The in vivo genetic activity profile of the monofunctional nitrogen mustard 2-chloroethylamine differs drastically from its bifunctional counterpart mechlorethamine
John P.H. Wijen,
Madeleine J.M. Nivard and
Ekkehart W. Vogel1
Department of Radiation Genetics and Chemical Mutagenesis, MGC, Sylvius Laboratories, Leiden University Medical Centre, Wassenaarseweg 72, 2300 RA Leiden, The Netherlands
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Abstract
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The property of forming crosslinks within DNA is seen as the major cause of the high carcinogenic, genotoxic and anti-neoplastic potency of bifunctional nitrogen mustards. To further investigate the importance for genotoxicity of a second reactive group in a molecule, the genetic activity profiles of the bifunctional nitrogen mustard mechlorethamine (MEC) and its monofunctional counterpart 2-chloroethylamine (CEA) were compared, using several in vivo end points in Drosophila. When post-meiotic male germ cells were alkylated by CEA and then transferred to nucleotide excision repair (NER)-proficient oocytes, no more than up to 4-fold increased forward mutation frequencies were induced. With oocytes deficient for XPG (DmXPG), frequencies were enhanced up to 50 times. For MEC mutation frequencies increased up to 40 times the background, whereas only a low hypermutability was observed when DmXPG were used instead of wild-type females, indicating that nitrogen mustard-induced monoadducts, in contrast to crosslinks, are efficiently repaired by the NER system. Specific locus mutations generated in the vermilion gene by CEA under NER conditions were almost exclusively base pair substitutions (93%). The high proportion of mutations at guanine positions indicates a strong contribution of N7-alkylguanine to the mutational spectrum. MEC induced 64% deletions and other DNA rearrangements in crosses of males with DmXPG females. The small portion of point mutations (36%) was further reduced to ~20% with NER+ females. Inactivation of NER had no potentiating effect on clastogenic events (chromosome loss) induced by CEA, which is in sharp contrast to the strongly enhanced forward mutation frequencies measured with DmXPG females. The weak genotoxic effectiveness of CEA under NER+ conditions is clearly due to efficient error-free repair of monoalkyl adducts. These results further support the concept that bifunctional nitrogen mustards exert their mutagenic activity through formation of DNA crosslinks and that DNA monoadducts make only a minor contribution to their genotoxic activity.
Abbreviations: BCNU, bis-chloroethyl nitrosourea; CAB, chlorambucil; CEA, 2-chloroethylamine; CL, chromosome loss; HMPA, hexamethyl- phosphoramide; ILD, intra-locus deletion; MEC, mechlorethamine; MEL, melphalan; MLD, multi-locus deletion; NER, nucleotide excision repair; RL, recessive lethal mutation; SCE, sister chromatid exchange; SLT, specific locus test.
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Introduction
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The ability to form intra- and interstrand crosslinks with DNA places bifunctional agents in a separate group among alkylating agents. The carcinogenic hazards associated with exposure to these agents are evident from their low TD50 values in rodents (1), their known carcinogenicity in humans (IARC) and their high ranking in the `Agent Score System'. In this analysis 234 chemicals were ranked on the basis of their genotoxic potency in at least two in vivo and three in vitro genotoxicity tests (2,3). A mechanistic evaluation revealed that among the 20 most potent genotoxic agents 12 were crosslinking alkylating agents (4).
Crosslinking agents of the nitrogen mustard type are among the oldest anti-neoplastic drugs used in chemotherapy and they are still in clinical use (5,6). By blocking DNA replication and by their clastogenic activity they are cytotoxic to cancer cells. There is much concern about their ability to cause secondary tumours in cancer patients treated with these covalent DNA-binding drugs (7). However, in view of the lack of better alternatives, their application against a terminal disease is still unavoidable.
