Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells

Mutation Research, 193 (1988) 43-51 DNA Repair Reports

43

Elsevier MTR 06260

Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells M. Bignami 1, A. Vitelli 1, A. Di Muccio 1, M. Terlizzese 1, A. Calcagnile 1, G.A. Z a p p o n i 2, P.H.M. L o h m a n 3, L. den Engelse 4 and E. Dogliotti 1 I Laboratory of Apphed Toxicology and 2 Laboratory of Comparative Toxtcology and Ecotoxicology, lstituto Superiore di Santtd, Viale Regina Elena 299, 00161 Rome (Italy), 3 Medzcal Btological Laboratory TNO, P.O. Box 45, 2280 AA R~jswijk (The Netherlands) and 4 Division of Chemtcal Carcmogenests, The Netherlands Cancer Institute, 121 Plesmanlaan, 1066 CX Amsterdam (The Netherlands)

(Received27 October 1986) (Revisionreceived11 August 1987) (Accepted 13 August 1987) Keywords: DNA adduct formation; Ethylnitrosourea;Diethyl sulphate; (Chinese hamster).

Summary DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TG r) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (oua r) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more oua r mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus. Correspondence: Dr. M. Bignami, Lab. Tossicologia Applicata, Istituto Superiore di Sanith, Viale Regina Elena 299, 00161 Rome (Italy). Abbreviations: CHO, Chinese hamster ovary; DES, diethyl

sulfate; DMSO, dimethylsulfoxide; dTp(Et)dT, thymidyl(3'5')thymidine; ENU, N-ethyl-N-nitrosourea;3-EtAde, 3-ethyladenine; 3-EtCyt, 3-ethylcytosine; 7-EtGua, 7-ethylguanine; 3-EtdThd, 3-ethyldeoxythymidine;3-EtGua, 3-ethylguanine; 3-EtThy, 3-ethylthymine; HPLC, high-performance liquid chromatography; HPRT, hypoxanthine-guanine-phosphoribosyltransferase; ouar, ouabain-resistant; O2-EtCyt, O2-ethylcytosine; O6-EtdGuo, O6-ethyldeoxyguanosine; O4-EtdThd, O4-ethyldeoxythymidine; O2-EtdThd, O2-ethyldeoxythymidine; O6-EtGua, O6-ethylguanine; O4-EtThy, O4-ethylthymine; O2-EtThy,O2-ethylthymine;O6-MeGua, O6-methyl. guanine; PBS, phosphate-buffered saline; 6-TGr, 6thioguanine-resistant.

Alkylating agents might exert their mutagenicity and cytotoxicity as a result of the transfer of an alkyl group to DNA (Vogel and Natarajan, 1983). Alkylation of D N A can lead to the formation of potentially miscoding or template-inactivating groups. For instance, alkylation at the 7 and 3 positions of purine bases is known to cause depurination of DNA. Apurinic sites are potentially mutagenic intermediates, probably also in mammalian cells (Loeb, 1985; Gentil et al., 1984). Alkylation of D N A at other positions may cause distortions of the helical structure a n d / o r block the template function. A consequence might be

0167-8817/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)

44 the alteration of genetic information unless timely repair occurs (Lawley, 1976). Lastly, alkylation of oxygen atoms of DNA bases may cause miscoding directly by fixing an anomalous tautomeric form. This has been demonstrated for the 0 6 position of guanine, the 0 2 and O 4 positions of thymine and the 0 2 position of cytosine (for a review see Saffhill et al., 1985). Since the pattern of DNA alkylation strongly depends on the chemical nature of the alkylating agent, the efficiency with which different biological effects are induced may vary from agent to agent. In the case of methylating and ethylating agents, several attempts have been made to correlate individual DNA adducts with mutation induction in mammalian cells (Newbold et al., 1980; Heflich et al., 1982; Beranek et al., 1983; Natarajan et al., 1984). The results of these investigations showed a good correlation between alkylation at the 0 6 position of guanine and the induction of forward mutations at the hypoxanthine-guanine-phosphoribosyltransferase (HPRT) locus in different mammalian cell systems. No correlation was found for the other DNA ethyl adducts examined. The investigations of Suter et al. (1980) and Fox and Brennand (1980), however, suggested that adducts other than O6-methylguanine (O6-MeGua) are relevant in the induction of reverse mutations at the HPRT locus in V79 cells. In this study, we have measured mutations induced by N-ethyl-N-nitrosourea (ENU) and diethyl sulfate (DES) at 2 different gene loci, i.e., HPRT and Na,K-ATPase, in Chinese hamster ovary (CHO) cells, a line unable to remove O6-al kylguanine (Goth-Goldstein, 1980; Dogliotti et al., 1987). In parallel, high-performance liquid chromatography (HPLC) analysis of DNA ethyl products was performed on CHO cells that had been subjected to the same mutagen exposure. Materials and methods

