary tract cancer, including cancer of the oropharynx, larynx and esophagus. To a large extent, these pathological effects related to alcohol may be attributable to AA, partly by the formation of free radicals leading to DNA damage and partly by the mutagenic effects of AA itself.
AA is classified as a probable human carcinogen as there is sufficient evidence in experimental animals for the carcinogenicity of AA (1). The genotoxic effects of AA in human cells in vitro include induction of single- and double-strand breaks, DNADNA and DNAprotein cross-links, chromosomal aberrations, sister chromatin exchanges and gene mutation (1). AA reacts with all deoxynucleosides with the exception of thymidine, and the order of reactivity is dGuo > dAdo > dCyd (3). The major stable DNA adduct formed after reduction under physiological conditions is N2-ethyl-2'-deoxyguanosine (4). The level of N2-ethyl-3'-dGMP adducts in granulocyte and lymphocyte DNA from alcoholic patients were 13- and 7-fold higher, respectively, than the corresponding levels in control subjects (5).
Squamous cell carcinomas of the esophagus and oral cavity have been suggested to be mainly attributable to the consumption of alcoholic beverages and to tobacco smoking. However, the relative impact of each major risk factor is difficult to assess as these patients are often exposed to both alcohol and tobacco. Somatic mutations of the p53 tumor suppressor gene have been shown to be particularly frequent in esophageal cancers, which suggests that p53 inactivation is the key event in esophageal carcinogenesis. The spectrum of p53 mutations identified in squamous cell carcinomas of esophagus (6,7) is in agreement with the etiological role of exogenous agents, such as cigarette smoke. It shows a predominance of G > T transversions and mutations occurring at A:T base pairs, but a lower frequency of G > A transitions at CpG sites compared with adenocarcinoma type of esophageal cancers. There is, however, no experimental proof for the involvement of alcohol, as there are no data available on mutational spectrum induced by AA in any mammalian genes apart from the tandem mutations induced by high concentrations of AA in shuttle vectors because of intra-strand cross-links (8). This is in contrast to the well-known transversion mutations induced by the major tobacco smoke carcinogen metabolite benzo[a]pyrene diolepoxide (BPDE) in vitro and animal models (9). Analyzing the spectrum of mutation induced by AA in somatic cells in vitro may, therefore, help to evaluate the relative importance of alcohol in the pathogenesis of esophageal cancer.
The p53 mutations in tumors have many features in common with the HPRT mutations in 6-thioguanine-resistant T lymphocytes, such as large target size with wide spectrum of mutations, non-random distribution of point mutations and predominance of missense mutations (10). Furthermore, BPDE has been shown to mainly induce GC > TA transversions with guanine on the non-transcribed strand (11), suggesting that preferential excision repair of BPDE adducts on the transcribed strand occurs in the HPRT gene in human primary T cells. We have demonstrated previously that AA induces a 316-fold increase of the mutant frequency at the HPRT locus in human lymphocytes, with a predominance of large 3'-flanking deletions (12). In the present study, we have analyzed a new collection of independent HPRT mutations, with emphasis on the point mutations.
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Materials and methods
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Mutant induction and selection
The culture media used for selection of HPRT mutants were as described previously (13). Lymphocytes (1.5x106 cells/ml) were purified from a buffy coat of a healthy blood donor and incubated in nutrient medium supplemented with 0.3% phytohemagglutinin for 20 h. To remove pre-existing in vivo HPRT mutants, cells were treated with HAT (hypoxanthine, aminopterin, thymidine) supplement (Gibco BRLTM) for 24 h, washed with phosphate-buffered saline (PBS) and suspended in growth medium to a cell density of 1.5x106/ml. To avoid evaporation, AA was diluted in 5 ml cold PBS and kept on ice. The cells were chilled in refrigerator for 15 min immediately before exposure to 2.4 mM AA. All cultures were kept in refrigerator for 2 h, and then moved to 37°C incubator with 5% CO2. After an additional 22 h, cells were washed with PBS, suspended in growth medium to a cell density of 1.5x106/ml and distributed on 24-well plates. Two 96-well plates were seeded with two target cells and 2x104 lethally irradiated feeder cells per well in growth medium for determination of relative cloning efficiency (relative survival) after treatment.
The remaining cells were subcultured and allowed for 8 days expression in growth medium, during which ~2x106 cells were kept in each well of 24-well plates by counting every second day. Cells from each subculture were then subjected to mutant selection on a half 96-well plate. Each microwell received 2x104 target cells and 1x104 lethally irradiated feeder cells in growth medium supplemented with 6-thioguanine (2 µg/ml). To avoid sibling mutants in the mutational spectrum, only one 6-thioguanine-resistant clone was collected from each microplate (subculture) for molecular analysis after 14 days growth.