Their ability to crosslink DNA is responsible for the high carcinogenic (reviewed in ref. 8,9) and genotoxic (reviewed in ref. 4) potency of bifunctional agents. This became obvious from a comparison of the carcinogenic and genotoxic properties of bifunctional agents with chemicals having only one active group (monofunctional). The carcinogenic potency of crosslinking agents in rodents, estimated as TD50 lifetime doses in mg/kg or mmol/kg body wt, is 10- to 1000-fold higher than their monofunctional analogues (10). The high significance of DNADNA crosslinkage for all biological end points is further substantiated by direct correlations between the carcinogenic potency of 17 crosslinking agents in rodents and their mutagenic and clastogenic effectiveness in Drosophila (10). In both the mouse and in Drosophila, crosslinking agents induce mutations at practically any stage of spermatogenesis, including the highly repair active stem cell spermatogonia. Such uniform correlations were not found for monofunctional agents. Furthermore, crosslinking agents are 143686 times more effective at inducing sister chromatid exchanges (SCE) compared with their monofunctional counterparts. This difference could not be explained by alkylation capacities with cellular DNA (1113). Thus there is sufficient evidence that addition of a second active group to a molecule increases its potency for adverse effects (tumours and genetic damage) by up to three orders of magnitude.
There are several mechanisms by which crosslinking agents may cause loss of heterozygosity, generating hereditable genetic diseases and/or cancers. The majority of trans- generational damage induced by bifunctional agents in post-meiotic germ cell stages are deletions: 8097% in Drosophila (4) and 6087% in the mouse (14,15). The 6087% for the mouse refers only to multi-locus deletions (MLD), but the remaining, unspecified 1340% of intragenic changes could also include deletions. Thus, the total percentage of deletions in the mouse could be as high as in Drosophila. These high percentages are explicitly found for crosslinking agents but do not occur after exposure to monofunctional agents. The formation of a deletion requires DNA strand breakage and is therefore a clastogenic event. An informative parameter to express the effectiveness of a carcinogen for clastogenic damage is the comparison of breakage-related chromosome loss (CL) with forward mutation induction in Drosophila, i.e. estimation of its relative clastogenic efficiency ICL/RL (16). Bifunctional agents all have ratios above two, indicating a high clastogenic potency. In addition to their potential for induction of deletions and other rearrangements, crosslinking agents are among the most efficient inducers of mitotic recombination (17).
To further deepen our insight into the mutagenic mechanism of crosslinking agents, the genotoxic efficiency of the bifunctional nitrogen mustard mechlorethamine (MEC) in germ cells and somatic tissue of Drosophila was systematically compared with that of its monofunctional analogue 2-chloroethylamine (CEA) (see Figure 1
). We analysed their molecular mutation spectra in the eye color gene vermilion, their response in a DNA repair assay and their relative clastogenic potency. Such an in vivo study has not been conducted before, because earlier reports in which a crosslinking mustard was compared with its half-mustard were restricted to the use of in vitro systems (13,18,19).

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Fig. 1. Chemical structures of the bifunctional nitrogen mustard MEC and the monofunctional analogue CEA.
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Materials and methods
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Chemicals and treatment of flies
CEA (CAS no. 870-24-6) was purchased from Fluka-Chemie (Buchs, Switzerland). MEC (CAS no. 51-75-2) was obtained from Aldrich-Chemie (Steinheim, Germany).
For oral treatment with CEA, adult males were fed CEA dissolved in 5% sucrose in phosphate buffer, pH 6.8. Eight layers of glass microfibre paper (Whatman GF/A) in a tube were soaked with 0.9 ml of this solution. Male flies were treated for 24 h. Treatment with MEC was performed by abdominal injection of 0.2 µl mutagen solution containing 0.7% NaCl. Injected males were allowed to recover for 1 day.
