Mutagenesis vol.17 no.5 pp.383–389, 2002
Induction of chromosomal aberrations by dacarbazine in somatic and germinal cells of mice
I.-D.Adler1,4, U.Kliesch1, I.Jentsch2,3 and M.R.Speicher2,3 1Institut
2Institut
fu¨r Experimentelle Genetik and fu¨r Humangenetik, GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany and 3Institut fu¨r Humangenetik, Technische Universita¨t Mu¨nchen, D-81675 Mu¨nchen, Germany
Dacarbazine (DTIC) is a chemotherapeutic agent that has been successfully applied to treat various types of cancer such as Hodgkin’s disease, malignant melanomas, soft tissue sarcomas and advanced neuroblastomas. Many of the patients are of reproductive age and express concern over the genetic risk of the treatment they receive. Therefore, DTIC was tested for its clastogenic effects in somatic and germinal cells of mice. In the bone marrow micronucleus assay DTIC induced micronuclei that increased linearly in the dose range 0–125 mg/kg. In a dominant lethal study DTIC gave a positive response at the dose of 500 mg/kg when conceptions occurred 5–16 days after treatment, corresponding to treated spermatids and early spermatozoa. The induction of heritable translocations was tested in that sensitive period. The observed translocation rate among the F1 progeny of male mice treated with 500 mg/kg DTIC was 2.13% (P < 00.1 against the historical control of 0.05%). Assuming linearity of the dose– response effect, the point estimate was used to calculate a doubling dose for the induction of heritable translocations of 12 mg/kg. Alternatively, an induced translocation rate of 41.6⫻10–6 per unit dose was calculated. Both figures indicate that an increased genetic risk may exist for male patients after chemotherapy with DTIC under the assumption that germ cells of mice and humans are equally sensitive to the clastogenic effects of DTIC. However, the genetic risk is restricted to conceptions within a period of 40 days after the end of chemotherapy, since the sensitive stages of spermatogenesis are spermatids and early spermatozoa.
Introduction Dacarbazine [5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide] (DTIC) is a widely used anticancer agent which requires metabolism by liver cytochromes P450. Recently the three human P450s (CYP1A1, CYP1A2 and CYP2E1) that catalyze the metabolism of DTIC were identified (Reid et al., 1999). Based on the kinetics of DTIC metabolism by mouse and human liver microsomes, the authors stated that ‘DTIC metabolism by humans is most likely equivalent if not greater than the metabolism by rodents’. In the murine intestine in vivo pretreatment of mice with the alkyltransferase inhibitor O6benzylguanine (BeG) was shown to block DTIC-induced apoptosis (Toft et al., 2000). This was explained by the authors by BeG inhibition of P450-dependent DTIC activation because 4To
for both DTIC and BeG the primary isoform of P450 is CYP1A2. The P450-dependent N-demethylation produces the active compound 5-(3-methyl-triazen-1-yl)imidazole-4carboxamide (MITC). In vitro DTIC is mutagenic by the formation of N7- or O6-methylguanine adducts in DNA, leading predominantly to GC→CG transversions and GC→AT transitions (Mudipalli et al., 1995). In transfected CHO cells with high expression of the human repair enzyme O6-methylguanine-DNA methylftransferase, the GC→AT transition rate was substantially decreased (Psaroudi and Kyrtopoulos, 2000). The spectrum of mutations induced by DTIC in vitro spanned from single base events such as substitutions (35%), deletions (30.5%) and insertions (19.4%) to large deletions (13.8%) of ⬎10 bp (Mudipalli et al., 1995). In vivo DTIC induced structural chromosome aberrations in mouse bone marrow cells in a dose-dependent manner (Al-Hawary and Al-Saleh, 1989). DTIC is often used in combination with other chemotherapeutic agents against advanced stage Hodgkin’s disease (Chim et