Environmental and Molecular Mutagenesis 21 :154-159 (1993)

Quantitative Relationship Between Ethylated DNA Bases and Gene Mutation at Two Loci in CHO Cells

P. Fortini, A. Calcagnile, A. Di Muccio, M. Bignami, and E. Dogliotti laboratories of Comparative Toxicology and Ecotoxicology (P.F., A. C., M.B., ED.) and Appl ied Toxicology

(A.D.M.), lstituto Superiore di Sanita, Rome, Italy

In a previous study we showed that the formation of 06-ethylguanine (06-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethyl- sulfate (DES) at the hypoxanthine-guonine- phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was ex- tended to another ethylating agent, ethyl meth- anesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N7 and O6 positions of guanine and at the N3 site of adenine were measured and the possible corre- lations with 6-thioguanine resistance (6-TG')

and ouabain resistance (owr ) mutations were investigoted. A good correlation between the levels of ethylation at O6 guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the ova' locus, the frequency of 06-EtGua adducts corre- lated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specific- ity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multi- component phenomena like mutation formation. 0 1993 Wiley-Liss, Inc.

Key words: molecular dosimetry, alkylating agents, DNA adducts.

INTRODUCTION 6-thioguanine resistance (6-TG') mutations by ethylni-

The identification of mutagenic lesions induced by chem- ical carcinogens in mammalian cells is not an easy task because of the multiplicity of chemical adducts produced on DNA and many of these reaction products are not relevant to the mutagenic or carcinogenic process. One of the most successful approaches has been to compare the mutagenic potency of chemicals belonging to the same chemical class, such as the alkylating agents, on the basis of DNA adduct formation. Different alkylating agents in fact show different mechanisms of DNA interaction; i.e., they differ in their relative affinity for akylation of DNA oxygen or nitrogen atoms producing a characteristic spectrum of lesions [for a review see Beranek et al., 19901. These differences have been used as a basis to compare mutagenicity of a series of methylating [Newbold et al. 1980; Beranek et al., 19831 and ethylating [Heflick et al., 1982; Natarajan et al., 1984; Van

trosourea (ENU) and diethylsulfate (DES). However, the same conclusion was not valid for ouabain resistance (oua') mutations, thus suggesting that other lesions were possibly involved in the fixation of ENU- and DES-induced muta- tions at the Na,K-ATPase gene locus [Bignami et al., 19881. In order to expand the knowledge about the mechanism involved in the induction of mutations at the two gene loci, the alkylation spectrum as well as mutation induction by a third alkylating agent, ethylmethanesulfonate (EMS) with DNA alkylation properties similar to DES, have therefore been analysed. The previously collected data on ENU and DES together with the present ones on EMS have been used to study the relationship between the amounts of 7-ethylgua- nine (7-EtGua), 3-ethyladenine (3-EtAde), and 06-ethyl- guanine (06-EtGua) produced by the three ethylating agents and mutation induction at both gene loci.

Zeeland it al., 19851 agents. The conclusion of all these reports is that the akylation at the Oh position of guanine is the main mutagenic lesion induced by alkylating agents at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene locus. Furthermore, the same alkyl adduct is strongly associated with mutations detected in mouse germ cells [Van Zeeland et al., 19851.

PreVioUs work developed in our laboratory also showed a good correlation between formation of this adduct and

0 1993 Wiley-Liss, Inc.

MATERIALS A N D METHODS

Cell Cultures

CHO cells (CHO-K 1 ) were cultured in Ham's F10 me- dium (Flow Labs.) supplemented with 10% fetal calf serum

Address reprint requests to M. Bignami, Laboratory of Comparative Toxi- cology and Ecotoxicology, Istituto Superiore di Sanita; Viale Regina Elena 299,00161, Roma, Italy.

DNA ond Gene Mutation in CHO Cells 155

(Flow Labs), penicillin kg/ml). Cultures were 95% relative humidity.

