DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis

Mutation Research, 231 (1990) 55-62

55

Elsewer MUT 02753

D N A adduct formation in mouse testis by ethylating agents" a comparison with germ-cell mutagenesis Albert A. van Zeeland 1, Anton de Groot 1 and Angelika Neuh~iuser-Klaus 2 1 Department of Ra&ation Genetics and Chemical Mutagenesis, Sylvius Laboratories, State Unwerstty of Leiden, Leiden (The Netherlands) and 2 Insntut fflr Siiugenergenenk, Gesellschaft fiir Strahlen- und Umweltforschung, Neuherberg (F R G.) (Received 23 October 1989) (Rewslon recewed 16 January 1990) (Accepted 17 January 1990)

Keywords DNA adducts; Ethylatmg agents; Germ-cell mutagenesls

Summary DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the 0 6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if or-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 or-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.

Most genotoxic chemicals cause more than one type of DNA adduct. However, this does not necessarily mean that all the various types of DNA adducts contribute to the mutagenic effects observed. Some of them might not be mutagenic at all. One approach to investigate the relative Correspondence: Dr. A.A van Zeeland, Department of RadlaUon Genetics and Chermcal Mutagenes~s, Sylxaus Laboratories, State Umverslty of Leiden, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands)

proportions of mutagenlc effects induced from various types of DNA adducts is to determine DNA adduct formation by related chemicals and to compare the frequency of their genetic effects on the basis of the frequency of DNA adduct formation. A correlation is expected to be found for those adducts which are important for the induction of genetic effects. Alkylating agents, and especially ethylating agents, have been used for this strategy because they all introduce ethyl groups into DNA, but the proportion of the vari-

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56 ous ethylated nucleotides IS very different depending on the type of ethylatlng agent (Singer, 1976; Swenson and Lawley, 1978; Beranek et al., 1980). When mutation reduction by ethylating agents m mammalian cells in culture is determined and compared with the frequency of various ethylation products in DNA, a strong relationship was found between the frequency of O6-ethylguanine and the frequency of gene mutations determined at the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene (Van Zeeland et al., 1985; Van Zeeland, 1988; Heflich et al., 1982; Mohn et al., 1984; Natarajan et al., 1984). Sirmlar observations were made m E coh when resistance to nalidtxic acid was determined (Mohn et al., 1984; Van Zeeland et al., 1985) and m Drosophila when induction of sex-linked recessive lethal mutations was compared with the potency of alkylating chemicals to react with O atoms in D N A (Vogel and Natarajan, 1979). However, all these observations were made under conditxons where D N A excision-repair mechanisms were functional. Under conditions where excision repair is deficient a strict correlation between the induction of gene mutations and the frequency of O6-alkylguanine does not exast any more, 1.e., when alkylatmg chemacals are used that have a preference for alkylatlng N atoms in DNA, more gene mutations are introduced than can be expected on the basis of the frequency of O6-alkylguanine in D N A (Vogel et al., 1986; Zdzlenicka and Simons, 1986). This paper describes measurements on D N A adduct formation by ethylating agents m D N A from mouse germ cells and in D N A from some other organs of the mouse. When these data are compared with the capacity of the various ethylatmg agents to reduce gene mutatmns in mature sperm of male mice, m general more mutations are obtained when chemicals are used that have a preference for reacting with N atoms m DNA. This ~s in line with the notion that late stages in the development of male mouse germ cells are excision repair-deficient (Sega, 1974). Materials and methods

[2-3H]Ethylnitrosourea (ENU) and [1-3H]di ethyl sulfate (DES) were obtained from New England Nuclear (Boston), whereas [1-3H]ethyl -