al., 1999; Engert et al., 1999) or against advanced malignant melanoma (Creagan et al., 1999; Kashani-Sabet et al., 1999; Yamazaki et al., 1999). Despite new combination treatment regimens, DTIC alone is the reference standard treatment for stage IV melanomas (Chapman et al., 1999). A combination of mesan, adriamycin, ifosfamide and DTIC (MAID) is used against advanced soft tissue sarcomas (Chevreau et al., 1999). DTIC alone is used against advanced neuroblastomas in the consolidation stage after initial combination chemotherapy (Kaneko et al., 1999). The introduction of combination treatments has transformed many of these tumours from incurable diseases to some with the highest cure rates. Therefore, many of these patients of reproductive age express the wish to have children but are concerned about the ensuing genetic risk. Several chemotherapeutic agents have been studied for germ cell effects with the specific locus test and the genetic risk of therapeutic exposure regimens has been determined (Ehling and Neuha¨user-Klaus, 1979, 1989, 1994; Ehling et al., 1988, 1999). For the occupational toxicants acrylamide and 1,3butadiene a genetic risk assessment was performed based on heritable translocation data (Adler et al., 1994; Pacchierotti et al., 1998). Presently, the clastogenic effects of DTIC were first determined in somatic cells in vivo, i.e. the mouse bone marrow micronucleus test, to confirm the data of Al-Hawary and Al-Saleh (1989) in our animal stock. After a positive response in bone marrow, whether clastogenicity was also induced by DTIC in mouse germ cells was tested using the dominant lethal test. Lastly, the heritable translocation assay was performed during the sensitive period of germ cell development as judged from the results of the dominant lethal assay. The results of the heritable translocation assay were used to calculate the genetic risk for patients undergoing DTIC treatment.
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Materials and methods Chemical, solvent and route of application DTIC (CAS no. 4342-03-4) was purchased from Sigma (Deisenhofen, Germany). DTIC was dissolved in corn oil at a temperature of 40°C. The solution was i.p. injected within 30 min of preparation. A dose-finding test was performed with 12 adult (102/El⫻C3H/El) F1 males randomly distributed to receive i.p. injections of 100, 200, 500 and 1000 mg/kg DTIC. The animals injected with the highest dose died within 24 h. All other animals survived. An initial 2 day phase of weight loss was overcome quickly and weight gains were in the control range after 6 days. Micronucleus test Animals Adult male and female (102/El⫻C3H/El) F1 mice from the GSF colony, aged 12–14 weeks and weighing 26–28 g, were employed. Each dose group consisted of 10 treated animals, five males and five females. Treatment Doses were chosen to avoid a severe depression in the ratio polychromatic erythrocytes (PCE):normochromatic erythrocytes (NCE). Animals were treated by i.p. injection with 0, 31.5, 62.5 or 125 mg/kg and bone marrow was sampled 24 h after treatment. The control group received corn oil only. The injection volumes were 0.1 ml/10 g body wt. Bone marrow preparation and micronucleus scoring The procedure for bone marrow preparation, staining and scoring was as described before (Adler, 1984). All slides were coded for microscopic analysis at 1250⫻ magnification. Per animal, 2000 PCE were scored for the presence of micronuclei and the means were expressed as micronucleated PCE (MPE) per 1000 PCE. The numbers of PCE were counted in fields that contained 2000 NCE to determine a shift in erythroblast proliferation. The values were expressed as percent PCE of the total erythrocyte counts. Micronucleated NCE (MNE) were also recorded but not included in the evaluation. Statistics The control data within the experiments were pooled when