MNER/MNER+ (mutability index)
In Drosphila, repair of pre-mutagenic lesion takes place in early male (paternal) germ cells, whereas spermatozoa and spermatids have lost the capability of repair (20,21). After fertilization, repair of DNA damage occurs in the oocyte, which is termed the maternal repair system. This is similar to the situation in the mouse (reviewed in ref. 22). These differences in the repair status of male and female germ cells form the basis for an efficient in vivo assay of the effect of repair on forward mutation induction. The procedure is to alkylate DNA in repair-inactive spermatozoa and spermatids, by treating adult males, and to transfer them to repair-competent oocytes or to oocytes with a deficiency for nucleotide excision repair (NER). This technique has two rather unique features. First, repair enzymes and other macromolecules in the oocyte, the cell where repair takes place after fertilization, remain unexposed by this technique. Second, the method enables quantitative comparisons with respect to the efficiency of repair, even in the absence of information on target tissue dose, because basically one and the same male germ cell population is mutagenized and split into two sub-groups only after treatment. The frequency of forward mutations induced in a NER background (MNER) (cross NER
x`exposed' NER+
) is divided by the mutation frequency fixed in the fertilized egg in the repair-proficient condition (MNER+) (cross NER+
x`exposed' NER+
), providing the MNER/MNER+ ratio. Forward mutations are measured as recessive lethal mutations (RL) in 700 loci of the X chromosome (23). Since repair status is the only variable condition in this system, an increased mutation yield with NER oocytes must be due to the repair deficiency. This system turned out to be a powerful instrument for the systematic comparison of groups of structurally related carcinogens (4).
For the test, mutagenized Hikone R males were individually mated with either [In(l) scSlLsc8R In(l) dl-49, y scSlsc8v; bw (=Inscy v; bw)] females (NER+) or females of genotype Inscy v; mus201D1, bw (NER) at a ratio of 1:4 virgin females (see ref. 24 for a description of the genetic markers). Two days later the males were mated with new virgin females. The mating period of the second brood was 3 days. The germ cells sampled by this technique were in the spermatozoa (brood 1) or spermatid stages (brood 2) at the time of the treatment. For further details see Vogel (25).
Relative clastogenicity index (ICL/RL)
Estimation of the relative clastogenicity index (16,26) was done by dividing the frequency of ring X chromosome loss (a measurement for DNA breakage events and SCEs; 2729) by the RL frequency (forward mutation test). The ring X chromosome loss test was performed by mating mutagenized ring X males [R1 (2) yB/BS Y y+] to y w spl sn3 females at a ratio of 1:4. Induction of ring X chromosome loss was also measured with repair-deficient y w spl sn3; mus201D1 females. Thus, CL/RL ratios were estimated with both the NER and wild-type female genotypes.
Specific locus test (SLT)
Treated male flies (Hikone R) were mated in mass cultures with virgin females proficient (Inscy v; bw) or deficient (Inscy v; mus201D1, bw or Inscy v; mus201D1) in NER, at a sex ratio of 1:1 (brood 1A). After 2 days the females were placed in new bottles (brood 1B) and the males were given new virgin females (brood 2A). Three days later the males were removed and the females were allowed to lay eggs for 2 more days (brood 2B). In this way offspring from treated spermatozoa and spermatids were collected.
The F1 female progeny was screened for induced vermilion mutants. The frequency of v mutants was calculated from the pooled data of broods 1 and 2 (not shown) because there were no significant differences between the two broods. Non-mutant F1 flies were crossed in mass matings and the F2 progeny were also screened for induced vermilion mutants (mosaics not detectable in F1). The number of F1 females set up was used to calculate approximate F2 mutant frequencies. As a control for exposure dose, RL frequencies were determined in parallel with each single SLT. Vermilion mutants induced by MEC were generated under NER and NER+ conditions. For CEA, vermilion mutants could be obtained only from crosses with NER females, because in experiments with NER+ females the mutation induction increased no more than three to four times the background level. Determination of a molecular spectrum under such conditions is not feasible.
Three different types of vermilion mutants were found, i.e. homozygous viable, male lethal and sterile mutants (30). From homozygous viable mutants a homozygous strain could be built up. Male lethal mutants had a recessive (hemizygous) lethal mutation which was not separable from the vermilion mutation. Therefore, the induced vermilion mutation was balanced over an Inscy, v chromosome in heterozygous females.
Characterization of vermilion mutants
Homozygous viable mutants were analysed by DNA sequencing. First, genomic DNA was isolated from ~1 g frozen flies (31) and the v gene amplified by PCR (32). The amplified DNA was digested with SacI and HindIII and cloned into pUC18. Sequencing on double-stranded DNA was performed manually (33) or with an automated laser fluorescence/DNA sequencer (Pharmacia/LKB, Woerden, The Netherlands). Each mutation was checked from a second independent clone to exclude amplification errors.