For estimation of the average cloning efficiency and mutant frequency, 2x105 cells from each subculture were mixed and subjected to limited dilution. Two microplates were made with two `mixed' target cells and 2x104 irradiated feeder cells per well without 6-thioguanine. Ten selection plates were made with 2x104 `mixed' target cells and 1x104 feeder cells per well. All plates were scored after 14 days and cloning efficiency was calculated from the proportion of negative wells assuming a Poisson distribution. Mutant frequency was obtained by dividing the cloning efficiency in the presence of 6-thioguanine with that in the absence of 6-thioguanine.
Detection of mutations in cDNA and genomic exons
From each sampled mutant clone, two aliquots of 3x104 cells were collected for genomic DNA analysis. The remaining cells were cultured for an additional 2 days in growth medium before they were washed and subjected to RNA isolation using Purescript kit (Gentra Systems, MN). The RNA precipitate was finally diluted in 15 µl RNA Hydration Solution and stored at 80°C until use.
For cDNA synthesis, 15 µl of the RNA solution was incubated for 1 h at 37°C with a 5-µl cDNA cocktail. The 20 µl reaction contained 15 mM TrisHCl pH 8.5, 60 mM KCl, 2.5 mM MgCl2, 1 mM of each dNTP, 1.6 µM reverse primer (5'-721GAT AAT TTT ACT GGC GAT GT702-3'), 1 U/µl RNAsin (Pharmacia Biotech) and 3 U/µl reverse transcriptase (Promega). The subsequent PCR amplifications including a nested PCR with a biotinylated primer were carried out as described (14).
The cDNA PCR product was checked in a 3.75% polyacrylamide gel, and single-stranded DNA was isolated using Dynal Beads (15). Sequencing reaction was carried out using Cy5TM AutoreadTM Sequencing Kit (Pharmacia Biotech), and run on an ALF Express (Pharmacia Biotech).
To identify mutations giving rise to splicing mutant, genomic exons that were skipped in the cDNA were amplified and sequenced. Eight microliters of a lysate prepared from 3x104 cells were used in a 50-µl PCR reaction as described previously (15). The PCR product was purified using MicroSpinTM (Amersham), and sequenced using ABI Prism BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit and a 377A Automated Sequencer (Applied Biosystems).
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Results
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The cloning efficiency of cells directly after the 24-h treatment with 1.2 mM AA was 50% of that in concurrent control cells, indicating a moderate relative survival of the treated cells. The average mutant frequency in AA-treated subcultures was 13.5x106 while the corresponding value in untreated subcultures was 4.9x106. A total of 126 induced and 63 control mutants were sampled for molecular analysis.
cDNA products were successfully obtained from 80 induced and 40 control mutants, and mutations were identified in all but six induced (7.5%) and one control mutants (2.5%). The relatively high frequency of unidentified mutations among induced mutants may be related to a high frequency of splice mutations causing truncated and unstable transcripts, and thereby, a recovery of the wild-type transcripts only. Indeed, the frequency of splice mutations appeared to be somewhat over-represented among induced mutants compared with spontaneous mutants, while the frequencies of nonsense and frameshift mutations were very similar between treated and untreated cultures (Table I
). Splice errors were recognized in 25 induced and 10 control mutants as skipping of whole exons, partial exclusion of exon sequence or partial inclusion of intron sequence (Table II
). Mutation in the splice donor or acceptor site flanking the affected exon was identified in all but seven induced and three control mutants. Frameshift or small deletion occurred in the coding sequence of 10 induced and five control mutants, and most of them at direct repeat sequences (Table III
). Base substitutions were identified in the HPRT cDNAs of 38 induced (Table IV
) and 21 control mutants (Table V
).
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Table I. Frequency distribution of different types of mutations identified in the HPRT gene in T cells exposed or unexposed to AA
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The distribution of different types of base substitutions identified in the coding or splice sequences is summarized in Table VI
. Transitions were more frequent than transversions among the 55 induced mutants while they were equally frequent among the controls. The induced transitions were mostly G > A changes in both coding and splice sequences, while the spontaneous transitions were dominated by C > T changes in the coding sequence (cytosine not a major target at splice sites). With regard to transversions, A > T changes represented the major type of induced base substitutions in the coding sequence, but no such change was found among controls. On the other hand, C > A transversions were found among controls only.
