Environmental and Molecular Mutagenesis 1657-63 (1990)

Acetaldehyde-Induced Mutation at the hprt Locus in Human Lymphocytes In Vitro Sai-Mei He and Bo Lambert Department of Clinical Genetics, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden Acetaldehyde (Aa) induces chromosomal aberration and sister chromatid exchange in a variety of test systems, but has not previously been evaluated for its ability to induce gene mutation in mammalian cells. We have studied the mutagenic effect of Aa at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in humon lymphocytes in vitro hy using the T-cell cloning technique and selection of mutant cell clones in medium containing thioguanine. Cells treated with 1.2 - 2.4 m M Aa for 24 hr or 0.2 - 0.6 m M Aa for 48 hr showed a dose-dependent decrease of cell survivcil and a 3- to 16-fold increase of the mutant Frequency. The inverse relationship between cell survival and mutant frequency was linear down to a relative survival of 15%, and showed a similar slope in the 24-hr and 48-hr treatment experiments. Forty-one mutant T-cell clones derived from cultures treated with 1.2 or 2.4 m M /\a and 15 from untreated

controls were expanded for D N A extraction and Southern blot analysis to study deletion mutation using a full length hprt cDNA probe, and clonal identity on the basis of T-cell receptor rearrangements. In the culture with a 16-fold increase of mutant frequency, 4 out of 10 independent mutants (40%) showed partial deletions extending beyond the 3’ coding sequences of the hprtgene. Two of 22 independent mutants derived from the other treated cultures with at most a 6-fold increase of mutant frequency, and 1 of 11 independent control clones showed rearrangement of the hprf gene, none of which affected the 3’end of the hprf gene. These results show that Aa is capable of inducing gene mutation at the hprt locus in human cells, and suggest that deletion mutation affecting the 3’-end of the gene may be o maior type of Aa-induced mutation of this locus.

Key words: T-cell cloning, thioguanine selection, T-cell receptor, Southern blot, gene rearrongement, gene deletion

INTRODUCTION Acetaldehyde (Aa) is a genotoxic compound to which humans are regularly exposed. It is formed in cells in vivo as a metabolite of ethanol oxidation and it occurs in common dietary components such as vegetables, fruit, and beverages. Aa is also present in ambient air due to vehicle emissions and tobacco smoke. anti in the occupational environment due to its use in the food and chemical industry [review in IARC, 19851. According to IARC [ 19871 there is sufficient evidence for a carcinogenic effect of Aa in experimental animals. Aa is highly reactive towards intracellular macromolecules, and shows genotoxic activity in a variety of test systems [review in Dellarco, 19881. With regard t o gene mutation, a weak effect has been reported in the Escherichin coli WP2 uvrA strain [Veghelyi et al.. 19781, while studies using Salmonella strains have been essentially negative. Aa induces sexlinked recessive lethals in Drosophila [Woodruff et al., I985 1, but no reports are available on its ability to induce 0 1990 Wiley-Liss, Inc.

gene mutation in mammalian cells. The genetic activity profile of Aa in mammalian cells is dominated by endpoints such as chromosome aberrations, micronuclei, and sister chromatid exchanges (SCE) [IARC, 19871. Thus, further understanding of the genetic toxicity of Aa calls for studies on its ability to induce gene mutation. We have used the human T-cell cloning technique to study the frequency of thioguanine-(TG) resistant cells after treatment with Aa in vitro, and Southern blot analysis to further characterize the nature of Aa-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprf) locus.

Received January

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1990; revised and accepted April 76, 1990

Address reprint requests to Dr. B . Lambert. Department of Clinical Genetics, Karolinska Institute. Karolinska Hospital, 103 01 Stockholm. Sweden.