Male lethal mutants were analysed cytogenetically. Salivary glands were isolated from female third instar larvae carrying the chromosome with the newly induced v allele and a wild-type chromosome. Large deletions containing the v locus could be seen as a loop in the wild-type chromosome (figure 1
in ref. 34). If no deficiency was detected, the mutant was analysed in the same way as sterile mutants.
Sterile mutant genomic DNA was isolated from a single fly and the v gene was amplified. In this way the v gene of the Inscy balancer and possibly the v gene with the induced mutation were both amplified. The v mutation of the balancer chromosome carries a small deficiency (15 bp) containing a PstI restriction site. Digestion of the PCR product would show if the vermilion mutation was an intra-locus mutation or a complete deletion of the vermilion gene (35).
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Results
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MNER/MNER+ mutability index
For the monofunctional CEA a significant induction of mutations under repair-proficient conditions is only seen for brood 1, representing exposed spermatozoa (Table I
). In the absence of NER activity in the females a strong hypermutability effect of around 7 was found for brood 1. Because a significant increase in RL was not observed in brood 2 (treated spermatids) in the presence of NER, an exact MNER/MNER+ mutability ratio could not be estimated. Nevertheless it is clear that the MNER/MNER+ ratio must have increased considerably (up to 50 times) in brood 2 compared with brood 1, indicating an important role of NER on DNA monoadducts induced by CEA. With the bifunctional MEC no or only a low effect of the repair deficiency was found for brood 1, whereas in brood 2 the MNER/MNER+ ratio was between 3.5 and 4.1. This low hypermutability as well as the brood difference is also seen for the aromatic nitrogen mustards chlorambucil (CAB) and melphalan (MEL). In brood 1 the MNER/MNER+ ratios for CAB and MEL were ~1 and in brood 2 they are 4.0 and 2.4, respectively (data not shown). The cause of this difference between the broods is that for the germ cell stages tested in brood 2 the time interval between adduct formation and mutation fixation is 23 days longer than for mature spermatozoa. This time-dependent increase in the mutability index from spermatozoa to late spermatids was also observed in fine brood fractionation experiments for other monofunctional agents and has been explained by an accumulation of apurinic sites by Vogel et al. (ref. 4 and references therein). The relatively low mutability indices estimated for the bifunctional counterpart of CEA show that premutagenic DNA lesions induced by MEC are less efficiently repaired by NER. As will be discussed later, this small increase in recessive lethal mutations induced by crosslinking nitrogen mustards can be attributed to unrepaired monoadducts.
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Table I. Determination of MNER/MNER+ indices for the bifunctional nitrogen mustard MEC and the monofunctional analogue CEA
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Maximum activity of CEA in the RL test
In a functioning NER system CEA is a very weak mutagen in adult feeding experiments, as illustrated by the low induction of recessive lethals of in general three to four times the background level (Table I
). For MEC it was shown that injection into the abdomen of the fly is a more efficient treatment procedure than feeding because it is inactivated in the intestinal tract of Drosophila (36,37). To investigate whether injection would increase the effectiveness of CEA, several doses of CEA were injected into adult male flies. As a reference the experiments with MEC were repeated. As can be seen in Figure 2
, injection of CEA did not improve the mutagenic effectiveness compared with feeding. Injecting concentrations >100 mM CEA were toxic to the flies. A clear doseresponse relationship together with the activity under repair-deficient conditions show that the target cells are reached. This indicates that the low induction of RL under wild-type conditions is due to repair and not due to metabolic inactivation of CEA in the intestinal tract.

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Fig. 2. Comparison of mutagenic activity in the RL test after injection or feeding of CEA or MEC under wild-type repair conditions. In contrast to MEC, CEA always shows a weak activity irrespective of the route of administration.
