Mutagenesis Advance Access originally published online on November 7, 2007
Mutagenesis 2008 23(1):51-56; doi:10.1093/mutage/gem042
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DNA repair deficiency and acetaldehyde-induced chromosomal alterations in CHO cells
Department of Agrobiology and Agrochemistry, University of Tuscia, Via S. C. De Lellis snc, I-01100 Viterbo, Italy
Induction of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) by acetaldehyde (AA) was evaluated in parental and different DNA repair-deficient Chinese hamster ovary (CHO) cell lines to elucidate the mechanisms involved in the protection against AA-induced chromosome damage. Cell lines employed included the parental (AA8), nucleotide excision repair (UV4, UV5, UV61), base excision repair (EM9), homologous recombination repair (HRR) (irs1SF, 51D1)-deficient and Fanconi-like (KO40) ones. The ranking of different cell lines for sensitivity to induction of CAs by AA was 51D1 > irs1SF > KO40 > UV4 > V33-EM9-AA8 > UV61-UV5 in a descending order. Cells deficient in HRR were most sensitive followed by Fanconi anaemia like (KO40) suggesting these pathways, especially HRR is very important for the repair of AA-induced lesions. These observations also suggest that interstrand cross links are primary biologically relevant DNA lesions induced by AA for induction of CAs. Only marginal differences were found between the cell lines for induction of SCEs. The possible mechanisms involved in AA-induced chromosomal alterations are discussed.
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Introduction |
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Alcohol is probably one of the major factors responsible for increased risk of head and neck cancer especially in central and East European countries (1
). An immediate product of ethanol metabolism is acetaldehyde (AA), which is a genotoxic compound to which humans are regularly exposed. In addition, AA is also an intermediate in the metabolism of sugars. AA results also from ethanol produced from carbohydrates through bacterial fermentation in the gastrointestinal tract (2) and is produced endogenously from sources such as tyrosine, β-alanine and deoxyribose phosphate during normal intermediary metabolism (3–5). It occurs in common dietary components such as fruits, vegetables, spices, flavourings and alcoholic beverages. It is also present in tobacco smoke, vehicle fumes, etc. AA is used in the manufacture of a number of industrial products such as acetic acid, dyes, explosives, lacquers, photographic chemicals, anti-oxidants, plastics and synthetic rubber (6,7).
AA is classified as a probable human carcinogen, (B2), by the US Environmental Protection Agency (8) based on the increased incidence of nasal tumours in rats and laryngeal tumours in hamsters after chronic inhalation exposure (9–11).
One of the DNA lesions resulting from the reaction of AA with deoxyguanosine is N2-ethyl-2'-deoxyguanosine (12). Another lesion of particular significance is the 1,N2-propano-2'deoxyguanosine adduct (13–15). The reactive proprieties of AA are due to the electrophilic nature of its carbonyl carbon that can react readily with nucleophiles in a variety of macromolecules, such as nucleic acids, protein and phospholipids, to form adducts, including cross links. In vivo and in vitro studies have suggested that AA can form DNA–DNA and DNA–protein cross links (16–19). Several types of DNA adducts formed by AA and their repair have been characterized. Nucleotide excision repair (NER), homologous recombination repair (HRR) and replication-coupled repair have been reported to be involved in the repair of interstrand cross links (ICLs) induced by AA or other cross-linking agents (20–24).
AA induces mutations, sister chromatid exchanges (SCEs), micronuclei and aneuploidy in cultured mammalian cells including human lymphocytes and gene mutations in bacteria (7,16,25–27). High frequencies of gaps, breaks and exchange-type aberrations in peripheral human lymphocytes derived from a patient with Fanconi anaemia (FA) after AA treatment have been reported (28).
The present study is an attempt to understand the relative importance of different mechanisms involved in the repair of DNA lesions induced by AA employing chromosomal aberrations (CAs) and SCEs as end points. In order to achieve this, we have employed Chinese hamster ovary (CHO) cell lines, deficient in different DNA repair pathways, such as NER, transcription-coupled repair (TCR), base excision repair (BER), non-homologous end joining (NHEJ), HRR and repair of DNA cross links (FA like). The rationale for this approach is that the defect in any particular DNA repair pathway will lead to higher frequencies of chromosomal alterations in the repair-deficient cells in comparison to parental cells (due to the presence of unrepaired DNA lesions) indicating the possible involvement of that pathway in the repair of AA-induced DNA lesions leading to chromosomal damage.