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MATERIALS AND METHODS Cells and Culture Media

Lymphocytes were isolated by Ficoll-paque (Pharmacia, Uppsala, Sweden) gradient centrifugation of buffy coats obtained by leukophoresis of peripheral blood from healthy male donors. A different blood donor was used for each experiment. The cells were preincubated at a density of 1 X lo6 cells/ml in nutrient medium (NM, which is RPMI 1640, Flow, supplemented with 150 IUiml benzylpenicillin, 150 p.giml streptomycin, and 10% fetal calf serum) over night, and stimulated with 1o/o phytohemagglutinin (PHA, HA 15, Wellcome, Dartford, England) for 23 hr before Aa treatment. The post-treatment cultivation and cloning procedures were carried out in growth medium (GM, which is NM supplemented with 0.6% PHA and 30-40% conditioned medium as a source of T-cell growth factor) [described in He et al., 19891. Aa Treatment and Mutant Expression

Aa (Merck-Schuchardt, Germany) was diluted in ice-cold sodium chloride solution (9 mg/ml) and immediately added to the cell cultures in a cold room. The volume of the flasks and medium was carefully standardized. The flasks were then filled with CO,, tightly sealed, and returned to the incubator. These precautions were taken to diminish variation in treatment concentrations, since Aa evaporates rapidly at room temperature due to its low boiling point (21°C). To ensure that enough number of independent mutants would be available for selection, as many as 40-100 X lo6 cells were used for each treatment concentration. After 24 or 48 hr incubation at 37°C in sealed flasks, the cells were washed with Hanks’ balanced salt solution (Flow) and resuspended in GM. A small cell sample was then taken from each flask to determine the treatment related cloning efficiency (CE) using the same procedure as for cells after the “expression phase” (see below). The cell cultures were then incubated for 7-9 days to allow for expression of the mutant phenotype. The cells were kept at approximately constant number and density during the expression phase, and a growth curve was established for each culture flask by counting the cells every 2-3 days. Cloning and Isolation of Mutants

The procedure for T-cell cloning was adopted from the methods of Albertini et al. [ 19821 and Morley et al. [ 19831 with some modifications and was essentially as described previously [He et al., 19891. After the expression phase, the cells from each culture were resuspended in fresh GM at the appropriate cell density and redistributed into 96-well round-bottom microtiter plates (Costar, Cambridge, MA). Two or three plates were used for determination of cloning efficiency (CE-plates) and eight or more plates were used

for TG selection (TG-plates). CE-plates received per well 4 target cells and 1-3 X lo4 X-irradiated (5,000 rad) lymphocytes (from different donors) as feeder cells in 200 p.1 GM. TG-plates received 3 X 10‘ target cells per well in 200 p1 GM containing 2.5 pgiml TG. The plates were incubated at 37”C, 85% humidity, and 5 % CO,. Growing cell clones were scored visually after 2 weeks. CE was calculated from the proportion of negative wells (Po) assuming a Poisson distribution of clone forming cells between wells, i.e., P,, = e-‘, and CE = xin, where n = number of cells seeded per well. The frequency of mutant cells (MF) was given by the CE in the presence of TG divided by the CE in the absence of TG. Clonal Expansion and Southern Analysis

A few clones from each TG-plate were expanded. The cells were resuspended in fresh GM without TG, mixed with feeder cells (1-3 x lo4 per well), and redistributed into new microtiter plates for at least 10 days further incubation. About 50% of the clones produced sufficient amount of cells (10-20 X lo6) for Southern analysis. DNA extraction and blot hybridization were carried out as described [He et al., 19891. In brief, approximately 10 p.g of genomic DNA was digested completely with the appropriate restriction enzyme, fractionated by electrophoresis on a 0.8% agarose gel, and transferred to a nylon membrane (Gene Screen Plus. Dupont, NEN Research Products, Boston, MA) by alkaline blotting. After prehybridization. the filter was hybridized over night with the appropriate 3’P-labeled probe and washed stringently (the final wash in 0.5 x SSC, 0.1% SDS at 65°C for 30 min) before autoradiography . For hprt analysis the restriction enzyme used was Pst- 1 . The probe was the Pst- 1 insert of pHPT 30 [Brennand et al., 19831, kindly provided by Dr. C. T. Caskey. The restriction fragment to exon assignment was based on data from Patel et al. [I9841 and Yang et al. [1984]. To study the T-cell receptor (TCR) rearrangement patterns, DNA was digested with Hind 111 and the blot was hybridized with a probe for TCR p [Yanagi et al., 19841 and TCR y [Lefranc and Rabbitts, 19851, kindly provided by Dr. T. W. Mak and Dr. R. J. Albertini (by the courtesy of Dr. T. H. Rabbits), respectively. Interpretations of the clonal identity vs. TCR rearrangements were based on the principles described by Nicklas et al. [1986, 19881.