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Relative clastogenicity index (ICL/RL)
It has long been known that the induction of CL represents a clastogenic event (26,28). Zijlstra and Vogel (26) reported a negative result in this assay for CEA, probably due to the relatively low concentration (4 mM) used. For induction of CL by monofunctional alkylating agents high doses are usually needed. This necessity for concentrations near toxic levels was most obvious in experiments on the clastogenic effects of ethylene oxide (38). Therefore, the experiments for CEA were repeated with higher doses (5100 mM). In addition, the CL test was performed using wild-type and mus201 females. CEA induced significant loss of the ring X chromosome only in brood 1 and at high doses (Table II
). A dose level of 200 mM CEA was associated with toxicity (data not shown). For 100 mM CEA and under wild-type repair conditions both end points (CL and RL) are significantly different from the control. Although a relative clastogenicity index (ICL/RL) can only be calculated for this concentration, it is clear that this ratio is above 1, which is different from the indices estimated for ethylating and methylating agents (see Discussion). Furthermore, a weak potentiating effect of mus201 on the clastogenicity of CEA was seen at a dose of 100 mM (CLNER/CLNER+ = 1.6 for 100 mM), but this effect was minor compared with the strong hypermutability effect on the induction of forward mutations (up to 50 times). A striking feature is a distinct effect of NER deficiency on ICL/RL obtained for crosses of NER+ malesxNER+ females as compared with NER+ malesxNER females. As a consequence of the substantial forward mutation induction by CEA with NER females all CL/RL ratios are below 1, as compared with ratios above 1 for wild-type conditions (Table II
). All these data show a strong hypermutability for RL and a relatively low hypermutability for CL, suggesting the involvement of different DNA lesions in the formation of chromosome breakage events versus forward mutations for CEA.
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Table II. The relative clastogenic efficiency of CEA in crosses of NER+ (exposed or control) males with NER+ or NER- females
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Specific locus tests (SLT)
The consequence of the weak activity of CEA in the RL test with repair-proficient females is that a sufficient number of CEA mutants could only be generated when repair was not active. For CEA 15 F2 mutants were found as against one F1 mutant (Table III
). This high frequency of F2 mutants compared with F1 mutants may be due to persistent lesions, rather than a block of replication. The frequency of F1 v mutants was 0.2x105 and that of F2 mutants was 16.3x105. Parallel to each SLT a RL test was performed, in order to control the exposure conditions. The RL frequency of 6.1x105 per locus is between the F1 and the F2 frequencies calculated for the SLT. The v frequency induced by MEC under NER-proficient conditions was 4.4x105. This is similar to the RL frequency of 5.1x105 per locus, which is 30 times the spontaneous frequency (see Table I
). The frequency of 9.4x105 v mutants for the F2 generation is higher than for F1 (4.4x105). In the SLTs where NER females were crossed with MEC-exposed males, two different types of females were used (Inscy v; mus201D1, bw and Inscy v; mus201D1). The two protocols did not lead to differences in mutant frequencies (data not shown) and the data were therefore pooled. The RL frequency (6.3x105 per locus) under repair-deficient conditions was again between the F1 (3.5x105) and the F2 v frequency (12.4x105).
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Table III. The number of vermilion mutants isolated after treatment of post-meiotic male germ cells with CEA or MEC
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Analysis of v mutations
All 16 vermilion mutants induced by CEA were homozygous viable. The single F1 mutant could not be analysed because the v fly was already dead when scored and single fly genomic DNA isolation did not succeed. This mutant is not included in Table IV
because it is not known what type of mutant (viable, lethal or sterile) it was. Of the 15 remaining mutants, 14 had a base pair substitution and one a frameshift through a 1 bp deletion. Two of the 14 base pair substitutions were transitions, both of GC
AT type. The remainder were transversions, of all possible arrangements.
In total 19 v mutants were induced by MEC under NER-proficient conditions. Homozygous viable, male lethal and sterile mutants were found among both F1 and F2 mutant flies. Of the 10 homozygous viable mutants eight were analysed. Three mutants had base pair substitutions and five carried an intra-locus deletion (ILD) of 427 bp. One mutant had a 3 bp insertion in addition to the deletion. The two non-analysed male viable mutants are classified as intra-locus mutations (see Figure 3
). All the male lethal mutants (cytogenetically checked) and the sterile mutants (checked by PstI digestion) were MLD. This is in accordance with other studies showing that almost all lethal and sterile mutants analysed are MLDs (30,39,40). This is due to a haplo-insufficient female fertility factor (hfs) and a recessive lethal locus (csk) adjacent to the v locus (4143). Therefore, the three mutants not analysed in the group of male lethal and sterile mutants were also considered to be MLDs (Figure 3
).