methane sulfonate (EMS) was obtained from A m e r s h a m (U.K.). Stock solutions were prepared as described previously (Van Zeeland et al., 1985). E N U stock solutions were 50 m M in phosphate buffer, p H 6. EMS stock solutions were 100 m M m phosphate-buffered saline (PBS, p H 7.2). DES stock solutions were 60 m M in 40% dimethyl sulfoxide (DMSO) in PBS. All stock solutions were prepared immediately before use. Male mice ( 1 0 2 / E l × C 3 H / E 1 ) F 1 were injected 1.p. with E N U , EMS or DES. 2 h after injection the liver, bone marrow and the testis were removed. After removal of the tumca albuginea, testis tubuli were transferred to PBS. G e r m cells were then released from tubuh by pushing out the contents with curved forceps. Using this procedure all germ-cell stages were liberated from the tubuli with the possible exception of part of the population of stem-cell sperm a t o g o m a that could remain attached to the walls of the tubuli. D N A was extracted from germ cells, empty testis tubuli, liver and bone marrow. D N A adduct formation was determaned after neutral and acid hydrolysis of D N A and separation of the vanous ethylation products using a high-performance liquid chromatography system as described previously (Beranek et al., 1980; Van Zeeland et al., 1985). Results and discussion

D N A adduct formation by ENU, EMS and DES was determmed 2 h after i.p. injection. With all 3 compounds the frequency of 7-ethylguanine and O6-ethylguanlne was determined at vanous exposure concentrations. In a number of cases other ethylation products were also measured. The results obtained with E N U (Table 1) show that D N A adduct formation in the hver is considerably higher than in bone marrow, germ cells and testis tubuh. The ratio of O6-ethylguanlne to 7-ethylguanine is relatively low at low exposure concentratlons which could be explained by the presence of methyltransferase activity, which besides methyl groups can also remove ethyl groups from the 0 6 position of guamne. At high exposures the ratio between O6-ethylguanine and 7-ethylguamne was quite simtlar in all organs studied, although the absolute frequencies of D N A adducts m the

57 TABLE 1 INDUCTION OF DNA ADDUCTS IN DIFFERENT ORGANS OF MICE BY ENU Organ

DNA adduct

Ethylatlon per 10 6 nucleotldes at various ENU exposures (mg/kg) 10

20

40

7-ethylguamne O6-ethylguamne 3-ethylademne O2-ethylcytosme 3-ethylguamne Ratio O6/7-EG

0.28 0.10 0.02 0 36

0 60 0 20 0 08 0.08 0 09 0 33

1 29 0.75 0 18 0.16 0.15 0 58

Tesus tubuh

7-ethylguamne O6-ethylguamne 3-ethylademne O2-ethylcytosme 3-ethylguanlne Ratio O6/7-EG

0 30 0.05 0.17

0.84 0 16 0 08 0 07 0.07 0 19

Bone marrow

7-ethylguamne O6-ethylguamne 3-ethylademne O2-ethylcytosme 3-ethylguanme RaUo O6/7-EG

0.45 0 09 0 08 0 20

Liver

7-ethylguanlne O6-ethylguamne 3-ethylademne O2-ethylcytoslne 3-ethylguanme OZ-ethylthyrmdme 3-ethylthyrmdme O4-ethylthynudme Ethyl T-p-T RaUo O6/7-EG

0 57 0.08 0 15 0.12 0.06 0.28 0 02 0 04 0.19 0 14

Germ cells

80 2 06 0.91

160

250

10 0 44

3.74 2 50 0 45 0 57 0 67 0 67

10.58 4 47 1 06 11 1 66 0 42

1.88 0.53 0 19 0 15 0 14 0.28

1 92 (0.4) 1.01 (0 13) 0.42 (0.15) 0 33 0 23 0.52

5.22 (1 18) 2.67 (0.44) 0 89 (0.44) 0 68 (0.07) 0.52 0 51

10 05 (2.76) 5 36 (0.53) 1 94 (0.83) 1.23 (0 01) 1.26 0 53

1 01 0.29 0.2 0.05 0 29

1 52 0 65 0.33 0.18 0.06 0 42

3 14 (0 14) 2 54 (0.28) 0 97 (0.1) 0 81 (0.39) 0 81

6 39 (1.03) 4 63 (0 23) 1 96 (0 2) 1.34 (0.33) 0 54 0 72

13 58 (2 6) 6.96 (0 01) 2.98 (0 22) 1 57 (0.01) 0 87 0 51

2 98 04 0 54 0.36 0.16 10 0.08 0.25 0.70 0 13

6.22 1.34 1 15 0.79 0 38 6 26 0.83 6.03 0.22

13 15 (3 49) 4.9 (1 59) 2 41 (0 72) 2 1 (0.29) 0 27

29.2 (6 37) 13 3 (3.03) 6 25 (2 31) 4.09 (1 2) 3 19 10 01 2 05 1 17 6.79 0.46

52 12 (8.71) 22 62 (2 79) 10.26 (3.04) 6.53 (1.26) 6 46 19 25 (1 85) 2 34 1 98 11 70 0 43