RESULTS Effects of Aa on Cell Survival and Growth

Initial dose-range experiments resulted in large variations in cell survival indicating that Aa-treatment requires special precautions due to the volatility of the compound. Therefore, subsequent treatment was carried out as described in the Materials and Methods section. A relative survival of

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Fig. 2. Cell growth during the post-treatment “expression” period in one 24-hr treatment experiment. Lymphocytes were isolated from male “buffy coat” at day 0. and stimulated with 1% PHA at day 1 . After 23 hr (day 2). the cells were treated with 0.6-2.4 mM of Aa for 24 hr. The cells were then cultured in GM and the increasing cell count was divided by the cell count present in the culture directly after the washing (day 3) and expressed as relative number of cells.

parent dose-related growth delay. This was followed by an exponential increase in cell numbers with a slope similar to that of the control cultures. The corresponding growth curves from the 48-hr treatment experiment using lower Aa concentrations showed little initial growth delay (data not shown).

Aa-Induced MF After the expression phase, cells were cloned in the presence or absence of TG and the mutant frequency, MF, was determined by dividing CE( +TG) with CE( -TG). Treated and untreated cultures showed similar CE( -TG) with an average of 8% (range 4-1296). This is considerably lower than the CE(-TG) of untreated cells before the expression phase (see above), and reflects the reduced cloning ability of cells after prolonged in vitro cultivation which is also indicated in Figure 2 by the decreasing growth rate of control cells during the late expression phase. Nevertheless, the “spontaneous” M F in untreated control cultures was similar in the different experiments with a mean of 4.9 X loph (Fig. 3), and there was no tendency of a reverse relationship between the M F and the CE( -TG) in either the untreated or the treated cultures. Therefore, the calculated M F b’ rives a reliable estimate of the mutagenic effect of Aa under these experimental conditions. An increase of M F was obtained in all the Aa-treated

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cultures, but variations between the 24-hr treatment experiments were large, and a clear dose-response relationship within one single experiment was only obtained by the 48hr treatment. The MF at 2.4 mM of Aa was 16-fold higher vs. 3.7 X in one of the than its control (6.0 X 24-hr treatment experiments, while the corresponding increase in another experiment was only 6-fold (1.9 x vs. 3.2 x On the other hand, the RS was 23% in the latter experiment as compared to only 12% in the former one. To a large extent, these variations reflect the difficulties in obtaining accurate and reproducible Aa concentrations. especially at high doses. Nevertheless, pooled data from the 24-hr treatment experiments showed a clear doserelated increase of MF, which was also supported by the result from the 48-hr treatment (Fig. 3). Moreover, when the induced MF was plotted against the RS, one and the same linear reverse-relationship between these two parameters was obtained in both types of experiments (Fig. 4). Since this relationship seemed to be independent of the treatment form one might propose that the RS is the best measure of effective mutagenic dose in these experiments. Southern Analysis

A total of 41 TG-resistant clones from Aa-treated cell cultures and 15 from untreated cultures were studied with regard to clonal identify and deletion mutation in the hprt locus. A clone was considered as unique when it showed a TCR rearrangement pattern clearly different from that of any other clone in the same experiment. Clones which were not distinguishable with any of the two TCR probes used