(100 U/ml), and streptomycin (100 incubated at 37°C in 5% CO, and

Chemicals

EMS (Kodak) was dissolved in dimethylsulfoxide (DMSO) shortly before use and quickly diluted in phos- phate-buffered saline (PBS) to the required concentrations. (1 -")EMS was obtained from New England Nuclear at a specific activity of 4.8 Ci/mmol in diethyl ether. Stock solu- tions were prepared in PBS just before use. The final spe- cific activity ranged from 14 to 5 1 mCi/mmol.

Determination of DNA Adducts by HPLC

For each experimental point, CHO cells at a density of 4 X 107/1.8 ml of PBS, pH 7.4, were treated with 3H-EMS (15,30,45,55 mM) in a total volume of 2 ml. After shaking for 30 min at 37"C, cells were centrifuged, washed once and lysed. DNA was isolated by repeated phenol-chloroform extractions and the quantitation of ethylated products by HPLC was carried out on a Hewlett-Packard instrument equipped with a Partisil 10-SCX cation-exchange column. The DNA was hydrolysed in HCll 0.1 N for 30 min at 70°C and injected together with the markers 7-EtGua, 3-EtAde, and 06-EtGua onto the column. Ethylated products were eluted by a 30-min linear gradient of 0.13-0.18 M ammo- nium formate, pH 4.0, in 12% methanol. Fractions (0.5 ml) were collected directly into scintillation vials, Lumagel (14 ml) was added and the radioactivity was determined in a Beckman liquid scintillation counter.

Cytotoxicity and Mutation Assay

CHO cells were treated with ianlabelled EMS under the same conditions described in the previous paragraph. After treatment with the alkylating agent, cells were washed and resuspended in complete medium. A dilution was plated to measure cell survival (100 cells/60 mm dish) and 7 days later cultures were fixed with methanol and stained with 10% Giemsa. To determine mutation frequency, 2 x lo6 cells were seeded in 100 mm dishes and allowed to grow for an expression time of 72 hr for oua' and 7 days for 6-TG'. During this period cells were subjcultured to maintain expo- nential growth. Cells were then trypsinized and replated at a density of lo6 or los cells per 100 mm dish for ouar and 6-TGr, respectively. The selective media, containing either 2 mM ouabain (Sigma) or 30 kM[ 6-TG, were changed after 1 week. From each cell suspension three parallel dishes were plated (100 cells/60 mm dish) to determine the cloning efficiency. The mutation frequency was expressed as the ratio of the number of mutant colonies to the number of clone forming cells.

RESULTS

Cytotoxicity and Mutation Induced by EMS

To avoid problems of extrapolation of data from best fitting curves, both mutation and DNA alkylation values were determined in the same EMS dose range. In the range of doses we used (15-55 mM), survival levels were greater than 10% (Fig. 1A); thus, determination of mutation fre- quencies was carried out at doses which were not exces- sively toxic. Linear doses responses were observed for the induction of both 6-TG' (r = 0.98) and ouar (r = 0.84) as a function of EMS exposure concentration (Figs. IB,C). In the case of oua' mutation data no significant improvement of the fit was found using a linear quadratic model. These figures are derived from at least three independent experi- ments.

Dose-Response Relationships for DNA Alkylation by EMS

In Figure 2 are shown the dose response relationships for the formation of ethylated bases in DNA after treatment of CHO cells with 'H-EMS. The data are derived from two independent experiments. The amount of total alkylation as well as the major ethylated DNA bases produced by EMS, i.e., 7-EtGua, 3-EtAde and 06-EtGua, all followed a linear dose-response (Fig. 2A-D). There is no apparent threshold in the 06-EtGua curve (Fig. 2D). which is in agreement with the absence of significant levels of DNA-alkyltransferase from this cell line [Goth-Goldstein, 1980; Bignami et al., 19891.