DNA was isolated 2 h after 1p m lecUon vmh [3H]ENU (speofic acUvlty was 20, 46, 39 or 360 mC1/mmole), nd, not detectable The numbers in parentheses are standard dewauons

l i v e r w e r e m u c h h i g h e r (Fig. 1). T h i s o b s e r v a t i o n suggests t h a t the h i g h level o f D N A a l k y l a t i o n in t h e liver Is d u e to m o r e e f f i c i e n t t r a n s p o r t a t i o n o f E N U t h r o u g h t h e b l o o d s t r e a m to this o r g a n a n d ~s n o t c a u s e d b y fast r e m o v a l o f a d d u c t s m o t h e r organs. Similar observauons were made with EMS a n d D E S ( T a b l e s 2 a n d 3). A l s o w i t h t h e s e c h e m icals t h e f r e q u e n c y o f D N A a d d u c t f o r m a t i o n in t h e liver was s u b s t a n t i a l l y h i g h e r t h a n in b o n e m a r r o w , g e r m cells a n d in testis tubuli. F o r all 3 c h e m i c a l s t h e f r e q u e n c y of a d d u c t f o r m a t i o n in D N A f r o m g e r m cells a n d in D N A f r o m e m p t y t u b u l i was v e r y similar, s u g g e s t i n g t h a t t h e r e is a n efficient transport of the chenucals through the t u b u h walls.

S i n c e t h e a l k y l a t i o n levels in t e s t i c u l a r D N A are relevant m studies where DNA adduct format i o n is c o m p a r e d w i t h t h e l e v e l o f m u t a t i o n i n d u c t i o n in o f f s p r i n g o f m i c e , a r a n k i n g o f t h e c h e m icals was m a d e w i t h r e s p e c t to t h e i r c a p a c i t y to p r o d u c e O 6 - e t h y l g u a n i n e o r 7 - e t h y l g u a m n e in test i c u l a r D N A . T h i s c o m p a r i s o n w a s m a d e at a r a n g e of e x p o s u r e levels at w h t c h d a t a a r e also a v a i l a b l e o n t h e i n d u c t i o n o f g e n e t i c e f f e c t s as d e t e r m i n e d in t h e s p e c i f i c l o c u s a s s a y ( E h l i n g , 1978). Fig. 2 s h o w s t h e r a n k i n g w i t h r e s p e c t to the c a p a c i t y o f t h e c o m p o u n d s to i n d u c e O 6 - e t h y l g u a n i n e in t e s t i c u l a r D N A . E N U is b y far the most potent followed by EMS and DES. The r a n k i n g w i t h r e s p e c t to t h e f o r m a t i o n o f 7 - e t h y l -

58 TABLE 2 I N D U C T I O N OF D N A A D D U C T S IN D I F F E R E N T O R G A N S OF MICE BY EMS Organ

D N A adduct

Ethylatlon per 106 nucleolades at vanous EMS exposures ( m g / k g )

Germ cells

7-ethylguamne O6-ethylguamne 3-ethylademne 3-ethylguanlne

Tesus tubuh

7-ethylguamne O6-ethylguamne 3-ethyladenme 3-ethylguamne

-

Bone marrow

7-ethylguanme O6-ethylguanlne 3-ethyladenme 3-ethylguamne

nd nd nd

Liver

7-ethylguamne O6-ethylguamne 3-ethylademne 3-ethylguamne

42.7 1.88 1.65 nd

50

100

175

4 04 nd nd nd

11 0 0 0

21 0 0 0

29 38 38 24

250

77 59 72 43

41.08 0 83 1 30 0 43

13 0 0 35 0 42 0.13

21 84 0 30 0 68 0.26

38 52 0 71 1 30 0 70

12.54 0 25 0.68 nd

0.56 1 60 nd

32 96 0 96 2 42 0 31

98 75 3.02 2 01

104 74 5 77 1.26 -

-

D N A was isolated 2 h after i p injection with [3H]EMS (285 m C t / m m o l e ) nd, not detectable.