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and showed the same hprt fragment pattern were considered to be replicates derived from the same mutant clone. Typical Southern blot autoradiographs of clonal DNA hybridized with the TCR y probe and hprt probe are shown in Figure 5A and B , respectively, and the results are presented in Table I. The 15 mutants obtained from untreated cultures represented 11 unique spontaneous mutations. A normal hprt pattern was found in all of them except one that showed deletion of hprt exons 2 and 3. Sixteen clones were obtained from the culture treated with 2.4 mM Aa for 24 hr, in which the MF was 16-fold higher than its corresponding control. Identical TCR patterns were found in two doublets and two triplets of clones. The hprt fragment pattern was normal in one of these doublets (89F6/4 and 7, Fig. 5 ) , and showed a deletion of fragments corresponding to exons 2-9 in the other doublet (89F6/10 and 20). One of the triplets (89F6/5, 8, and 11) had a deletion of hprt exons 7-9, while the other triplet (89F6/16, 18, and 19) showed a deletion of hprr exons 4-9 that was identical to a deletion in another independent mutant (89F616). No hprt alterations were found among the remaining five unique mutants. Thus, in this culture a total of 4 out of 10 independent mutations (40%) were associated with gross deletion extending through the 3'-end of the hprt gene. All other clones studied were derived from cultures treated with 1.2 or 2.4 mM Aa for 24 hr, in which the MF

Acetaldehyde-Induced hprt Mutation

61

Fig. 5. A: Southern blot patterns of Hind 111-digested DNA from eight Aa-induced mutants in the culture showing a 16-fold increase of mutant frequency, hybridized with a TCR probe. Ti y. Two of the mutants (89F61 7,4) shared the same Ti y and Ti p pattern, indicating their common mutational origin. Among the other four clones showing the same Ti y pattern (89F611 1,10,8,5), one (89F6110) could be distinguished from the others by its different Ti p pattern. The remaining clones (89F616.3) showed unique Ti y patterns. B: Southern blot patterns of Pst-I digested mutant DNA, hybridized with a full-length hprt probe. The unselected

clone (wt) shows the expected wild-type pattern and number of fragments produced by Pst-l from the X chromosome-linked hprt gene and autosomal hprr pseudogenes (+). The other eight lanes contained DNA from the same set of mutants as in A . The three mutants (89F611 I .8.S), sharing the same TCR patterns showed all deletion of hprr exons 7-9. while the doublet (89F6/7,4)and 89F613 had the normal hprt pattern. The other independent mutant (89F616) had deleted hprt exons 4-9, and 89F6110 had only exon I remained.

TABLE 1. Summary of Origin and Nature of hprt Mutants

was low (3- to 6-fold over background) in most of the experiments. The majority (88%) of the analysed mutants represented unique mutations (Table I) which indicates that the MF was about the same as the estimated MF in these cultures. In the single experiment where a high (16-fold) increase of MF was obtained, 10 out of 16 of the analysed mutants were unique (Table I). Thus, at most a 10-fold increase of MF was obtained in these experiments, which indicates that Aa is a comparatively weak mutagen in this system. A considerable portion (40%) of the mutations in the culture with a 16-fold increase of MF were large deletions extending beyond the 3'-end of the gene. Thus, the mutagenicity of Aa resembles that recently reported for formaldehyde. In a system similar to ours but using cells of a human lyrnphoblast line, Crosby et al. 119881 found that repetitive treatment with low dose of formaldehyde was needed to achieve more than 10-fold increase of MF, and almost half of the induced mutations were large deletions. Repeated Aa-treatment over several days turned out to be less practical in our system, as such prolonged in vitro cultivation before cloning gave rise to very low cloning efficiencies. On the other hand, we found that treatment with low concentrations of Aa for 48 hr was more effective with regard to mutation induction than high concentrations

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was 3 to 6 times higher than the corresponding control. These 25 clones were found to represent 22 independent mutations on the basis of their TCR patterns. Only two showed gross hprt alterations, one (88F18/5) had a novel band at 1.9 kb and the other one (88F42/4) a deletion of exon 1 (Table I). Thus, in this set of Aa-treated cultures, the frequency of hprt rearrangement was the same as in the control (9%). and no large deletions affecting the 3'-end of the gene were observed.