Chemicals ENU (Pfaltz and Bauer, Waterbury, CN), crystallized from ethanol, and DES (Fluka AG, Chemische Fabrik, Buchs, Switzerland) were dissolved in dimethylsulfoxide (DMSO) (Sigma Chemical Co., St. Louis, MO) shortly before use

and quickly diluted in phosphate-buffered saline (PBS) to the required concentrations (final concentration of DMSO: 0.5%). Ethyl-2-3H-nitrosou rea (3H-ENU; specific activity, 2.56 Ci/mmole) was obtained from New England Nuclear (Boston, MA) and diluted in ethanol to 2.5 mCi/1.3 ml. The radiochemical purity was 75% as determined by HPLC on a Serva RP-18 reverse-phase column eluted by a water : methanol gradient. Di-(ethyl-23H) sulfate (3H-DES; specific activity 770 mCi/mmole) was obtained from New England Nuclear. For cell treatments, aliquots of 3H-ENU or 3H-DES (2.5 mCi) from a freshly opened vial were used. The mutagen concentrations were adjusted by the addition of unlabeled ENU or DES. Standards of the following ethyl derivatives were synthesized according to published methods with minor modifications: 3-ethylguanine (3EtGua), 7-ethylguanine (7-EtGua), O6-ethyldeoxyguanosine (O6-EtdGuo), O2-ethyldeoxythymidine (O2-EtdThd), O4-ethyldeoxythymidine ( 0 4EtdThd), 3-ethyladenine (3-EtAde), O2-ethylcytosine (O2-EtCyt), 3-ethyldeoxythymidine (3EtdThd) and 3-ethylcytosine (3-EtCyt) (for references, see Den Engelse et al., 1986). The ethyl ester of thymidilyl[3'-5']thymidine (dTp(Et)dY) was a kind gift of Dr. J.H. van Boom (Leiden, The Netherlands). Ouabain (Sigma) was dissolved in cell culture medium (see below) by 1 h incubation at 56 ° C. The medium was subsequently sterilized by filtration through a 0.2-/~m filter. The final concentration in the medium was 2 mM. 6-Thioguanine (Sigma) was dissolved in 0.1 M NaOH: the final concentration in the medium was 30 raM. Cell cultures CHO cells (CHO-K1; mycoplasma screened; Flow Labs, McLean, VA) were cultured in Ham's F10 medium (Flow Labs.) supplemented with 15% newborn bovine serum (NBS; Flow Labs), penicillin (100 U/ml) and streptomycin (100 #g/ml). Cultures were incubated at 37 °C in a controlled environment with 5% CO 2 and 95% relative humidity. Determination of DNA adducts by HPLC For each experimental point, CHO cells in log growth were concentrated by centrifugation to a