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Table VI. Distributions of different types of base substitutions in the HPRT coding and splice sequences among AA-induced and control mutants
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The overall frequency of mutations at G:C base pairs (transitions or transversions) was lower among induced mutants than among control mutants (Table VI
). Mutations at sites where guanine was on the non-transcribed strand represented however the vast majority of mutations at G:C base pairs among induced mutants (26/30), while no such strand preference was seen among spontaneous mutations (9/19, P = 0.008). The corresponding increase in the frequency of mutations at A:T base pairs among induced mutants was mainly a result of mutations at sites where adenine was on the non-transcribed strand (15/25). Again, there was no such strand preference among control mutants (3/7).
The strand specificity of induced mutations at G:C base pairs was mainly because of GC > AT transitions identified in the coding sequence (Table VI
). This is particularly true if mutations at CpG sites are excluded. GC > AT transitions at CpG sites is generally associated with spontaneous deamination of 5-methylcytosine generating thymine (16). Among the 21 control mutants, only one C > T transition could be attributable to such a spontaneous process at a CpG site (position 151; Table V
). Among the 38 mutations identified in the coding sequence of AA-treated cells (Table IV
), however, CpG sites were involved in three out of four C > T transitions (position 508), but only two out of 14 G > A transitions (position 143). Some specific sequence context could be seen among the remaining 12 G > A transitions induced in the coding sequence. Five of them occurred at AGGG, AGG, AAGG or AAG sequences and seven at TG dinucleotide or TG repeats (Table IV
). In addition, among the eight G > A mutations in the splice sequences, seven occurred at AG, AAG or AAGG sequences and one at TG dinucleotide (Table II
). Two out of three mutations at AG dinucleotide coincided with the AG consensus splice acceptor sequence (IVS4-1). Thus, 5'-AGG-3' or 5'-AAG-3' may be potential target sequences for AA-induced DNA damage.
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Discussion
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To our knowledge, this is the first study on the mutational spectrum induced by AA in human gene and in human primary somatic cells. The possibility of using primary T lymphocytes for mutational analysis has only been utilized in a few studies regarding mutations induced by BPDE (11), 1-nitrosopyrene (17) and styrene oxide (14). In contrast to these previous studies, we have removed the pre-existing in vivo HPRT mutants before in vitro treatment and selected independent (unique) mutants from different subcultures for molecular analysis. The mutational spectra we obtained should thus be considered as the true in vitro spontaneous or induced spectra in the T cells of the blood donor without any in vivo background mutants or in vitro sibling mutants. The size of control data set was, however, as small as in all previous studies because of the lower control mutant frequency. Pooling with previous control data is not appropriate because of the differences in study design, neither is comparison of the control or induced spectrum with the in vivo HPRT mutation database (18) because of the mixed or undefined background exposures.
The only previous report of point mutations induced by AA is that by Matsuda et al. (8) who studied the supF gene in shuttle vector plasmids replicated in human fibroblast cell lines. The predominant type of mutation found in double-stranded plasmids were specific tandem base substitutions (GG > TT or CC > AA) that could arise from intra-strand cross-links between adjacent guanine bases. The AA concentrations used in these experiments were, however, 1001000-fold higher than that in our study. This may explain why no GG > TT tandem mutations were found in our collection of HPRT mutants and only a few such mutations exist in the p53 database (19). The mutant frequency in the AA-treated cultures in the present study was 3-fold over the concurrent controls. This mutant induction was obtained at the low-toxicity level of 50% relative survival and is in agreement with that obtained previously at similar toxicity level of AA in cells from another donor (12).
We found the mutations induced by AA to be mainly G > A transitions with guanine in the non-transcribed strand. Another major type of base substitution induced by AA may be A > T transversion, with adenine in the non-transcribed strand. It has been proposed that the DNA damage induced by AA is repaired by nucleotide excision repair pathway (8). This is based on binding of xeroderma pigmentosum (XP) complementation group A protein to AA-treated DNA, higher sensitivity of excision repair-deficient XP cells compared with repair-proficient normal cells, and a higher frequency of AA-induced mutations of the shuttle vectors in XP cells than in normal cells. In repair-proficient diploid human fibroblasts (20), the pre-mutagenic lesions formed by BPDE were lost from the transcribed strand of the HPRT gene faster than from the non-transcribed strand. This preferential repair of BPDE adducts could be supported by the strand specificity of mutations induced by BPDE in the HPRT gene of human primary lymphocytes (11). Thus, the strand bias we observed in the distribution of AA-induced mutations in the HPRT gene may be related to preferential repair of DNA adducts induced by AA on the transcribed strand. Taken together, our results are in agreement with the high reactivity of AA with deoxyguanosine and deoxyadenosine (3) and suggest a preferential repair of such adducts on the transcribed strand of the HPRT gene.