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Fig. 3. The nature of vermilion mutations induced by CEA (NER only) and MEC (NER+ and NER) in post-meiotic male germ cells of Drosophila.
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Under repair-deficient conditions 14 MEC mutants were isolated. One mutant was found in a recessive lethal test performed in parallel with a specific locus test (non-mutant F1 females used; see Materials and methods). Therefore, this mutant was excluded from calculation of the v frequency, but included in the mutation spectrum. Five of the nine male viable mutants were base pair substitutions. The other four mutants were: a deletioninsertion of 11 and 3 bp, respectively; a deletion of 14 bp; a duplication of ~800 bp; a frameshift through a 1 bp deletion. The five male lethal or sterile mutants were all analysed by PstI digestion and all of them appeared to be MLDs. The three mutation spectra are summarized in Figure 3
.
All intra-locus rearrangements induced by MEC were grouped together, as shown in Figure 4
. We included one mutant (MEC 3) which was found by chance in the mutability test under repair-deficient conditions, therefore, this mutant was not included in the spectrum. Irrespective of the repair status, almost all sequence changes in the eight ILD mutations induced by MEC possess repeats near the deletions (marked by grey boxes). The importance of short direct repeats for deletion mutagenesis is strengthened by similar findings across different species (30,4447). Part of the repeat found in mutant 38 is formed by an insert. Two deletions (mutants numbers 34 and 38) show a small insertion of 3 bases (in parentheses and underlined) additional to the deletion, which is repeated in the non-deleted sequence in the near vicinity (58 bp) of the insert.

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Fig. 4. Deletions induced by MEC under NER+ and NER conditions. Mutants are shown by number. Lower case and double underlined letters depict the deletion, upper case letters show border sequences. Additional insertions (in parentheses) are underlined together with their repeats in the non-deleted sequence. Nearby repeats are marked by gray boxes.
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Discussion
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Impact of DNA repair on mutagenicity
Compared with its bifunctional counterpart, CEA shows only marginal activity when mutagenized male germ cells are transferred to repair-competent eggs. With MEC high recessive lethal frequencies of ~8% (data not shown) can be reached, which on a per millimole basis is ~200 times higher than the activity of CEA. For CAB RL frequencies as high as 12% have been found on the X chromosome. That CEA reaches the target cells is evident from the results using repair-deficient eggs and the doseresponse relationship seen in Figure 2
. The low genotoxic effectiveness of CEA was predicted, considering the fact that it is a type 1 monofunctional agent (4). This category of monofunctional alkylating agents generally has a high SwainScott s value due to their high nucleophilic selectivity (48). Although no s value is known for CEA, we assume it to be ~1, since it is comparable with the monofunctional reactivity value of MEC (s value 1.18) (49). CEA is therefore expected to predominantly alkylate nitrogen atoms in the DNA. Other type 1 agents, like ethylene oxide, propylene oxide, butylene oxide and propylene imine, often show low mutation induction in post-meiotic stages of male Drosophila and those so far tested generally a very low mutation frequency in repair-proficient pre-meiotic stages in mouse and/or Drosophila (22). Efficient error-free repair of N-alkylation damage is the cause of this low genotoxic potency. The high hypermutability ratios for CEA in this study and of other monofunctional agents of category 1 (10,25) strongly support this conclusion. It would therefore be interesting to investigate the mutagenic activity of CEA in excision repair-deficient knock-out mice. The selectivity of CEA for alkylation of nitrogen positions is further substantiated by the large number of base pair changes on G positions, 11 of 14 (Table IV
). This suggests a strong preference for N7-G alkylation. That CEA induces low levels of O-alkylation is indicated by the low number of GC
AT and AT
GC transitions. Alkylation on O6-guanine or O4-thymine, respectively, can produce these transitions (50,51).