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Materials and methods |
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Cell lines
Parental CHO cells (AA8) and their mutants deficient in different DNA repair pathways, i.e. NER (UV4 and UV5), TCR (UV61), HRR (irs1SF and 51D1), NHEJ (V3-3), BER (EM9) and repair of DNA cross links (KO40) were used (Table I, 29
–36). All these cell lines were originally isolated and kindly provided by L. H. Thompson.
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Cell culture and treatment
EM9 and UV61 cells were cultured in F10 medium (GIBCO Italy); AA8, 51D1, V3-3 and KO40 cells were cultured in MEM (Minimal Essential Mediun) (GIBCO Italy) and UV4, UV5 and irs1SF cells were cultured in D-MEM (Dulbecco's Modified Eagle Medium) (GIBCO Italy). All the media were supplemented with 10% heat-inactivated foetal calf serum (GIBCO) and antibiotics (Biowhittaker, Cambrex Italy). 5-Bromodeoxyuridine (BrdUrd) was purchased from Sigma and AA from Carlo Erba and stored at –20°C. Considering that AA evaporates at 20°C, a preliminary study on the induction of SCEs and CAs was conducted in AA8 cell line to find out the proper regime of treatment in terms of temperature of treatment and concentration of AA. Immediately before treatment, AA was diluted in cold (+4°C) phosphate-buffered saline solution (PBS) in glass tube to obtain the final concentration of 120 mM. This was made fresh every time and maintained on ice till use. The following protocol, allowing the scoring of both CAs and SCEs in the first mitosis after treatment (37
,38), was employed. Exponentially growing cells were grown in medium containing BrdUrd (6 µg/ml) for one cell cycle prior to treatment. The cells were washed two times with PBS, transferred to fresh medium at room temperature (18°C), supplemented with 2% Hepes Buffer (Biowhittaker, Cambrex Italy), and then treated with six different final concentrations (0.3, 0.6, 1.0, 1.8, 2.5 and 3.6 mM), and to prevent evaporation of AA, the caps of the cultured flasks remained closed during the whole incubation period. The selection of the concentrations used was made following the results obtained in preliminary experiments. After 2 h of treatment the cells were washed two times in PBS and allowed to recover for different times depending on the cell line to compensate for cell cycle delay encountered in the mutant cells.
Cytological preparations
Colcemid (0.2 µg/ml) was added for the final 3 h before cell harvesting. Cells were then detached from the flasks, collected by centrifugation, exposed to hypotonic salt solution (1% citric acid) for 8 min and fixed in methanol:acetic acid (3:1) for at least 1 h at 4°C. Cells in fresh fixative were dropped onto clean slides and allowed to dry. Air-dried slides were processed according to the Flourochrome Plus Giemsa method (39) to evaluate cell cycle progression and the frequencies of both CAs and SCEs.
Scoring of CAs and SCEs was made on 100 and 25 randomly selected complete M2 metaphases from each culture, respectively. In some mutant cell lines, lesser number of cells were scored due to the low mitotic indexes, especially at higher doses (Table II). To determine the proliferation, 100 metaphases from each culture were scored with regard to staining pattern to determine the percentages of second mitoses (M2). Mitotic index (MI) was calculated on 1000 cells. All analyses were carried out on coded slides.
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Statistical analysis was made employing Student's t-test for determining the significance of differences between the frequencies of CAs following treatment with different concentrations of AA.
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Results |
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Chromosome aberrations
The spontaneous frequencies of CAs varied between cell lines, the DNA repair-deficient cells having higher frequencies than the parental cells (AA8), which had one aberration per 100 cells. In all, 6, 4, 3, 4, 3, 34 and 23 aberrations per 100 cells were observed in EM9, V3-3, UV61, UV4, UV5, KO40, 51D1 and irs1SF cells, respectively (Table II). Cell lines deficient in HRR had higher spontaneous aberrations (chromatid breaks and exchanges) in comparison to the other cell lines.