If the extent of substitution at the various DNA sites is expressed relative to the amount of 7-EtGua (binding level of 7-EtGua = l), ratios of 0.063 and 0.030 are obtained for 3-EtAde and 0'-EtGua, respectively, and these values are in good agreement with literature data [Natarajan et al., 1984; Singer, 19851.

Relationship Between Mutation Induction and Individual Ethylated DNA Bases

To investigate possible correlations between individual ethylated DNA bases and mutation induction at the HPRT and Na,K-ATPase gene loci, mutation frequencies were plotted as a function of each individual adduct formed dur- ing exposure to EMS, ENU, and DES. Data for EMS analy- sis are taken from the curves shown in Figure 1B,C, while data relating to ENU and DES are taken from Bignami et al. [1988].

In Figure 3 are shown the relationships between induction of 6-TG' and the amount of 7-EtGua, 3-EtAde and 06- EtGua in DNA (Figure 3A,B,C, respectively). In the case of 7-EtGua, a straight line with different slope was obtained for each of the three ethylating agents indicating that this lesion is not involved in mutations at this locus (Fig. 3A). When

156 Fortini et al.

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Fig. 1. A: Survival of CHO cells after a 30 min exposure to EMS. B: Induction of 6-TG' mutations after a 30 min exposure to EMS. The sponta- neous mutation frequency was 1.9 X The data are fitted by the linear regression line y = 5.6 + 1.48 x , r = 0.98. C: Induction ofoua'mutations after a 30 min exposure to EMS. The spontaneous mutation frequency was 0.07 x lo-'. The data are fitted by the linear regression line y = -0.8 + 0.23 x, r = 0.84.

3-EtAde was analysed, EMS and DES mutational data fell on the same line, but ENU data produced a line with a different slope (Fig. 3B). The data points fell on the same straight line only when 6-TG' mutations induced by the three ethylating agents were plotted as a function of the amount of 06-EtGua, suggesting therefore that this lesion is the main ethylation product responsible of the mutation at the HPRT gene locus (Fig. 3C).

The same analysis was perfomled in the case of oua6 mutation induction (Fig. 4A-C). In this case three different slopes for the three ethylating agents were found when mu- tations were plotted as a function of both 7-EtGua and 3-EtAde (Fig. 4A,B). However, also in the case of 06- EtCua no single line fitted all the data, with ENU and EMS having the same slope and DES a different one (Fig. 4C). In conclusion in the case of the comparison EMS/ENU, our data suggest that 06-EtGua is the base principally involved in the induction of mutations at the NdK ATPase gene locus. However, in the case of DES a more complex corre- lation exists between ouar mutations and DNA alkylated bases and a single lesion does not seem to be capable of reconciling the curve fitting analysis.

DISCUSSION

The results of this comparative study on the mutagenicity of ENU. EMS and DES as a function of specific ethylated bases confirm that 06-EtGua levels correlate with mutant frequency at the hprt gene by these ethylating agents. This conclusion is derived from a direct comparison of mutation induction as a function of each individual ethylated base. A unique linear dose response is produced only for 06-EtGua. For two of these compounds, ENU and EMS, a good corre- lation between O6-EtCua formation and mutation frequency at the oua' locus was also observed. 06-alkylguanine has been shown to induce miscoding in in vitro systems [Love- less, 1969; Saffhill et al., 19851 leading to misincorporation of thymine opposite the modified guanine residue. Its muta- genic properties in vivo have been clearly identified by adduct-directed mutagenesis studies where a single 06-alkyl- guanine was introduced in the genome of a phage or a plasmid molecule able to replicate in bacteria [Loechler et al., 1984; Hill-Perkins et al., 19861 or in mammalian cells [Ellison et al., 19891. Analogous to the in vitro data, 06- methyl- and ethyl-guanine induced exclusively GC to AT transitions at the genome site where the adduct was origi- nally located.