The predominance of G > A transitions at non-CpG sites and transversions at A:T base pairs in the AA-induced mutational spectrum in human somatic cells provides an experimental support to the involvement of alcohol drinking in the pathogenesis of esophageal cancer. Table VII
shows the strand- and CpG-specific mutational spectra in the coding and splice sequences in the HPRT gene of AA-treated and untreated cells. This is to be compared with the corresponding mutational spectra in the p53 gene in squamous cell carcinomas and adenocarcinomas of esophagus in Europe and North America (Table VII
), which we extracted from the online p53 database (19). The proportion of single base substitutions identified in the splice sites of the p53 gene is, however, low (three out of 129 squamous cell carcinomas and four out of 130 adenocarcinomas) compared with that in the HPRT gene (Table VI
). This may partly be related to the fact that most p53 mutational screenings in tumors are carried out at the genomic level rather than at the cDNA level. The most striking observation is that AT > TA transversions, with A on the non-transcribed strand, occurred in cells treated with AA and in squamous cell carcinomas only. Apart from the high frequency of GC > TA transversions in squamous cell carcinomas, which is most probably attributable to tobacco smoke, the overall spectrum of squamous cell carcinomas appeared to be quite similar to the AA-induced spectrum. The total frequency of GC > AT transitions in squamous cell carcinomas was 31.8%, which is not so much different from that in AA-treated cells (47.3%).
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Table VII. Distribution of single base substitutions in the coding and splice sequences in the HPRT gene of AA-treated T lymphocytes and untreated cells, compared with the corresponding mutational spectra in the p53 gene in squamous cell carcinomas and adenocarcinomas of esophagus in Europe and North America. Data on p53 mutations are extracted from the online database (19)
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GC > AT transitions at non-CpG sites, with G on non-transcribed strand, occurred, however, more frequently in the HPRT gene of AA-treated cells than in the p53 gene of squamous cell carcinomas (36.4 versus 6.2%, Table VII
). Conversely, the frequency of AA-induced GC > AT transitions at CpG sites of HPRT coding region was lower than that seen in the p53 gene of squamous cell carcinomas of esophagus (9.1 versus 20.2%). This is in accordance with the known difference in the occurrence of CpG-site mutations between the two genes. There are only eight CpG dinucleotides within the 657-bp coding region of the human HPRT mRNA, and deamination-mediated GC > AT transitions have been reported to occur at four of them, mostly at C151 and C508 (21). Unmethylated cytosine, amino acid alteration not affecting enzyme function or unsaturated mutational spectrum reported to the HPRT database (18) may explain the complete lack of mutations at the remaining four sites. Mutations at CpG sites account for 33% of HPRT single base substitutions in newborns, 5% in normal adults and 15% in LeschNyhan syndrome patients with germline mutations (21). In contrast, over 50% of single base substitutions are found at CpG sites in the p53 gene of adenocarcinomas of esophagus (Table VII
), which is equally frequent as transitions at CpG dinucleotides in LiFraumeni syndrome patients with p53 germline mutations (22). This high frequency of somatic mutations at CpG sites in the p53 gene may be related to the fact that CpG sequences in the p53 coding region are highly methylated, and methylated CpG sequences are particularly prone to modification by exogenous carcinogens as well as endogenous events (16).
The frequency of p53 mutations was much higher in smokers than in non-smokers, and in drinkers than in non-drinkers (6). In contrast to these European and North American patients, a more recent study on Chinese esophageal cancer patients revealed high mutation prevalence among both smokers and non-smokers, with 50% of the p53 mutations being G > A transitions (23). Although some specific dietary carcinogens may be involved in such high-risk regions of esophageal cancer, our findings indicate that the predominance of G > A transitions in these esophageal cancer patients may be at least partly attributable to AA through alcohol drinking. Considering the high prevalence of alcohol-related cancers, even a small increase in cancer risk as a result of AA is of great importance, especially in those individuals who exhibit a higher risk for other reasons. Of particular interest is that both alcohol dehydrogenase and aldehyde dehydrogenase are highly polymorphic among Orientals and have been shown to affect esophageal cancer risk (24). Another enzyme involved in AA detoxification is glutathione S-transferase M1, which is commonly deleted in all ethnic populations and shown to be an additional constitutional risk factor for laryngeal cancer among alcoholics (25). A study of the relationship between drinking and smoking habits, polymorphism in genes involved in the metabolism of alcohol and tobacco carcinogens, and mutational spectra in the HPRT and p53 gene may further reveal the significance of AA on the development of human cancers.
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Notes
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1 To whom correspondence should be addressed Email: saimei.hou{at}cnt.ki.se 
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Acknowledgments
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This study was supported by the Swedish Cancer Foundation and Swedish Match Medical Research Fund.
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Received January 23, 2001;
revised January 23, 2001;
accepted May 25, 2001.