The results on the frequencies of CAs per 100 cells, SCEs per cell, MI as well as percentages of second mitoses (M2) are presented in Table II. A reduction on MI of 
50% was found at the dose 1 mM for irs1SF; at 1.8 mM for KO40, 51D1, EM9 and V3-3; at 2.5 mM for UV61, UV4 and UV5 and at 3.6 mM for AA8.
A reduction of 30–50% in proliferation (as deduced from the frequencies of M2) was found at the dose of 0.6 mM for irs1SF; at 1 mM for UV5 and V3-3; at 1.8 mM for EM9 and 51D1; at 2.5 mM for UV61, KO40 and AA8.
For the induction of CAs, there was a concentration-dependent increase in all cell lines except UV5 and UV61 in which only at the highest doses (i.e. 1.8 and 2.5 mM, respectively) there was a statistically significant increase in CAs (Table II).
As expected for typically S-dependent agents [i.e. the cells need to pass through an S phase for visualizing aberrations (40)] like AA, the types of CAs induced were only of the chromatid type, chromatid exchanges being the most frequent type of aberrations followed by chromatid breaks and isochromatid breaks. The largest increase in chromatid exchanges was found in 51D1. UV5 was less sensitive for the induction of CAs in comparison to parental and other cell lines.
In order to evaluate the relative sensitivity for induction of CAs in the different cell lines, a comparison was made at the dose 0.6 mM of AA that elicits similar reduction (<13%) of the proliferation, except for the irs1SF cell line where the reduction of proliferation was >40%. At this dose, the ranking of sensitivity for CAs was done based on the sensitivity formula (FAb) calculated in the following way: the increase of abnormal cells (Ab) in the mutant cell line (X) over the corresponding control (C) in comparison to the increase of abnormal cells in the parental cell line over their control (C):
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It resulted in the following ranking (from 0.21 to 27.28 times) (Table III):
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Although this ranking is derived for this dose (0.6 mM), the situation does not change when all values up to 1 mM are taken together for total frequencies on induced aberrations (from 0.27 to 31.9 times, Table III). When comparison is made at a dose of 1.8 mM, where the data are available for all cell lines except irs1sf and V3.3, the ranking still remains the same (from 0.63 to 67.1). HRR-deficient cells (51D1 and irs1SF), FA-like (KO4O) and NER-deficient UV4 cells were more sensitive than the parental cell line as well as other mutant cell lines studied.
Sister chromatid exchanges
The results on the frequencies of SCEs induced by AA in different cell lines are presented in Table II. In all cell lines, except EM9, there was a concentration-dependent statistically significant increase in the frequencies of SCEs. The EM9, a cell line which is known to have high-level baseline frequencies of SCEs (35), responded with a non-statistically and dose-independent increase in the frequencies of SCEs. In irs1SF, instead, the increase was minimal, but significant.
In order to evaluate the sensitivity to the induction of SCEs in the different cell lines, a comparison was made at the same concentration used to determine the sensitivity to the induction of CAs where the reduction of the proliferation was similar. At this dose (0.6 mM), the sensitivity formula for the SCEs (FSCE) [increase in AA-induced frequencies of SCEs in the mutant cell line (X) over its control (C) in comparison to the increase in AA-induced SCEs in the parental cell line AA8 over its control (C)] was applied.
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Only marginal differences in sensitivity (from 0.68 to 1.68 times) (Table III) were found among the different cell lines, with a ranking of:
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The ranking order changes at other doses. In fact, it has to be noted that at the highest dose (3.6 mM) tested only in KO40 cell line, there were mitoses in M2 but they were highly damaged and not scorable. At the dose of 2.5 mM only in AA8, UV61 and KO40, it was possible to score SCEs. Whereas in UV4, UV5 and EM9 cell lines, SCEs could be scored up to 1.8 mM. Though there were M2 mitotic cells in 51D1 cell line at 1.8 mM, these were highly damaged and SCEs could not be scored. In other cell lines, there were also difficulties in scoring SCEs at high doses due to the increased chromosomal damage making it difficult to count the SCEs (Table II). The differences between cell lines for induction of SCEs by AA were not as large as observed for CAs.