homogeneous as determined by the binomial dispersion test (Snedecor and Cochran, 1967). Significant differences between treatment and control groups were determined by the Mann–Whitney U-test (Sachs, 1984). Dominant lethal test (102/El⫻C3H/El) F1 males were treated i.p. with 250 or 500 mg/kg DTIC. These doses were chosen as the maximum tolerated dose and one dose below. To each dose group, a concurrent control group of males was i.p. injected with corn oil. Each group consisted of 40 males. They were mated 4 h after treatment at a ratio of 1:1 to untreated virgin females of the same stock, aged 12–16 weeks. Females were changed every 4 days for a total of seven (250 mg/kg) or eight (500 mg/kg) 4 day mating intervals. Females were inspected for the presence of a vaginal plug every morning. At pregnancy days 14–16 the females were killed and uterus contents were inspected for live and dead implants (Ehling et al., 1978; Bateman and Epstein, 1971). The numbers of dead implants were compared between each control and treated mating group using the χ2 test (Sachs, 1984). Dominant lethality (DL) was expressed as %DL ⫽ [1 – (live implants per female in the experimental group/live implants per female in the control group)]⫻100. Dominant lethality was compared on a male-to-male basis by the Mann– Whitney test using average values of live implants from all females per male (Chanter et al., 1989). Heritable translocation test Parental generation C3H/El male mice from the GSF colony were 10–12 weeks old at the time of treatment and weighed between 25 and 28 g. A group of 50 males were treated with 500 mg/kg DTIC and mated 4–17 days after treatment to untreated virgin 102/El females from the GSF colony at a mating ratio of 1:2. Pregnant females were allowed to come to term. Litters were counted and sexed at birth and weaned at the age of 3 weeks. The experiment was repeated to raise sufficient numbers of offspring. Selection of translocation-suspect F1 animals by litter size reduction Progeny of both sexes were mated at the age of 10–12 weeks within the experimental groups, avoiding brother–sister mating. To determine possible translocation heterozygotes by reduced fertility a sequential decision procedure of eliminating pairs with normal litters was employed (Adler, 1980). Up to three litters were observed before pairs with reduced litter size or pairs without any litter were separated. Confirmation of translocation-suspect F1 males Suspect F1 males were mated to four or five (102/El⫻C3H/El) F1 females from the colony. Females were killed at mid-pregnancy to determine the
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frequency of dead implants. Males with an average of three or more dead implants per pregnant female were subjected to cytogenetic confirmation of the translocation in testes preparations. Individuals thus identified as translocation heterozygotes were bred again until at least eight male offspring were weaned to maintain the translocation line. Translocation heterozygotes were subjected to karyotype analysis in Giemsa banded bone marrow preparations. Confirmation of translocation-suspect F1 females Suspect F1 females were mated to (102/El⫻C3H/El) F1 males from the colony and were again allowed to have up to four litters. Male F2 progeny from small litters of suspect F1 females were weaned and subjected to meiotic chromosome analysis at maturity. Translocation heterozygote females were confirmed by the presence of at least one translocation heterozygote individual among 8–10 F2 males. The translocation F1 females or their translocationcarrying F2 offspring were subjected to karyotype analysis of Giemsa banded bone marrow chromosomes. Meiotic chromosome analysis In order to keep the translocation-suspect males for further breeding, they were unilaterally orchidectomized under brief ether narcosis. Meiotic