DISCUSSION Our results show that Aa induces mutations at the hprt locus in human lymphocytes. However, the induced MF

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He and Lambert

for 24 hr. The former treatment condition did not cause a detectable growth delay and therefore cells could have passed through one or may be even two DNA-replication cycles in the presence of Aa. In contrast, treatment with high concentrations for 24 hr caused considerable cell death and prolonged growth delay making DNA-replication during the treatment unlikely. Thus, Aa-induced mutagenesis seems to be most efficient when Aa is present during DNAreplication. The above conclusion is consistent with our previous observations with regard to Aa-induced SCE in human lymphocytes, which suggested that the SCE-inducing effect of Aa is to a large extent dependent on the continuous presence of the compound in the medium during DNA-replication [He and Lambert, 1985; Lambert and He, 19881. Moreover, these studies suggested that Aa may remain within cells for considerable time periods, possibly by the formation of reversible Shiff bases with amino groups in proteins and DNA, and react with DNA during successive replication cycles. If one assumes that Aa exerts its mutagenic effect during the entire incubation time, the 48-hr treatment gave rise to twice as many mutations as the 24-hr treatment at comparable dose levels (mM X hr, Fig. 3). Little is known about the types of DNA damage caused by Aa in intact cells. It has been reported to form DNA interstrand cross-links in human lymphocytes [Lambert et al., 19851, and DNA-protein cross-links in rat nasal mucosa in vivo [Lam et a]., 19861. Binding and adduct formation of Aa to nucleosides, DNA and other macromolecules have been demonstrated in vitro [review in Dellarco 19881. It is not known if and to what extent these lesions, if formed in living cells at non-lethal concentrations, are involved in the genotoxic effect of Aa. While Aa is a potent clastogenic and SCE-inducing agent at relatively non-toxic concentrations, it appears to have a comparatively weak mutagenic effect. This may indicate that the types of Aa-induced lesions involved in the chromosomal events are not exactly the same as those inducing the gene mutations. Nevertheless, the possible predominance for large gene deletions among the Aa-induced hprt mutations in the experiment showing a 16-fold increase of mutant frequency (Table I) indicates that there may be a common pathway for the generation of gene mutation and chromosomal endpoints similar to that suggested for the deletions caused by formaldehyde. also a cross-linker [Crosby et al., 19881. The relatively weak mutagenicity obtained in this study could be related to the use of the lzprt locus. The low response of the liprt locus (as compared to other autosomal loci) to a clastogen such as x-rays has been proposed to be due to the inability to recover hprt mutants containing deletions affecting adjacent essential genes on the hemizygous X chromosome [DeMarini et al.. 19891. It is also noteworthy, that all of the independent, large deletions induced by Aa in this experiment had the 5' break-

point within the gene, and the 3' breakpoint in the flanking region. Xu et al. [ 19891 recently reported a predominance of deletion mutation in the 3'-end of the hprt gene in spontaneous and x-ray induced Chinese hamster cell mutants. and suggested the possible existence of hot spots for deletions in this region of the hamster gene. In the experiments showing a low or moderate increase of MF, there were few mutants (9%) with abnormal Southern patterns and none with large deletions extending through the 3'-end of the gene. Whether this is due to a dose dependence for the types of mutations induced, or simply reflects the spectrum of spontaneous mutations is not clear. The former phenomenon was observed in a study of gpt (xanthine guanine phosphoribosyl transferase)-mutations in a Chinese hamster cell line after treatment with mitomycinC , where point mutations dominated at the lower concentration and large deletions at the higher dose [Tindall and Stankowski, 19881. Obviously, further studies of the types of the DNA damage and the nature of mutations induced by Aa are needed to understand the mechanism behind its genotoxic effects.

ACKNOWLEDGMENT This study was supported by The Swedish Cancer Society, The Swedish Environmental Protection Board, The Swedish Tobacco Company Research Fund, The Swedish Fund for Animal Care, and King Gustav V:s Jubilee Fund.