Apparently, DNA crosslinks are poorly repaired, taking into account the high mutagenic potency and the absence or low hypermutability of bifunctional agents in NER cells (10,25; this study). There is suggestive evidence that the low hypermutability of MEC (MNER/MNER+
1.34.1) stems from the contribution of monoadducts when NER is not active in the oocyte. This hypothesis is supported by the molecular nature of MEC-induced mutants. As expected, this crosslinking agent induced mainly deletion mutations (4). Furthermore, under NER conditions point mutations make up 36% of the mutation spectrum, but this portion drops to 20% when NER is functional and a substantial part of the monoadducts are repaired. Quantification of the hypermutability for MEC in the SLT is not possible because different concentrations were used and distinct germ cell stages were sampled in the mass crosses. Hypermutability ratios as high as 2.44.1 in brood 2 were observed for the bifunctional nitrogen mustards MEC, CAB and MEL. In contrast, no hypermutability effect was seen with the crosslinking agent hexamethylphosphoramide (HMPA) and three bifunctional chloroethyl nitrosoureas, e.g. with bis-chloroethyl nitrosourea (BCNU) (4). Consistent with this lack of hypermutability is the low number (48%) of base pair substitutions in the molecular spectra of HMPA (52) and BCNU (53), compared with 20% for MEC. The reason for this difference between nitrogen mustards and BCNU and other bifunctional nitrosoureas is the way the crosslink is generated. The first reaction in the formation of a crosslink by BCNU is attack by a carbonium ion at an oxygen position, preferably the O6 of guanine. Monoadducts on this position are substrates for alkylguanine transferases but are poorly repaired by the NER system. This first alkylation is followed by slow elimination of the chloride ion and subsequent alkylation of a second base generating an intra- or interstrand crosslink (8).
Impact of functionality on clastogenicity and mutation spectrum
In the past, determination of relative clastogenic efficiency provided useful information in two respects. First, a clear distinction could be made between monofunctional agents and agents capable of crosslinking DNA (16,26). Second, the CL/RL ratio allowed predictions in terms of the proportion of base pair substitutions versus rearrangements to be expected at the DNA sequence level (10,54).
The ratio of chromosome loss to forward recessive lethal mutations is always below 1.0 for monofunctional methylating and ethylating agents. For bifunctional agents this ratio is generally >2 (Figure 5a
). At a dose of 100 mM CEA, a significant induction of CL was observed, yielding a CL/RL ratio of 2.9 when DNA repair was active (Table II
). Although the small increases seen for CL at doses of 5 and 50 mM CEA were statistically not significant, meaning that an exact classification on the basis of the CL/RL ratio is not possible, it seems clear that the CL/RL ratios are always higher than 1 for CEA. Other monofunctional agents, such as ethylene oxide, propylene oxide and ethylene imine, have ratios between 1.2 and 3.1 (16,26,38). With CL/RL ratios >1 these agents form an intermediate group because, unlike methylating and ethylating agents, on the basis of this parameter they cannot be separated from crosslinking agents (4).


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Fig. 5. The strong correlation between two in vivo end points, i.e. the relative clastogenicity index (CL/RL) and the molecular spectrum of mutations. The ratio of CL to forward mutations and the ratio of base pair substitutions to the sum of ILD and MLD and other rearrangements are depicted. (a) Three model substances (4) are shown: methylmethane sulfonate (type I) and N-ethyl-N-nitrosourea (type II) are both monofunctional and HMPA (type III) is a polyfunctional agent. Data from Pastink et al. (31), Nivard et al. (39) and Aguirrezabalaga et al. (52). (b) The mutagens investigated in this study, CEA and MEC.
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The reason for the relatively high clastogenic efficiency of ethylene oxide and ethylene imine could be their ability to transfer a hydroxyethyl or aminoethyl group to the phosphate backbone of the DNA (5557). The consequence of this alkylation reaction is that the phosphodiester bond in the DNA backbone could be broken, resulting in a single-strand break. It is not known whether CEA can also alkylate DNA phosphates, but a similar adduct to that produced by ethylene imine, an aminoethyl adduct, is expected (58).