chromosome analysis was performed in testes preparations obtained according to the method of Evans et al. (1964). At least 25 cells at diakinesis–metaphase I were scored per animal in Orcein stained slides (100⫻ magnification, phase contrast) for the presence of translocation multivalents. Bone marrow chromosome karyotyping Bone marrow preparations were obtained according to the standard preparation procedure (Adler, 1984) and stained by the trypsin–Giemsa method (Gallimore and Richardson, 1973). Presumed break points were assigned to bands according to the standard mouse karyotype (Evans, 1989). Mouse multiplex fluorescence in situ hybridization (M-FISH) Mouse M-FISH was performed as recently described (Jentsch et al., 2001). In brief, mouse chromosome-specific painting probes generated by flow sorting (Rabbitts et al., 1995) were amplified by DOP–PCR (Telenius et al., 1992). To facilitate the generation of the complex probe mix, for each of the five fluorochromes used (SpectrumGreen, Cy3, Cy3.5, Cy5 and Cy5.5) a DNA pool was made. Each DNA pool consisted of all painting probes which in our labeling scheme are labeled with the same fluorochrome. For example, the Spectrum Green pool consisted of painting probes for chromosomes 3, 6, 8, 11, 12, 13, 15, 19 and X, the Cy3 pool of chromosomes 1, 2, 4, 9, 11, 12, 16, 19 and Y, and so on (for details see Jentsch et al., 2001). The DNA pools were labeled by DOP–PCR. After overnight ethanol precipitation the probe mixture and the slides were denatured and the hybridization mixture was added to the slide. The slides were incubated overnight at 37°C. Following hybridization, post-hybridization washes and blocking with 3% BSA for 30 min at 37°C, anti-digoxigenin rabbit antibody (1:500) was added to the slides and incubated for at least 45 min in a moist chamber at 37°C. After washing (3⫻5 min each) in 4⫻ SSC/Tween at 45°C, a second layer of antibodies consisting of anti-Cy5.5 rabbit antibody (1:300; Amersham Pharmacia Biotech) and avidin–Cy3.5 (1:300; Amersham Pharmacia Biotech) was applied and again incubated for at least 45 min in a moist chamber at 37°C. After final washes (3⫻5 min each in 4⫻ SSC/Tween at 45°C) slides were counterstained with 4⬘,6-diamidino-2-phenylindole (DAPI) and mounted in p-phenylenediamine dihydrochloride antifade solution. A Leica DMRXA-RF8 epifluorescence microscope equipped with special filter blocks (Chroma Technology, Brattleboro, VT) and a Sensys CCD camera (Photometrics/Kodak KAF 1400 chip) was used. Both the camera and microscope were controlled with Leica Q-FISH software (Leica Microsystems Imaging Solutions, Cambridge, UK) and images were analyzed using the Leica MCK-Software package (Leica Microsystems Imaging Solutions; Eils et al., 1984). Statistics Fisher’s exact test was used to determine statistical differences between translocation in the experimental F1 groups and the historical control group (Sachs, 1984).
Results Micronucleus test The micronucleus test gave a positive response (Table I). The dose–response effect between 0 and 125 mg/kg DTIC can be described by the linear equation y ⫽ 0.2 ⫹ 0.016D. The result confirms that DTIC is a clastogen in mouse bone marrow cells.
Chromosomal aberration induction by dacarbazine
Table I. Results of the mouse bone marrow micronucleus test with DTIC Dose (mg/kg)
Interval (h)
0 31.5 62.5 125.0 aP
MNPE/2000 PE
24 24 24
MNPE/1000 PE (mean ⫾ SE)
Males
Females
1, 2, 2, 3, 3 10, 10, 18, 21, 28 21, 23, 28, 29, 31 35, 46, 47, 48, 52
1, 2, 2, 3, 4, 5 8, 9, 10, 13, 16 13, 20,26, 27, 35 30, 33, 35, 44, 54
1.3 7.3 12.8 21.2
⫾ ⫾ ⫾ ⫾
PE (%)
0.5 0.7a 0.8a 1.3a
48.4 46.3 47.4 48.5
⬍ 0.01 (Mann–Whitney test).
Table II. Dominant lethal test with DTIC at 250 and 500 mg/kg Mating intervala I
II
III
IV
V
VI
VII
Dose (mg/kg) 250 0b 500 0 250 0b 500 0 250 0b 500 0 250 0b 500 0 250 0b 500 0 250 0b 500 0 250 0b 500 0