45 density of 4 x 107/1.8 ml of PBS, pH 7.4. Cell suspensions (4 × 107 cells/sample) were exposed to 3H-ENU (1, 1.5, 2, 4, or 6 mM) or 3H-DES (0.5, 1, 1.5, 2, 3, 4, 6 or 8 mM) in a total volume of 2 ml. After shaking for 30 rain at 37 ° C, the cells were washed by centrifugation and lysed. DNA was isolated by repeated phenol-chloroform extractions (Maniatis et al., 1982). Quantitation of ethylated products was performed as described by Beranek et al. (1980) and Den Engelse et al. (1986) with minor modifications. Briefly, the HPLC analysis was carried out on a Hewlett-Packard instrument, equipped with either a Partisil 10-SCX cation-exchange column (250 x 4.6 mm; Whatman, Clifton, N J) or a Kieselgel 60 RP-18 reverse-phase column (250 × 4 mm). The supernatant of the neutral hydrolysate was injected together with synthetic markers for 3-EtGua, 7-EtGua, 3-EtAde, 3-EtCyt and O2-EtCyt onto the cation-exchange column and eluted by a 30-rain linear gradient of 0.13-0.18 M ammonium formate in 12% methanol in water (adjusted to pH 4.0 with formic acid). The sediment of the neutral hydrolysate was hydrolysed enzymatically and injected together with the markers O4-EtdThd, O2-EtdThd, O6-EtdGuo, 3-EtdThd and dTp(Et)dT onto the RP-18 column. Elution was performed by a 2-step linear gradient of methanol in water (20-35% methanol in 20 min, then to 90% in 10 rain). Fractions (0.5 ml) were collected directly into scintillation vials, Lumagel (14 ml per fraction) was added and the radioactivity was determined in a Beckman liquid scintillation counter.

an expression time of 48-72 h for ouabain resistance (oua r) and 7-10 days for 6-thioguanine resistance (6-TGr). During these periods cells were subcultured when necessary to maintain exponential growth. After the expression period, cells were trypsinized and replated in 100-mm dishes with 10 ml of selection medium (see Chemicals) at a density of 106 or 105 cells per dish for oua r and 6-TG r, respectively. The selection medium was changed after 1 week. 10 days later the cells were fixed and stained. From each cell suspension 3 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 cloneforming cells.

Cytotoxicity and mutation assays

Analysis of ethylated DNA

Parallel to the experiments with labelled ethylating agents, the same CHO cell population was treated with unlabeUed mutagens under exactly the same conditions used with labeled compounds. CHO cells in log phase were exposed to ENU or DES as described in the previous paragraph. After treatment, cells were washed by centrifugation and resuspended in complete medium. A dilution of the cell suspension was plated to determine cell survival (100 cells in 60-mm dishes) and 7 days later the cultures were fixed with methanol and stained with 10% Giemsa. In order to detect mutations, samples of 2 x 106 cells were reseeded in 75-cm2 flasks and allowed to grow for

Table 1 shows the DNA ethylation pattern of CHO cells exposed for 30 min to either 3H-ENU or 3H-DES. The extents of all ethylated products are expressed relative to 7-EtGua (binding level of 7-EtGua = 1.0). The extent of the substitution at various DNA sites after ENU treatment is in good agreement with literature data (Den Engelse et al., 1986, for review). When the ethylation pattern after DES treatment of CHO cells was compared with data obtained in HeLa cells (Sun and Singer, 1975), a good agreement was observed in the case of O6-ethylguanine (O6-EtGua) and ethylphosphotriesters and a reasonable agreement in the case of 3-EtAde and 3-ethylthymine (3-EtThy). At

Statistical analysis In an attempt to determine which ethylation products were responsible for mutation induction, the amount of each individual adduct produced by both ENU and DES was compared to the number of induced mutations obtained on the same cell population. Several DNA adduct level and mutation frequency determinations at the same ENU and DES doses were paired and analyzed by linear regression method. The slopes of separate regression lines calculated on ENU and DES data were statistically compared using the t-test (Crow et al., 1960; Cooley and Lohnes, 1971; Armitage, 1971). Results

46 TABLE 1 E T H Y L A T I O N P R O F I L E S OF D N A OF CHO CELLS EXPOSED TO 3H-ENU A N D 3H-DES Site of D N A ethylation

ENU

DES

Gua 7

1.000

1.000

3 0 6

0.087_+0.001 a 0.6 _+0.1

ND c 0.028 _+0.005

3 Cyt

0.23_+0.08

0.105± 0.008

0 2

0.14+0.07

Ade < 0.001 b

Thy 3 0 2 0 4

< 0.001 0.31 +0.1 0.14+0.008

< 0.001 < 0.001 0.009+0.002

Phosphate dTpdT

0.20 + 0.04

0.024 + 0.002

The amounts of the ethylatlon products are expressed relatwe to the amount of 7-EtGua. a Mean + SD. b Values preceded by < were below the limits of detection. c N D = not determined.