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Discussion |
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Recent studies have shown that spontaneously occurring CAs in human lymphocytes are predictive of future cancer outcome irrespective of exposure to carcinogens (41
). Since several steps, such as mutations in oncogenes, defects in DNA repair, structural and numerical CAs, etc., are involved in the outcome of cancer, it is difficult to discern a single important step in carcinogenesis. The current consensus, however, is that chromosome instability is the major step for the ultimate origin of human cancer (42), stressing the importance of chromosome abnormalities in the process, as suggested by Boveri as early as 1914 (43,44). In addition, CAs in peripheral blood lymphocytes can serve as a useful marker in biomonitoring studies aimed at establishing possible correlations between DNA polymorphisms in genes controlling metabolizing and DNA repair enzymes to the susceptibility to cancer (45). These considerations stress the importance of CAs as an important biomarker in studies to unravel the mechanisms of action of mutagenic carcinogens.
In vitro studies have shown that AA induces both mono-adducts and ICLs in the DNA. Recently, numerous studies have been undertaken to identify the important repair pathways involved in the removal of DNA adducts, especially ICLs induced by AA and other cross-linking agents, such as crotonaldehyde, mitomycin C (MMC), cyclophosphamide, nitrogen mustard, photoactivated 4''-hydroxymethyl 1-45', 8 trimethylpsoralen, etc. (20–26). The mechanisms by which DNA ICLs are repaired in mammalian cells are not very clear. In bacteria and yeasts, NER and recombination are required for the removal of ICLs and DNA double-strand breaks (DSBs) are known to be produced as repair intermediates in yeast cells (21). In mammalian cells, in addition to these above-mentioned pathways, evidence has been presented for a DSB-dependent activation of FA/BRCA (breast cancer) pathway during repair of ICLs mainly operating during the S phase of the cell cycle (24). Involvement of NER in a recombination-independent and error-prone pathway has also been proposed for repair of ICLs (26).
There are surprisingly only few studies on clastogenicity of AA in mammalian cells, in spite of its importance as a primary metabolite of alcohol in human and its employment in the manufacture of many industrial products. AA has been shown to induce CAs, SCEs and mutations in human lymphocytes (16,17,25,27,28) as well as aneuploidy in Chinese hamster cells in vitro (46). The observation that lymphocytes from FA patients responded with higher frequencies of CAs than from normal individual indicates that AA-induced DNA cross links may be the important adduct for the increased sensitivity, as FA cells are known to be defective in repairing DNA ICLs. (28) The present study was undertaken to probe into the pathways involved in the repair of DNA damage induced by AA in CHO cells employing mutant cell lines deficient in different repair pathways, such as NER, TCR, BER, NHEJ, HRR and FA like (francG) using chromosomal alterations (CAs and SCEs) as end points.
Pattern of chromosomal alterations induced by AA
AA induced exclusively chromatid type of aberrations (breaks and exchanges), acting like an S-dependent agent. In general, AA induced more chromatid exchanges than breaks as commonly observed following treatment with DNA cross-linking agents (47,48).
SCEs represent exchanges between sister chromatids during DNA replication at identical sites reflecting homologous recombination. AA was found to induce SCEs in a dose-related manner in all cell lines studied, except in EM9, 51D1 and irs1SF. The background level of SCEs is very high in EM9 cells (46.8 per cell) and about seven additional SCEs were found at all doses employed. This small increase in the frequencies of SCEs will fall within the scoring errors unavoidable at such high background frequencies. This may indicate that BER-repairable AA-induced DNA lesions are not involved in the induction of SCEs by AA. Though HRR is considered to be the pathway for formation of SCEs, in HRR-deficient cell lines (irs1SF, 51D1) the background SCE levels were comparable to that of the parental cells (AA8), suggesting that the spontaneously occurring SCEs do not originate by HRR. There was only a doubling of the frequencies of SCEs due to AA (0.6 mM) in HRR-deficient mutant cells. This increase may be due to the exchanges between sister chromatids, not related to HRR-mediated SCEs, but a reflection of those associated with chromatid breaks as envisaged by Revell (49) in his hypothesis of formation of chromatid breaks. This could also be a statistical chance association between the two events (50). Such an association between a chromatid break and an SCE at the site of a break has been found in vivo in rats treated with 7-12-dimethylbenz(a)anthracene (51). Similar observations have been made on the occurrence of SCEs at chromatid break sites in chicken DT40 cells and their HRR-deficient mutants (52).