guanine (Fig. 3) shows that EMS is the most potent followed by DES and ENU. It has been shown that the 3 chemicals behave very differently with respect to the reduction of gene mutations when assayed in the specific-locus assay, using the

same strain of mice as in our study. E N U is a strong inducer of gene mutations in spermatogonia, whereas the induction of gene mutations m post-spermatogomal stages is much lower and the mutations are primarily mosaics (Ehling and

TABLE 3 I N D U C T I O N OF D N A A D D U C T S IN D I F F E R E N T O R G A N S OF MICE BY DES Organ

D N A adduct

Ethylauon per 106 nucleoudes at various DES exposures (mg/kg) 28 1

48.3

56 1

96.6

112 3

193 2

7.6

2 25 0 078 nd

90

4 25 0 189 nd

21 3

4.4

1.85 nd nd

7.2

3 96 nd nd

18.6

Germ cells

7-ethylguamne O6-ethylguamne 3-ethylademne

Testis tubuh

7-ethylguamne O6-ethylguanme 3-ethylademne

0 75 nd nd

Bone marrow

7-ethylguanme O6-ethylguanlne 3-ethylademne

0 123 0 286

nd 0.202

7-ethylguamne O6-ethylguamne

2.11 0 131

18.39 0.65

Liver

2 82

D N A was isolated 2 h after Lp. rejection with DES (34 or 330 mCl/mmole), nd, not detectable

11.84 0 85

59

7-Efhylguanine

60

40

f/) ¢1

A

in Tesficular

DNA

{

•

-0

~z 40

.+-

0

¢1

20

Z 0 0 CL

25

O~

r-

20.

0

_~B 50

.

"0

15.

¢-

•*-

100

.

.

.

~,

ENU

A

EMS

a

DES

2o 200

250

o

10

"6 7: ~

o 0

.

.

.

50

100

150

200

250

E x p o s u r e C o n c e n t r a t i o n (rnl~)

Fig 3 E x p o s u r e dose r e l a t i o n s h i p in the m o u s e F r e q u e n c y of 7 - e t h y l g u a n m e in testlcular D N A as a function of the e x p o s u r e c o n c e n t r a t i o n of E N U , E M S a n d D E S D a t a from g e r m cells a n d tesus t u b u h are pooled.

10'

I.M

o

L_

150

.

30

O= 50

100

150

200

250

ENU Exposure concentration (mg/kg) Fig 1. Exposure dose relationship m the mouse Frequency of ethylatxonproducts (A, 7-ethylguamne, B, O6-ethylguanme)m vinous organs of the mouse as a function of the ENU exposure concentrauon.(zx)germ cells, (v) testis tubuh, (El)bone marrow, (A) hver Neuh~iuser-Klaus, 1984; Van Zeeland, 1988; Favor et al., 1990a). EMS induces very few mutations and DES induces no mutations in spermatogonia. However, both are active in mature sperm (Ehling and Neuh~iuser-Klaus, 1988; Van Zeeland, 1988).

O~-Efhylguanine "u

in Tesficular

DNA

6

o -~ 5. o

z

ENU EMS

o

o_ o

2

[]

DES

1

.~ Ow

0

50

100

150

200

250

E x p o s u r e C o n c e n t r a t i o n (rnM)

Fig. 2. E x p o s u r e dose relaUonshtp m the mouse. F r e q u e n c y of O 6 - e t h y l g u a m n e m testicular D N A as a function of the exp o s u r e c o n c e n t r a t i o n of E N U , E M S a n d D E S D a t a from g e r m cells a n d testis tubula are pooled.