Pregnant females
Total implants
Live implants
Dead implants (DI)
n
%
n
Per female
n
Per female
n
Per female
38 39 28 35 31 34 29 37 37 37 29 36 39 35 27 35 35 36 25 38 32 35 27 33 34 33 28 31
95.0 100 70.0 87.5 77.5 87.2 74.4 92.5 92.5 94.9 82.8 90.0 97.5 89.7 81.8 87.5 87.5 92.3 75.8 95.0 80.0 89.7 81.8 82.5 90.0 84.6 84.9 77.5
445 456 308 404 334 361 312 436 411 404 295 381 426 390 292 385 381 387 269 409 331 392 295 363 355 351 292 348
11.7 11.7 11.0 11.5 10.8 10.6 10.8 11.8 11.1 10.9 10.2 10.6 10.9 11.1 10.8 11.0 10.9 10.8 10.8 10.8 10.3 11.2 10.9 11.0 10.4 10.6 10.4 11.2
394 415 278 367 295 339 264 403 367 371 249 346 380 353 248 348 345 351 243 386 288 357 267 331 335 310 268 323
10.4 10.6 9.9 10.5 9.5 10.0 9.1 10.9 9.9 10.0 8.6 9.6 9.7 10.0 9.2 9.9 9.9 9.8 9.7 10.2 9.0 10.2 9.9 10.0 9.9 9.4 9.6 10.4
51 41 30 37 39 21 47 33 44 33 46 35 46 37 44 37 36 36 26 23 43 35 28 32 20 41 24 25
1.3 1.1 1.1 1.1 1.3 0.6 1.6c 0.9 1.2 0.9 1.6d 1.0 1.2 1.1 1.6d 1.1 1.0 1.0 1.0 0.6 1.3 1.0 1.0 1.0 0.6 1.2 0.9 0.8
Early DI Per female
Late DI Per female
Dominant lethals (%)
1.16 0.90 0.89 0.97 1.26 0.59 1.52 0.0 1.11 0.84 1.55 0.94 0.95 0.94 1.56 1.0 0.86 0.97 0.96 0.6 1.31 1.00 1.0 1.0 0.50 1.09 0.75 0.77
0.18 0.15 0.18 0.09 0.0 0.03 0.10 0.06 0.08 0.05 0.04 0.06 0.23 0.11 0.07 0.05 0.17 0.03 0.08 0.0 0.03 0.0 0.03 0.0 0.09 0.15 0.11 0.03
5.1 6.0 13.0 16.8 1.0 10.4 –8.0 7.1 2.0 5.0 19.3 1.0 –8.1 7.7
aMating intervals of 4 days. bOnly 39 males mated 1:1. cP dP
⬍ 0.01 (Mann–Whitney test). ⬍ 0.05 (Mann–Whitney test).
Dominant lethal test The dominant lethal test gave a positive response only with the higher dose tested (500 mg/kg, Table II). The frequencies of dead implants per female were significantly increased above the concurrent controls during mating intervals II–IV. These sensitive 5–16 days after treatment of the males correspond to early sperm and late and mid spermatids (Adler, 1996). Preimplantation loss (total implants in the control minus total implants in the treatment group) was not detected. The results show that DTIC is a germ cell clastogen. Heritable translocation test The heritable translocation assay was performed in two experiments spaced 4 months apart in order to raise enough progeny. A total of 516 F1 progeny were tested for fertility reduction and 10 translocation heterozygotes were identified (Table III).
An additional female carried a deletion of the X chromosome and was included in the calculation of the translocation frequency. One female carried two independent reciprocal translocations (Table IV) and was counted as only one event in the calculation of the translocation frequency. Most translocation chromosomes and break points were identified by karyotyping of G-banded bone marrow chromosomes. In three cases, the karyotype analysis could not reveal the chromosomes involved and M-FISH was performed. These cases were the male DAC860 with t(8;17), which had a very distal break in chromosome 8 (Figure 1), the sterile male DAC989 with t(12; 15) and the female DAC970 with two translocations, t(14;17) and t(4;8) (Figure 1). The overall results between the two repeats were not significantly different (2.33 versus 1.85% translocation carriers). The mean observed translocation rate was 2.13%, 385
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Table III. Results of the heritable translocation test with DTIC Experiment no.
F1 males
F1 male translocation carriers (%)
F1 females
F1 female translocation carriers (%)
Total F1 progeny
Total F1 translocation carriers (%)
I II I ⫹ II
162 136 298
6 (3.7) 2 (1.5) 8 (2.7)
138 80 218
1 (0.7) 2a (2.5) 3 (0.9)
300 216 516
7 (2.33) 4 (1.85) 11 (2.13)b
aOne female with two bP ⬍ 0.01 against the
reciprocal translocations and one female with a deletion of the X chromosome. historical control of five translocation carriers among 9890 progeny (0.05%) (Fisher’s exact test).