equal levels of total DNA alkylation the amounts of O6-EtGua, O4-ethylthymine (O4-EtThy) and dTp(Et)dT were substantially higher after exposure to ENU than after alkylation with DES. Two adducts, O2-ethylthymine (O2-EtThy) and O2-EtCyt, were detected after ENU treatment but were below the detection limit in the case of DES. Radioactive profiles of DES-treated DNA revealed 2 unidentified peaks in the enzymatic hydrolysate which were just above the limit of detection (twice the background). In the samples derived from DES-treated cells, radioactivity coinciding with the marker peaks of 3-EtGua and 3-EtCyt could not be quantitated due to the overlap with the larger peak of 7-EtGua. For the same reason we were unable to quantitate 3-EtCyt in the case of ENU-treated DNA. Determinations of D N A adduct levels were performed in the dose range used for mutation assays. Dose response curves for 7-EtGua, 3EtAde, O6-EtGua, O4-EtThy and dTp(Et)dT are shown in Fig. 1. In the range of doses considered here a linear relationship was found to fit the

experimental data. ANOVA test applied to regression analysis showed no statistically significant deviations from linearity. Moreover regression analysis of log-transformed data confirmed this result showing slopes not significantly different from unity.

Mutagenicity and cytotoxicity Exposure to either ENU or DES resulted in a dose-dependent reduction in survival and dose-dependent increases in 6-TG r and oua r mutation frequencies (Fig. 2). At the same molar dose, DES was found to be more toxic than ENU, confirming the observation that agents with high s-values (s = Swain-Scott substrate constant) (DES: s = 0.64) are more effective in cell killing than agents with low s-values (ENU: s = 0.26) (Natarajan et al., 1984). With both compounds linear dose-response curves were obtained for the induction of 6-TG r and oua ~ mutants (ANOVA test and regression analysis of log-transformed data). In the range of data considered here no significant improvement of the fit was found using a linear quadratic model instead of a simple linear model, although a departure from linearity for high doses was suggested by the analysis of a wider dose range. As shown in Fig. 2, at equimolar concentrations ENU is a more powerful inducer of 6-TG r mutants than DES. In contrast, no such difference was observed between ENU and DES for the induction of oua r mutations.

Comparison of mutatton frequency wtth spectflc DNA ethylation products To analyze whether a particular DNA adduct might be responsible for induction of mutations at these 2 loci, mutation frequency data obtained after treatment with ENU and DES were compared with the amounts of each individual adduct formed during the same mutagen exposure. In the statistical approach used for this comparison, replicated frequencies of ENU- or DES-induced mutation and the corresponding levels of ethylated DNA adducts at each dose level were paired. Separate linear regression lines were calculated on ENU and DES data and the slopes of these 2 lines were compared to determine whether they differed significantly. Fig. 3 shows

47 180-

150-

7-

3- EtAde

EtGua 15-

100-

/

10-

5-

&

10

8-

4-

15-

dTp(Et)dr

O~,-Errhy

/

610-

4. 5-

2. I-

1-

r

2

i

i

4

i

6

i

i

i

8

r

2

i

i

i

4 6 8 ConcentraOon(raM)