It has been found in cultured mammalian cells (Hefhch et al., 1982; Van Zeeland et al., 1985), in Drosophila (Vogel et al., 1986), and m E cob (Van Zeeland et al., 1985) that at equal frequencies of O6-ethylguanine, mutation induction by these ethylating agents is sirmlar, suggesting that O6-ethylguanine is the major mutagenic lesion introduced. This observation might also hold for mutations in mouse spermatogonia, where ENU induces much more O6-ethylguamne than EMS and DES and is also much more mutagenic. However, m post-meiotic stages of germ cells in the mouse the ranking of mutagenic potency changes dramatically. EMS and DES are more mutagenic than ENU, which means that the ranking of mutagenic potency in post-meiotic stages seems to follow the ranking with respect to the capacity to induce 7-ethylguanine rather than O6-ethyl guanine. A possible explanation of these observations could be differences in the capacity of the various cell types for D N A excision repair (Sega, 1974; Sega et al., 1976). This is a repair process which, among other lesions, is able to remove apunnic sites from DNA. These apurimc sites will be generated in ethylated D N A because of the chemical instability of N-alkylated purines and because of the presence in some cell types of specific D N A glycosylases which can remove Nalkylated bases from DNA, thereby generating abasic sites. It is known that apurimc sites are mutagenic and that D N A polymerases prefer-

60

entlally insert an adenine opposite an apurinic site (Schaaper et al., 1983). Under excision repair-proficient conditions these apurinic sites are removed and are therefore present only for short periods of ume and at low frequencies. Under excision repair-deficient conditions apurinic sites will accumulate with time and cause sigmficant mutagemc effects in addition to the effects caused by O-alkylations. Tins model is supported by observations in post-meiotic cells of Drosophila (Vogel et al., 1985) and in cultured mammalian cells (Zdzienicka and Slmons, 1986), where it has been shown that mutation induction by alkylating agents, which preferentially alkylate N atoms in DNA, is much higher in excision repair-deficient strains than in excision repair-proficient strains. In contrast, alkylating agents which induce high levels of O-aikylations, such as ENU, are hardly more mutagenic in excision repair-deficient condinons when compared to excision repair-proficient conditions. If the compound-specific spectra obtained for the mouse are caused by similar mechanisms in m a m m a h a n cells in culture and in Drosophila, the following model to explain the total data set emerges. In rephcating cells for all monofunctional alkylatlng agents O6-alkylguanine is the major mutagenic lesion. N-alkylation products become significantly mutagenic under condlnons where excision repair is deficient o r / a n d intact repair is saturated. Hypermutability in excision repair-deficient conditions of alkylating agents is greater for alkylating agents with a strong preference for N atoms in DNA. The lesions responsible for this hypermutabihty effect might be a p u n m c sites caused by chemical instability of N-alkylated purlnes m DNA. These apurimc sites are not removed from D N A when excision repair ~s deficient. Stem-cell spermatogoma are mitotically dividing cells, in which an alkylatlng agent with a preference for high levels of O6-al kylguanine ~s expected to be mutagenic (ENU). Post-spermatogomal cells, however, do not rephcite D N A until after fertihzatIon. This means that mutation fixation is also delayed until after fertilization. In mature sperm, which are excision repair-deficient (Sega, 1974), apurlmc sites generated due to instability of N-alkylated purmes can accumulate with time to relatively high levels and become mutagenlc after fertilization. Ttus model

is consistent with the observation that alkylating agents that preferentially alkylate N atoms m D N A (EMS, DES, MMS) are especially more mutagenic in mature sperm of the mouse. The peak of the mutagenic effect of these chemicals is observed about 1 week after treatment and not immediately. This is in line with the hypothesis that a certain amount of time is necessary in order to accumulate significant amounts of apurlmc sites. Sega et al. (1974, 1978) noticed that the induction of dominant lethal mutations by EMS did not correlate with D N A alkylatlons in sperm D N A but did correlate very well with the alkylation level of protanune, since both dominant lethals and protamlne alkylations increased with time until a m a x i m u m was reached about 10 days after treatment. These investigators therefore proposed a causal relationship between dominant lethal induction and protamine alkylatlons. An alternative explanation could be that not protamine alkylations or D N A alkylations as such are responsible for induction of dominant lethal mutations, but that apunnic sites which also accumulate with time are the responsible D N A lesions. The consequence of this model is that the molecular nature of the mutants induced in spermatogonia by E N U is expected to be different from those induced in mature sperm. Mutations observed In spermatogonia are expected to be pnmarily G C to A T transitions (mispairlng of O6-alkylguanxne) or A T to G C transitions (mispairing of O4-alkylthymidlne). Since in opposite a p u n m c sites an A usually is inserted, the mutations observed in the mouse about 1 week after treatment are expected to be primarily AT to TA or G C to T A transversions depending whether the a p u n m c sites accumulate at A or at G positions. Spectra of mutations induced by the alkylating agents E N U , EMS and DES in Drosophila have been determined in wild-type and in repair-deftcient condiuons and are consistent w~th this model (Nivard et al., 1989; Pastink et al., 1989, Sierra et al., 1989). The shape of the exposure dose relationship for the induction of O6-ethylguanine in testicular D N A by E N U (Fig. 2) appears to be non-hnear. At low exposure levels the frequency of O6-ethyl guamne is lower than expected on the basis of linearity, although measurable levels of O6-ethyl -