REFERENCES Albertini RJ, Castle KL, Borcherding WR (1982): T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood. Proc Natl Acad Sci USA 79:6617-6621. Brennand J. Konecki DS. Caskey CT (1983): Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyl transferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts. J Biol Chem 258:9593-9596. Crosby RM, Richardson KK, Craft TR. Benforado KB, Liber HL, Skopek TR ( 1988): Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. Coli. Environ Mol Mutagen 12: 155- 166. Dellarco VL ( 1988): A mutagenicity assessment of acetaldehyde. Mutat Res 195: 1-20. DeMarini DM, Brockman HE, de Serres FJ, Evans HH, Stankowski LF, Hsie AW ( 1989): Specific-locus mutations induced in eukaryotes (especially mammalian cells) by radiation and chemicals: a perspective. Mutat Res 220: 11-29, He S-M, Holmberg K, Lambert B, Einhorn N (1989): Hprt mutations and karyotype abnormalities in T-cell clones from healthy subjects and melphalan-treated ovarian carcinoma patients. Mutat Res 210:353358. He S-M, Lambert B (1985): Induction and persistence of SCE-inducing damage in human lymphocytes exposed to vinyl acetate and acetaldehyde in vitro. Mutat Res 158:201-208. International Agency for Research on Cancer (IARC) (1985): IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Ally1 Compounds. Aldehydes, Epoxides and Peroxides. VOI 36, IARC. Lyon. pp 101-132.

Acetaldehyde-Induced hprt Mutation International Agency for Research on (Cancer (IARC) (1987): IARC Monographs on the Evaluation of (Carcinogenic Risks to Humans. Suppl 6. IARC, Lyon, pp 21-23. Lam CW, Casanova M, Heck HD’A (1986): Decreased extractability of DNA from proteins in the rat nasal mucosa after acetaldehyde exposure. Fund Appl Toxic01 6541-550. Ldmbert B, Chen Y, He S-M, Sten M (1985): DNA cross-links in human leucocytes treated with vinyl acetate and acetaldehyde in vitro. Mutat Res 146:301-303. Lambert B, He S-M (1988): DNA and chromosome damage induced by acetaldehyde in human lymphocytes in vitro. Ann NY Acad Sci 534369-376. Lefranc M-P, Rabbitts TH (1985): Two tantemly organized human genes encoding the T-cell g constant-region sequences show multiple rearrangement in different T-cell types. Nature 3 16:464-466. Morley AA, Trainor KJ, Seshadri RS (1983): Cloning of human lymphocytes using limiting dilution. Exp Hematol 11:418-424. Nicklas JA, O’Neill JP, Albertini RJ (1986): Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes Mutat Res 173:67-72. Nicklas JA, O’Neill JP. Sullivan LM, Hunter TC, Allegretta M, Chastenay BF. Libbus BL. Albertini RJ (1988): Molecular analysis of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: ll. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vibo. Environ Mol Mutagen 12: 27 1-284. Patel PI, Nussbaum RL. Framson PE, Ledbetter OH, Caskey CT, Chinault

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C (1984): Organization of the hprt gene and related sequences in the human genome. Somatic Cell Mol Genet 10:483-493. Tindall KR, Stankowski LF Jr (1988): The predominant mutational change at the gpt locus in AS52 cells differs with dose following mitomycin-C treatment. Environ Mol Mutagen 11 (suppl l1):lOS. Veghelyi PV, Osztovics M, Kardos G, Leisztner L, Szaszovszky E. Igali S, Imrei J (1978): The fetal alcohol syndrome: Symptoms and pathogenesis. Acta Pediatr Acad Sci Hung 19:171-189. Woodruff RC, Mason JM, Valencia R, Zimmering S (1985): Chemical mutagenesis testing in Drosophila, V. Results of 53 coded compounds tested for the National Toxicology Program. Environ Mutagen 7:677-702. Xu 2, Yu Y, Hsie AW, Caskey CT. Rossiter B, Gibbs RA (1989): Deletion screening at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinase hamster cells using the polymerase chain reaction. Teratogen Carcinogen Mutagen 9: 177-187. Yanagi Y, Yoshikai Y, Leggett K, Clark SP, Aleksander I, Mak TW (1984): A human T-cell specific cDNA clone encodes a protein having extensive homology to immunoglobulin chains. Nature 308: I45 - 149. Yang TP, Patel PI, Chinault AC, Stout JT, Jackson LG, Hildebrand BM, Caskey CT (1984): Molecular evidence for new mutation at the hprt locus in Lesch-Nyhan patients. Nature 310:412-414.

Accepted by-

J.J. McCormick