0

i

i

2

i

J

4

6

8

Fig. 1. Frequency of several ethylated products in DNA of CHO cells as a function of the exposure concentration to E N U (O) and DES (A). Each point represents the mean of 3 independent observations. The solid lines represent the linear regression lines: 7-EtGua, ENU: y = ( 2 . 9 + 0 . 3 ) x + ( 1 . 6 + 0 . 8 ) , r = 0 . 9 5 ; 7-EtGua, DES: y = ( 1 8 . 9 + l . 9 ) x - ( 6 . 5 + 7 . 5 ) , r = 0 . 9 3 ; 3-EtAde, ENU: y = (0.7+0.1)x+(0.49+0.32), r = 0.87; 3oEtAde, DES: y = (2.1 +0.29)x+(0.92+ 1.13), r = 0.89; O6-EtGua, ENU: y = (1.6+0.21)x +(0.74+0.53), r =0.93; O6-EtGua, DES: y = (1.26+0.12)x-(1.08+0.47), r = 0.95; O4-EtThy, ENU: y = (0.4+0.04)x-(0.01 + 0.12), r = 0 . 9 7 ; O4-EtThy, DES: y=(0.25+0.018)x-(0.11+0.07), r = 0 . 9 8 ; dTp(Et)dT, ENU: y = ( 0 . 8 4 + 0 . 1 ) x - ( 0 . 1 7 + 0 . 2 8 ) , r = 0.96; dTp(Et)dT, DES: y = (0.80 + 0.06)x- (0.45 + 0.26), r = 0.96.

the relationship between mutation induction at the Na,K-ATPase (left side) and HPRT (right side) gene loci and O6-EtGua formation in CHO cells. Both the confidence intervals of the regression lines generated for the ENU- and DES-induced 6-TG r mutations (Fig. 3, right side) and statistical comparison of the regression coefficients indicated that the difference between the slopes of the regression lines is insignificant; therefore formation of O6-EtGua might be quantitatively correlated with mutations induced at the HPRT locus. When the same analysis was applied to oua r

mutation data, a significant difference (p < 0.01) between the slopes of the regression fines for ENU and DES was observed, i.e., at comparable amounts of O6-EtGua, DES induced many more oua r mutants than ENU. The analysis of the slopes for other ethylated products (7-EtGua, 3EtAde, O4-EtThy and phosphotriesters) is shown in Table 2. The difference between the slopes of the regression fines for ENU and DES indicates the absence of a relationship between DNA alkylation at the 7 position of guanine, the 3 position of adenine or the phosphates and induction of 6-TG r mutants. The formation of O4-EtThy,

48

.100

100 EN

DES

q%

I

10

'

50

"1

(o

•1

c:

C 0 r~

0.1 0

0 Concenfrafion (mM)

Fig. 2. Induction of 6-TG r (e) and oua' (A) mutaUons after a 30-mm exposure to E N U or DES. The solid lines represent the linear regression lines for the mutauon frequency for 6-TG r (ENU: y = (24.2 + 2 . 5 7 ) x - (12.4 + 6.4), r = 0.95; DES: y = (13.1 5: 1 . 3 ) x - (1.38 _+2.7), r = 0.94) and for oua r (ENU: y = (3.6_+ 0.52)x - (1.06 5: 1.21), r = 0.92; DES: y = (3.33 5: 0.62)x - (0.48 5:1.35), r = 0.84). The broken lines represent the survival data (zx), these hnes were fitted by eye. Each point represents the mean of 3 independent observations_+ standard deviations. The spontaneous mutation frequencies were 8 x 10-7 for oua r and 0.7-1 × 10-5 for 6-TG'.

however, appeared to be correlated to mutation induction at this locus. As regards oua r mutations, none of the ethyl

adducts revealed an association with mutations induced at the Na,K-ATPase gene locus although phosphotriesters were weakly correlated.

TABLE 2 C O M P A R I S O N OF THE SLOPES OF L I N E A R REGRESSION LINES OF M U T A T I O N I N D U C T I O N AS A F U N C T I O N OF E A C H I N D I V I D U A L ETHYLATION P R O D U C T Site of substitution 3-EtAde (slope 5: SD)

7-EtGua (slope 5: SD)

O4-EtThy (slope + SD)

dTp(Et)dT (slope + SD)

O6-EtGua (slope + SD)

1.90 + 0.27 0.63 _+0.22 0.01

0.69 + 0.17 0.08 5:0.02 0.01

5.93 + 1.96 4.44 + 1,37 n.s. b

3.78 5:1.12 0.82 + 0.32 0.01

0.56 + 0.15 0.82 + 0.24 n.s.

2.81 + 0.25 1.56 + 0.17 0.01

1.37 + 0.33 0.18 + 0.02 0.01

7.73 + 2.56 19.15 5:3.04 0.01

4.74 _ 1.61 3.59 5:0.51 n.s.