Table IV. Results of the karytype analyses for the F1 animals with reduced fertility recoved in the heritable translocation test F1 code
Litter size (mean ⫾ SE)
Translocation chromosomes
Presumed break points
DAC823m DAC855m DAC860m DAC883m DAC905m DAC907m DAC989m DAC1045m DAC862f DAC970f DAC1045f DAC759f DAC1049m
3.8 ⫾ 0.7 Sterile 5.0 ⫾ 1.2 Sterile 4.3 ⫾ 2.1 4.5 ⫾ 0.9 Sterile 4.0 ⫾ 1.4 4.0 ⫾ 1.5 2.0 ⫾ 0.0 6.0 ⫾ 1.1 3.4 ⫾ 1.4 Sterile
t(1;16) t(17;Y) t(8;17) t(3;16) ct t(12;13) t(12;18) t(12;15) t(4;12) t(1;14) t(4;8) ⫹ T(14;17) Del X XO XXY
1 A5; 16 C3.3 17 D, YC Very distal (M-FISH) 3 H3, 16 A2 12 F1, 13 A4 12 F1, 18 C (M-FISH) 4 B3, 12 F1 1 D, 14 D1 4 D2.2, 8 A3; 14 E3, 17 E2 (M-FISH) A2
f, female; m, male; M-FISH, multicolor fluorescence in situ hybridization used for karyotyping.
which is significantly different from the historical control in our laboratory of 0.05% (P ⬍ 0.001). Discussion The present results of the micronucleus test with DTIC show that the chemotherapeutic agent is a clastogen in somatic cells of mice. The results of the dominant lethal test show that it is also a clastogen in mouse germ cells. The dominant lethal effect was observed in mating intervals II–IV. These mating intervals correspond to the spermatogenic stages of spermatids and early testicular sperm. Assuming that human germ cells have the same sensitivity pattern as mouse germ cells, one can recommend that conception should be avoided within a period of 40 days after the end of chemotherapy (Adler, 1996). In order to determine the frequency of chromosomal aberrations induced by DTIC that are compatible with fetal survival, the heritable translocation assay was performed during the stages most sensitive to dominant lethal induction by DTIC. Among a total of 516 F1 offspring, 11 carriers of translocations (one of which was actually a carrier of an X chromosome deletion) were observed, which yields a translocation rate of 2.13%. It may be noteworthy that more male than female translocation carriers were recovered, i.e. eight males and four females. Four F1 males were completely sterile. Of these, three were found to be translocation carriers and one showed a 41 XXY karyotype (Table IV). One female with reduced fertility showed a 39 XO karyotype. These animals with the numerical sex chromosome abnormalities cannot be explained by the DTIC treatment since only post-meiotic stages of male germ cell development were treated. The results with M-FISH showed that this technique is useful when translocated parts of specific chromosomes cannot be identified with the G-banding technique. The application 386
of M-FISH was helpful in revealing the translocation of the male DAC860 to involve chromosomes 8 and 17 and showed that the female DAC970 carried two independent reciprocal translocations between chromosomes 4 and 8 and chromosomes 14 and 17. However, with M-FISH it is almost impossible to assign the break points of the respective translocation to particular chromosome bands. It is noteworthy that four of the 11 translocations observed involved chromosomes 12 and all breaks were located in band 12F1. The ratio of four seemingly identical break points among a total of 22 break points in this study is suggestive of a DTIC-related hot-spot. So far, this region has not been identified as a hot-spot for chromosomal breakage in our translocation studies or in the compilation of translocation break points (3/548 break points) by Beechey (1996). The difference is highly significant (P ⬍ 0.001, Fisher’s exact test). The distal region of mouse chromosome 12 carries an accumulation of 42 genes for heavy chain immunoglobulins (Chromosome 12 Committee, 2000) and shows homology to the human chromosome region 14q32.33, also carrying immunoglobulin heavy chain genes (Mouse Genome Database, 2000; mouse chromosome 12 linkage map with human homologies). The significance of these details remains to be elucidated. In order to determine the genetic risk from DTIC therapy for patients one can calculate the doubling dose (DD) under the assumption of a linear dose–response effect. The doubling dose is the spontaneous translocation rate (spont TR) multiplied by the experimental dose (exp D) divided by the induced translocation rate (ind TR) (DD ⫽ spont TR⫻exp D/ind TR). The induced translocation rate is the observed translocation rate (2.13%) minus the spontaneous translocation rate (0.05%), which amounts to 2.08%. For the present data obtained with 500 mg/kg we obtain the following equation: 0.0005⫻500/ 0.0208 ⫽ 12. In other words, a dose of 12 mg/kg DTIC
Chromosomal aberration induction by dacarbazine
Figure 1. Translocation karyotypes: (a) DAPI banding; (b) M-FISH. Translocation chromosomes are indicated by arrows. Both translocation carriers were identified by meiotic chromosome analysis. DAC860 was a semi-sterile F1 male with a translocation between chromosomes 8 and 17 which was not recognized in Giemsa banded karyotypes. DAC970 was a semi-sterile F1 female with two translocations. The translocation between chromosomes 4 and 8 was recognized in Giemsa banded karyotypes but the translocation between chromosomes 14 and 17 was only found with M-FISH. For technical details of M-FISH see Jentsch et al. (2001).