Nature of DNA repair involved in AA-induced chromosomal alterations
Several DNA adducts induced by AA have been identified and characterized. Among these, ICLs have been considered to be important mutagenic lesion. Studies on the repair of DNA ICLs have implicated several types of repair processes, such as NER, NER coupled with recombination, NER in an recombination-independent and error-prone pathway, homology-directed repair, DSB-dependent activation of the FA/BRCA pathway and replication-coupled repair (see Introduction for references). At chromosomal level, it has been reported increased frequencies of AA-induced CAs in the lymphocytes of a FA patient in comparison to normal individuals indicating the possible involvement of induced ICLs (28), as FA cells are known to be defective in the repair of ICLs (53,54). Based on the above observations, we would confine our discussion on the relative involvement of different repair pathways of ICLs, which are involved in protection of chromosome damage induced by AA.
Among the cell lines deficient in NER, UV4 was most sensitive and UV5 and UV61 were least sensitive to AA treatment (at comparable dose levels of 0.6 and 1.0 mM). Thus, it is possible that the higher sensitivity of UV4 cells to ICLs is due to their defect in HRR as well as NER (23). TCR appears not to play any major role in the repair of ICLs induced by AA.
Cell lines defective in HRR (51D1, irs1SF) were sensitive to AA on the basis of the induced frequencies of CAs (31.9- and 9.52-fold increase over controls, respectively) and inhibition of cell cycle progression (Table II). 51D1 cells were more sensitive than the irs1SF cells. This may be due to different mutated proteins in HRR pathway in these two cell lines. One can conclude that HRR is an important pathway for the repair of AA-induced ICLs which lead to CAs.
FA-like cells (KO4O) were also sensitive (2.96-fold) to AA suggesting that this pathway may be also important for repair of ICLs. Interestingly, a 3-fold sensitivity to MMC has been reported for this cell line (36). Results from cell-defective BERs indicate no major role for this pathway in repairing the DNA adducts induced by AA.
Cells defective in NHEJ of DNA DSBs (V3-3) were sensitive to some extent to AA as evidenced by the high frequencies of CAs and inhibition of cell cycle progression (Table II). Since DSBs have been reported to be generated as intermediaries in the repair of ICLs and since NHEJ operates at all phases of cell cycle, these results are not surprising. According to the model proposed by Bender et al. (55) for the formation of CAs in S phase, DSBs are generated during the repair of chemically or UV-induced lesions and unrepaired or mis-repaired DSBs give rise to chromatid aberrations. Thus, both HRR and NHEJ should be important for repair of DSBs in the S phase. It is therefore expected that cells deficient in these two repair pathways should respond with high frequencies of CAs following AA treatment. Between these two pathways, HRR appears to be more important. Sasaki et al. (52) have proposed a model for pathways of repair of ICLs induced by MMC, invoking two types, namely HRR pathway (CAs and SCE prone) and Mre11 pathway (CAs and SCE less prone) according to which HRR-deficient cells either die due to toxicity or rescued by other pathways such as Mre11 to explain the lower levels of MMC-induced CAs and SCEs observed in DT 40 chicken cells deficient in HRR. If we consider that ICLs are the important AA-induced DNA lesions in CHO cells, then HRR-deficient cells respond differently from chicken DT 40 cells, which may suggest existence of differences between rodent and avian cells in handling DNA damage such as ICLs.
Taken together all the data, HRR seems to be the most important pathway for repair of ICLs induced by AA in mammalian cells as judged by the frequencies of CA cells deficient in this pathway of repair. Though the role of other pathways such as NER seem to be necessary as initial steps, ultimately HRR appears to play a major role in the repair of the DSBs generated as intermediaries in this process.
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Acknowledgments |
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This paper is dedicated to the memory of our friend and colleague, Prof. Fernando Dulout, University of La Plata, Argentina.
Research described in this article was supported in part by Philip Morris USA Inc. and Philip Morris International and University of Tuscia, Viterbo, Italy. Conflict of interest statement: none declared
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Notes |
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* To whom correspondence should be addressed. Tel: +39 0761 357206; Fax: +39 0761 357242; Email: palitti{at}unitus.it
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References |
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Received on June 18, 2007; revised on September 24, 2007; accepted on September 24, 2007.
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