0.82 5:0.89 3.39 5:0.87 0.01

6-TG r

ENU DES p value a ( < ) OUQ

r

ENU DES p value ( < )

" The p value represents the statistical significance of linear regression slope differences. b Not significant.

49

20 t

-~

~'

15~

DES

~

c lO"

j/

J

~I0

)I

.''"'DES

"~" 5

//

0

1

•

2

3

4

o..~"

co

5

6

.'"%

0

I

NU

2

3

4

5

6

Q 6 E i'hylguanlne(E Chyla lions/ 10(nucleosldes)

Fig. 3. 95% confidence intervals for the regression lines. DES: broken line, open symbols; ENU: continuous line, closed symbols.

Discussion

Our results show that the extents to which O6-EtGua and O4-EtThy are being formed in ENU- and DES-treated CHO cells might correlate with the frequency of induced mutations at the HPRT locus. A positive correlation between alkylation at the 0 6 position of guanine in DNA and mutation induction by a series of alkylating agents has been shown in microorganisms and in rodent cells in culture (Heflich et al., 1982; Natarajan et al., 1984; Van Zeeland et al., 1985). Although these correlations are statistical, there are several indications that both O6-alkylguanine and O4-al kylthymine are miscoding and mutagenic in in vitro and in vivo systems (Gercham and Ludlum, 1973; Loechler et al., 1984; Saffhill et al., 1985; Singer et al., 1986). Similarly a role for O4-EtThy in the induction of mutational damage in bacteria and mammalian cells treated with ethylating agents has been proposed (Guttenplan and Hu, 1984; Mattern et al., 1985; Hu and Guttenplan, 1985; de Kok et al., 1985). The potentially miscoding lesions O2-EtCyt and O2-EtThy (Singer et al., 1979; Saffhill, 1985) might well contribute to ENU-induced mutagenicity, but a statistical analysis was impossible since these adducts could not be detected in the DNA of DES-treated CHO cells. In contrast with mutations at the HPRT locus,

no correlation was found between the frequencies of ENU- and DES-induced mutations in the Na,K-ATPase gene and amounts of any individual adduct, with the possible exception of ethylphosphotriesters. DES-induced mutations at the Na,K-ATPase locus occurred at higher frequencies than expected from the extents of O6-EtGua and O4-EtThy (when compared with ENU-induced mutations at the same locus). Studies with a wider range of ethylating agents might give more information on the lesions responsible for the appearance of oua r mutations. Furthermore, it is important to consider several limitations inherent in our approach. Primary DNA modifications not included in our HPLC analysis, such as 1-ethyladenine or 3-EtCyt, cannot be ruled out as causal lesions for mutagenicity of ethylating agents. More importantly, an assumption of the molecular dosimetry studies is that DNA damage is randomly distributed. On the contrary, there are several indications that alkylating agents might possess different affinities to react with particular parts of the genome or with relevant DNA sequences. For example, heterogeneity of O6-al kylguanine formation by alkylating agents in different sequences (Briscoe and Cotter, 1985; Milligan et al., 1986) or chromatin fractions (Faustman and Goodman, 1981; Nehls and Rajewsky, 1985) has been reported. Moreover, a possible explanation that might be envisaged for the apparent lack of correlation with O6-EtGua and O4-EtThy in the case of oua r mutations is related to the mechanism by which oua r mutations arise. Recently oua r has been demonstrated to be acquired by cultured cells either by amplification of the gene (Ash et al., 1984) or by induction of an ouabain-inducible K-transport system that results in increased levels of specific mRNA (English et al., 1985). The first possibility is strengthened by the observation that chemical carcinogens could indeed induce such amplification (Schimke, 1984). A phenomenon other than base-pair substitutions at one of a limited number of sites in the part of the gene containing the structural information for the Na,K-ATPase might therefore be responsible for the oua ~ phenotype. Considering the limited information, the molecular analysis of oua r mutants induced by different alkylating agents will be of obvious interest. This

50

would allow to determine whether the same or different genetic alterations are involved in the appearance of mutations at this locus.