The CL/RL ratios for MEC, CAB and MEL range from 2.5 to 4.6, as predicted for crosslinking agents (16). Clear differences between mono- and bifunctional nitrogen mustards occur, however, if `mutagen dose' is considered. Bifunctional analogues are more powerful clastogens, which is obvious from the 100- to 5000-fold higher activity per millimole exposure in the CL test compared with CEA (4). Tokuda and Bodell (13) compared the induction of SCEs in 9L cells treated with MEC or with the two monofunctional analogues bis(ethyl)-2-chloroethylamine and bis(methyl)-2-chloroethylamine. In concordance with our results they found that, on a molar basis, MEC was 471686 times more effective at inducing SCEs. These differences could not be explained by increased alkylation of DNA since the reaction of MEC with 4-(p-nitrobenzyl)pyridine, used to estimate alkylation capacities with cellular DNA, was only 1.7- to 4-fold greater than those of the monofunctional mustards. Also, in the micronucleus assay with Chinese hamster cells bifunctional CAB had a stronger ability to break chromosomes than its half-mustard analogue (19).
The high clastogenic efficiency of bifunctional agents finds further expression in the high proportion of deletion mutations and other rearrangements generated in the vermilion locus (Figure 5a
). The spectrum of MEC consisted of 80% deletions under repair-proficient conditions (Figure 5b
). CAB also induces ~90% deletions (to be published elsewhere). This is in accordance with mouse specific locus test results. The main mutational events induced by crosslinking nitrogen mustards in post-meiotic germ cells of the mouse are also rearrangements, mostly deletions (14,15). In contrast, with monofunctional CEA almost exclusively base pair substitutions (93%) are found in Drosophila when NER is deficient. With MEC, 64% deletions and other rearrangements but only 36% base pair substitutions are induced in NER females. This shows that the lesions responsible for the high mutagenic effectiveness of MEC and other bifunctional nitrogen mustards cannot be monofunctional adducts. These monoadducts are efficiently repaired by the NER system, as is obvious from the high hypermutability index of CEA. In Chinese hamster cells mutation spectra also differ considerably between mono- and bifunctional nitrogen mustards. CAB induced ~80% deletions, whereas ~70% of mutations induced by the half-mustard were point mutations (19).
The clear shift from high CL/RL ratios (23) under repair-proficient to lower ratios (<1) under repair-deficient conditions observed for CEA implies the involvement of different lesions for the two genetic end points. For forward mutations (RL), DNA adducts on nitrogen positions are most likely responsible for their induction, because these lesions are well repaired by NER, as is evident from the strong hypermutability response with mus201 females. For breakage-related effects, adducts on the phosphate backbone of the DNA might be the main cause. These lesions are apparently poorly repaired by the NER system. In accordance with the relatively low clastogenicity of CEA under deficient conditions is the high percentage of base pair substitutions (see Figure 5b
). A low contribution of rearrangements among vermilion mutants correlating with low clastogenicity was also found for ethylene oxide under NER-deficient conditions (38).
In conclusion, the genetic activity profile of monofunctional CEA differs considerably from its bifunctional counterpart MEC. Monofunctional CEA is weakly mutagenic because it induces lesions which are well repaired. Bifunctional nitrogen mustards display their mutagenic activity through the formation of DNA crosslinks, DNA monoadducts making only a minor contribution to their mutagenic activity. These findings further substantiate previous concepts that the major activity responsible for the high genotoxic potency of polyfunctional agents in germ cells of mice and Drosophila (4,59) as well as their high carcinogenicity in rodents (1) and humans (IARC) is related to their ability to form crosslinks with DNA.
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Notes
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1 To whom correspondence should be addressed Email: e.w.vogel{at}lumc.nl 
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Acknowledgments
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We would like to thank Sharda Bisoen, Ineke Bogerd and Corrie van Veen for their adept technical assistance. This work was financially supported by the Stichting Koningin Wilhelmina Fonds, contract RUL 95-1043, and by the EC contract ENV4-CT97-0505.
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Received December 30, 1999;
revised April 27, 2000;
accepted July 3, 2000.