induces as many translocations as exist spontaneously. Under the assumption that mouse and human germ cells are equally sensitive to DTIC we can calculate a risk factor. The calculated doubling dose has to be related to the therapeutic i.v. dose of 25.6 mg/kg (Chapman et al., 1999), which results in a risk factor of 2.13. This indirect calculation of a genetic risk due to DTIC chemotherapy indicates that the background risk to sire a child is increased to twice the spontaneous level during the sensitive period of spermatogenesis. We can also use the experimental data for a direct estimate of risk by calculating the induced translocation rate per unit dose. The induced translocation rate of 0.0208 is divided by the experimental dose of 500 mg/kg. This results in 41.6⫻10–6 translocations induced by 1 mg/kg DTIC. If we multiply this figure by the therapeutic dose we arrive at
1064.9⫻10–6 translocations due to human chemotherapy with DTIC. If we consider the spontaneous translocation incidence in humans of 879⫻10–6 (Sankaranarayanan, 1982) we see that the risk of giving birth to a child with a translocation is the additive value of the induced plus the spontaneous translocation rate or ~2000 among 106 live births. These figures may be helpful in consultations with patients if a conception has taken place during the sensitive period after the end of chemotherapy. It is to be noted, however, that the genetic risk following DTIC treatment will only last until the sensitive stages of spermatogenesis have been eliminated from the system, i.e. in humans for ~30–57 days (Adler, 1996). The above calculation is only an example for one of the clinical doses used. Similar calculations are possible for the various treatment regimens which span from single DTIC 387
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treatment to repeated DTIC treatments at various intervals to combination treatments. In repeated treatment schedules the genetic risk of the individual doses may have to be added if they hit the same sensitive germ cell population, i.e. in the present case repair-incompetent spermatids, which lasts for 22–23 days in humans (Adler, 1996). If the intervals between treatments are longer, the risk of the individual dose will persist for a longer period. Other chemotherapeutics in combination treatments may have similar effects or may affect different stages of germ cell development and cause a different mutation spectrum. In the Dartmouth regimen for the treatment of metastatic melanoma, DTIC is combined with cis-platinum, carmustine and tamoxifen (Chapman et al., 1999). While no data exist on the germ cell mutagenicity of carmustine and tamoxifen, cis-platinum was reported to induce specific locus mutations in spermatids and the doubling dose was determined to be 2 mg/kg (Ehling, 1994). In the MAID regimen to treat advanced soft tissue sarcomas, DTIC is combined with doxorubicin and ifosfamide (Chevreau et al., 1999). The latter is also a germ cell mutagen in spermatids and a doubling dose for the induction of specific locus mutations of 49.3 mg/kg was published (Ehling et al., 1998). Since post-meiotically induced specific locus mutations are predominantly intergenic or intragenic deletions, they represent the same genetic end point as translocations, namely clastogenicity. The genetic effects of these combined treatments will at least be additive, if not synergistic. It seems important to quantify the genetic risk for more chemotherapeutic agents, for dose fractionation and especially for successful combinations of chemotherapeutic agents by similar animal experiments. Acknowledgements We gratefully acknowledge the technical help of Helga Gonda, Isa Otten and Martin Skerhut in performing the animal experiments.
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