Despite the clear correlation between 06-EtGua and mu- tation induction by EMS and ENU at both gene loci, exami- nation of the mutational spectra of these two compounds has revealed important differences. EMS has been shown to produce almost exclusively GC to AT transition mutations [Lebkowski et al., 1986; Ashman and Davidson, 19871, consistent with a major role for 06-EtGua, while only 50% of ENU-induced mutations are this type of base pair substi- tution [Eckert et al., 1988; Bronstein et al., 19911. Transi- tions and transversions at AT base pairs cover the remaining 50% of the total base pair changes induced by ENU. A likely source of this type of mutations are 04-ethylthymidine and 0'-ethylthymidine which represent 2 and 7% of ENU alky- lation respectively. The repair of these ethyl adducts appears to be slow and this might enhance the probability of muta- tion formation. Our correlation study which would indicate

DNA and Gene Mutation in CHO Cells 157

TOTAL A L K Y L A T I O N

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Fig. 2. Plots of the i.otal DNA alkylation level, 7-EtGua, 3-EtAde, and 06-EtCua as a function of the exposure to EMS. A: Total DNA alkylation level (y = -1.63 + 1.38 x, r = 0.98). B: 7-EtGua (y = -0.62 + 0.56 x. r = 0.98). C: 3-EtAde (y = 0.03 + 0.031 x, r = 0.97). D: 06-EtGua(y = 0.015 + O.O15x, r = 0.94).

A

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Fig. 3. Induction of 6-TG'mutations as a function of the individual ethylated DNA bases 7-EtGua (A), 3-EtAde (B) and 06-EtGua (C) induced by ENU. EMS, and DES. The single line fitting ENU. EMS and DES mutation data as a function of the amount of 06-EtGua is represented by the equation y = 4.05 + 10.2 x. r = 0.97.

that the unrepaired 06-EtGua is the main contributor to EMS and ENU-induced mutations at both gene loci does not re- flect the complexity of ENU muta,genesis. This type of anal- ysis might not be sensitive enough to identify multi-compo- nents in the genesis of the mutagenic response.

EMS- and ENU-induced mutations correlated with the level of 06-EtGua also at the ouar gene locus. These data allow to conclude that the lack of correlation between 06- EtGua formation and ouar mutations is restricted to DES and is not a specific feature of this gene locus. No information is

158 Fortini et al.

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0 50 100 150 200 250 300

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Fig. 4. and 06-EtGua (C) induced by ENU, EMS, and DES.

Induction of ouar mutations as a function of the individual ethylated bases 7-EtGua (A), 3-EtAde (B),

available on DES-induced mutational spectrum. This ethy- ACKNOWLEDGMENTS lating agent is thought to react with DNA like EMS through a partially bimolecular substitution reaction (S,2) which exhibits partial tendency towards a unimolecular (S, 1)

This work has been supported by the European Economic Community, contract No. EV-4V-0044 I (A).

mechanism. The relative amounts of the various ethylated bases produced in DNA by DES and EMS is in fact similar with a slightly increased proportion of 3-EtAde produced by DES. The genetic effects of DES have been extensively analysed in both prokaryotes and eukaryotes [for a review see Hoffmann, 19801. Considering these data in their total- ity, DES is an efficient inducer of base-pair substitutions and a poor inducer of frame-shift mutations. The data on bacterial systems [Eisenstark and Rosner, 19641 and yeast [Prakash and Sherman, 19731 suggest that DES induces predominantly GC to AT transitions but also AT to GC transitions and AT to TA transversions [Stewart et al., 1972a,b]. The molecular mechanisms of mutation formation by DES might therefore involve, besides 06-EtGua, other types of DNA lesions. For example, 3-EtAde damage by blocking replication might lead to AP site formation and possibly to mutations at AT sites. The analysis of mutations at the ouar locus allowed us to identify an 06-EtGua-inde- pendent subset of DES-induced mutations. The sensitivity of this gene locus might be due to the small size of the target

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Accepted by- A.V. Carrano