Acknowledgements The authors are grateful to Ir. A.A.M. Hart for helping with statistical analysis and to L. Gargano, M.C. D'Ascoli, U. Cervelli and R.J. de Bry for expert technical help. This work has been supported by the European Economic Community (Contract No. 503 ENV I s) and by the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Medicina Preventiva (Contract No. 83.02880.56).

References Armitage, P. (1971) Statistical Methods in Medical Research. Blackwell Scientific Publications, Oxford. Ash, J.F., R.M. Fineman, T. Kalka, M. Morgan and B. Wire (1984) Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection, J. Cell Biol., 99, 971-983. Beranek, D.T., C.C. Weis and D.H. Swenson (1980) A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography, Carcinogenesis, 1,595-606. Beranek, D.T., R.H. Heflich, R.L. Kodell, S.M. Morns and D.A. Casciano (1983) Correlation between specific DNAmethylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells, Mutation Res., 110, 171-180. Briscoe, W.T., and L.E. Cotter (1985) DNA sequence has an effect on the extent and kinds of alkylation of DNA by a potent carcinogen, Chem.-Biol. Interact., 56, 321-331. Cooley, W., and Lohnes (1971) Multivariate Analysis, J. Wiley and Sons, New York. Crow, E.L., F.A. Davis and M.W. Maxfield (1960) Statistics Manual. Dover Press, New York. de Kok, A.J., A.A. van Zeeland, J.W.I.M. Simons and L. Den Engelse (1985) Genetic and molecular mechanisms of the in vitro transformation of Syrian hamster embryo cells by the carcinogen N-ethyl-N-nitrosourea, II. Correlation of morphological transformation, enhanced fibrinolytic activity, gene mutations, chromosomal alterations and lethality to specific carcinogen-induced DNA lesions, Carcinogenesis, 6, 1571-1576. Den Engelse, L., G.J. Menkveld, R.J. De Brij and A.D. Tates (1986) Formation and stability of alkylated pyrimidines and purines (including imidazole ring-opened 7-alkylguanine) and alkylphosphotriesters in liver DNA of adult rats treated with ethylnitrosourea or dimethylnitrosamine, Carcinogenesis, 7, 393-403. Dogliotti, E., A. Vitelli, M. Terlizzese, A. Dl Muccio, A. Calcagnile, U. Saffiotti and M. Bignami (1987) Induction

kinetics of mutations at two genetic loci, DNA damage and repair in CHO cells after different exposure t~mes to Nethyl-N-nitrosourea, Carcinogenesis, 8, 25-31. English, L.H., J. Epstein, L. Cantley, D. Housman and R. Levenson (1985) Expression of an ouabam resistance gene in transfected cells, J. Biol. Chem., 260, 1114-1119. Faustman, E.M., and J.l. Goodman (1981) Alkylation of DNA in specific hepatic fractions following exposure to methylnitrosourea or dimethylnitrosoamine, Tox. Appl. Pharmacol., 58, 379-388. Fox, M., and J. Brennand (1980) Evidence for the involvement of lesions other than O6-alkylguanine in mammalian cell mutagenesis, Carcinogenesis, 1,795-799. Gentil, A., A. Margot and A. Sarasin (1984) Apurlmc sites cause mutations in simian virus 40, Mutation Res., 129, 141-147. Gercham, L.L., and D.B. Ludlum (1973) The properties of O6-methylguamne in templates for RNA polymerase, Biochim. Biophys. Acta, 308, 310-316. Goth-Goldstein, R. (1980) Inability of Chinese hamster ovary cells to excise O6-alkylguanine, Cancer Res., 40, 2623-2624. Guttenplan, J.B., and Y.C. Hu (1984) Mutagenesis by N-nitroso compounds in Salmonella typhtmunum TA102 and TA104: evidence for premutagemc adenine or thymine DNA adducts, MutaUon Res., 141, 153-159. Hefhch, R.H., D.T. Beranek, R.L. Kodell and S.M. Morris (1982) Induction of mutations and slster-chromatid exchanges in Chinese hamster ovary cells by ethylating agents. Relationship to specific DNA adducts, Mutation Res., 106, 147-161. Hu, Y.C., and J.B. Guttenplan (1985) Evidence for a major premutagenic ethyldeoxythymidine-DNA adduct in an in vivo system: N-nitroso-N-ethyhirea-treated Salmonella typhtmurtum, Carcinogenesis, 6, 1513-1516. Lawley, P.D. (1976) Carcinogenesis by alkylating agents, Chemical carcinogens, Am. Chem. Soc. Monogr., 173, 83-244. Loeb, L. (1985) Apurinic sites as mutagemc intermediates, Cell, 40, 483-484. Loechler, E.L., C.L. Green and J.M. Essigmann (1984) In vivo mutagenesis by O6-methylguanine built into a unique site m a viral genome, Proc. Natl. Acad. Sci. (U.S.A.), 81, 6271-6275. Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mattern, I.E., F.P. Olthoff-Smit, B.L.M. Jacobs-Meljsing, B.E. Enger-Valk, P.H. Pouwels and P.H.M. Lohman (1985) A system to determine base-pair substitution at the molecular level, based on restrictxon enzyme analysis; influences of the muc genes of pKM101 on the specificity of mutation induction m E. coh, Mutation Res., 148, 35-45. Milhgan, J.R., L. Catz-Biro, S. Hirani-Hojatti and M.C. Archer (1986) Effect of DNA structure on methylation by methylnitrosourea and acetoxynitrosodimethylamine, Proc. Am. Ass. Cancer Res., 27, abstr. 349. Natarajan, A.T., J.W.I.M. Simons, E.W. Vogel and A.A. van Zeeland (1984) Relationship between cell killing, chro-