61 100 x

80.

[~

60' 40' u

3 E o

= --

20' 0 0

2 0a-efhylguclnine

4 per

6

10 s n u c l e o f i d e s

Fig. 4. Dose mutatton re]aUonshap for ENU-lnduced mutations m the specihc-locus assay of the mouse The induced mutation frequency determined by Favor et al. (1990b) is plotted against

the frequencyof O6-ethylguamnem germ cells

guanine are present (Table 1). This observation is in hne with the shape of the exposure-response curve for the mduction of specific-locus mutations in spermatogonia (Favor et al., 1990b). At low exposure levels mutation induction is lower than expected on the basis of linearity. A plot of the dose determined as O6-ethylguanine per nucleotide in D N A of germ cells versus induction of specific-locus mutations as determined by Favor et al. (1990b) is given m Fig. 4. In contrast to the dose exposure relationship (Fig. 2), which is nonlinear, the dose to mutation relationship appears to be linear, which is expected if O6-ethylguanine is the major lesion responsible for mutation induction by ENU. The small threshold which might be present in Fig. 4 could be caused by the fact that O6-ethylguanine frequencies were determined 2 h after i.p. injection. At the time of mutation fixation, the frequency of O6-ethylguanine in spermatogonia might be lower, due to removal of part of this lesion by O6-alkylguanine transferase. This repair effect is expected to be more pronounced after exposure to low amounts of ENU. We have previously shown that mutation induction at the hprt locus in cultured Chinese hamster cells by various ethylating agents is the same when compared at equal frequencies of 0 6ethylguanine (Van Zeeland et al., 1985). This means that one could calculate how many O6-eth ylguanine residues on average are required in a gene to induce a mutation. We assume that most mutations induced by alkylating agents are single base-pair changes in the coding region of the gene

involved. We further assume that the number of base pairs in the coding region is C, and that after an exposure E the frequency of O6-ethylguanine is D ethylations per nucleotide. If it is assumed that the frequency of mutations after exposure E is M, the number of O6-ethylguanine residues in the coding region after exposure E could be calculated using the formula 2C × D. The number of O6-ethylguanine residues in the coding region of a gene which are necessary to induce one mutation would then be: N = (2C × D)/M. In the case of the hprt gene C = 654 base pairs and when D = 2 X 1 0 - 6 ethylations per nucleoude, M = 2.4 )< 1 0 - 4 mutations per surviving cell (Van Zeeland et al., 1985). This results in a value of N = 10.9 for the hprt gene, i.e., in this gene an average of 10.9 O6-ethylguamne residues are necessary in the coding region to produce one mutauon. However, one has to keep in mind that the V79 Chinese hamster cells used for these studies do not contain O6-al kyltransferase activity, which means that O6-ethyl guanine is a stable D N A adduct that persists at least until the first round of D N A replicaUon. A similar calculation could be made for mutauon induction by ENU in spermatogonia of the mouse. However, the average size of the coding regions of the 7 genes used m the specific -locus assay is not known. If one assumes an average size of 1000 base pairs the value of N would be 13.2 which is very close to the number found in hamster cells. At very low exposure levels of ENU m the mouse, where part of the O6-ethylguanine adducts maght be removed by D N A repair, a higher initial level of O6-ethylguanine is probably necessary to induce a mutation. Acknowledgements

This work was supported by the Dutch Cancer Society (Project IKW 85-64) and the Commission of the European Communities (Contract EV4V0047-NL(GDF)). References

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