51 mosomal aberrations, sister-chromatid exchanges and point mutations induced by monofnnctional alkylating agents in Chinese hamster cells, Mutation Res., 128, 31-40. Nehls, P., and M.F. Rajewsky (1985) Differential formation of O6-ethylguanine in the DNA of rat brain chromatin fibers of different folding levels exposed to N-ethyl-N-nitrosourea in vitro, Cancer Res., 45, 1378-1383. Newbold, R.F., W. Warren, A.S.C. Medcalf and J. Amos (1980) Mutagenicity of carcinogenic methylating agents is associated with a specific DNA modification, Nature (London), 283, 596-599. Saffhill, R. (1985) In vitro miscoding of alkylthymines with DNA and RNA polymerases, Chem-Biol. Interact., 53, 121-130. Saffhill, R., G.P. Margison and P.J. O'Connor (1985) Mechanisms of carcinogenesis induced by alkylating agents, Biochim. Biophys. Acta, 823, 111-145. Schimke, R.T. (1984) Gene amplification, drug resistance and cancer, Cancer Res., 44, 1735-1742. Singer, B., R.G. Pergolizzi and D. Grunberger (1979) Synthesis and coding properties of dinucleoside diphosphates containing alkyl pyrimidines which are formed by the action of carcinogens on nucleic acids, Nucleic Acids Res., 6, 1709-1720.

Singer, B., S.J. Spengler, H. Fraenkel-Conrat and J.T. Kusmierek (1986) O4-Methyl, -ethyl, -isopropyl substituents on thymidine in poly(dA-dT) all lead to transition upon replication, Proc. Natl. Acad. Sci. (U.S.A.), 83, 28-32. Sun, L., and B. Singer (1975) The specificity of different classes of ethylating agents towards various sites of HeLa cell DNA in vitro and in vivo, Biochemistry, 14, 1795-1802. Suter, W., J. Brennand, S. McMillan and M. Fox (1980) Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 Chinese hamster cells, independently of cytotoxic and mutagenic responses, Mutation Res., 73, 171-181. Van Zeel~ind, A.A., G.R. Mohn, A. Neuhiiuser-Klaus and U.H. Ehling (1985) Quantitative comparison of genetic effects of ethylating agents on the basis of DNA adduct formation. Use of O6-ethylguanine as molecular dosimeter for extrapolation from cells in culture to the mouse, Environ. Hlth Perspect., 62, 163-169. Vogel, E., and A.T. Natarajan (1983) in: A. Hollaender (Ed.), Chemical mutagens: principles and methods for their detection, Plenum Press, New York, pp. 295-336.