Mutanon Research, 260 (1991) 99-104 © 1991 Elsevier Science Pubhshers B.V 0165-1218/91/$03 50 ADONIS 016512189100084B
99
MUTGEN 01646
Genotoxicity evaluation of five tricyclic antidepressants in the wing somatic m u t a t i o n and r e c o m b i n a t i o n test in Drosophilamelanogaster Nancy van Schaik 1,2 and Ulrich Graf 1 1 Insntute of Toxwology, Swiss Federal Insntute of Technology and Unwerstty of Zurwh, CH-8603 Schwerzenbach (Switzerland) and 2 Department of Genetics, University of the Wltwatersrand, 2050 Johannesburg (R S.A ) (Received 3 September 1990) (Accepted 18 October 1990)
Keywords. Tncychc antidepressants, Genotoxactty, Somatic cells; Drosoplula, Arnltnptyhne, Destpramme, Irmprarmne; Nortrlptyhne, Protnptyhne
Summary Five tricyclic antidepressants were tested for genotoxicity using the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Three-day-old larvae trans-heterozygous for 2 linked recessive wing hair mutants (multiple wing hairs and flare) were fed the test compounds in water mixed with a standard dry food for 48 h. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can be the consequence of either somatic mutation or mitotic recombination. Desipramine and imipramine were clearly genotoxic at concentrations above 1 mM whereas amitriptyline, nortriptyline and protriptyline were not genotoxic at concentrations up to 100 mM. This seems to implicate the nitrogen atom at position 5 in the 7-membered ring of the tricyclic molecule as being responsible for the genotoxic property of the compounds in Drosophila.
Depression is the most common form of mental disease, estimated to affect at least 1 in 10 persons at some time. Clinicians divide the depressions into several distinct disorders but the drugs of choice for the treatment of all of them are the tricyclic antidepressants (TCAs) (Paykel and White, 1989). These drugs have been used widely over the past 30 years and, although considerable information is available about side effects and toxicity at both therapeutic doses and overdose,
Correspondence: Dr. U. Graf, Institute of Toxicology, ETH and University of Zurich, P.O. Box 550, CH-8603 Schwerzenbach (Switzerland)
much less attention has been paid to their possible genotoxicity. What reports exist often give an unclear picture. For example, Balbi et al. (1980) reported that amitriptyline, desipramine, imipramine and nortriptyline were all negative in the Salmonella/microsome test for strains TA1534, TA1537, TA98, and TA100 either with or without $9, while Kawachi et al. (1980) reported that imipramine was positive in TA100 with activation as well as in the Bacillus subtths rec assay. Filippova et al. (1975) found amitriptyline to be negative m the Drosophila CIB sex-linked recessive lethal test but Saroten (a proprietary name for the same compound) positive in the same test. With human lymphocyte cultures, both amitriptyline
100
and imipramine have been reported to mcrease chromosomal aberrations at concentrations above 4 times plasma levels and at upper plasma levels, respectively, by Saxena and Ahuja (1988), whereas Fu and Jarvlk (1977) found no effect for imipramine at similar concentrations. Because of the paucity of reformation on certain TCAs and the conflicting evidence on others, we decided to test this group of compounds in the Drosophila wing somatic mutation and recombination test. In addition, the comparison of the results in this test of a number of chemically slrmlar compounds may help to identify chemical properties responsible for genotoxicity. Materials and m e t h o d s
Chemical compounds The 4 tricyclic antidepressants amitrlptyline hydrochloride (CAS No. 549-18-8), desiprarmne hydrochlorlde (CAS No. 58-28-6), imipramine hydrochlonde (CAS No. 113-52-0) and nortriptyline hydrochloride (CAS No. 894-71-3) were purchased from Sigma (St. Louis, MO, U.S.A.). Protnptyhne hydrochlonde (CAS No. 1225-55-4) was a gift
AMITRIPTYLINE
li
CH--CH2--CH2--N--(CH3)2 C
°PROTRIPTYLINE
I
CH2--CH2--OH2--NH--CH3 C
J~
NORTRIPTYLINE
CH--CH2--CH2--NH--CH3 N
IMIPRAMINE
i
CH2--CH2--CH2--N--(CH3)2 N
DESIPRAMINE
i
CH2--CH2--CH2--NH--OH3
Fig 1. Structural formulas of the 5 tncychc antidepressants *Protnptyhne has an additional double bond m the 7-membered ring between C10 and C l l
from Dr. E. Bachmann (Institute of Toxicology, ETH and Umverslty of Zurich, Schwerzenbach, Switzerland). The structural formulas of the 5 compounds are shown in Fig. 1. For larval feeding the compounds were d~ssolved in distilled water. Larval feedmg Eggs were collected for 8 h in standard culture bottles containing extra live yeast. Three days later, the larvae were removed from the bottles using a 20% sodium chlonde solution. They were then put into plastic wals containing a specml medium with the test compounds. The solutions of the various compounds were used to prepare Drosophila Instant Medium (Formula 4-24, Carolina Biological Supply Co., Burlington, NC, U.S.A.). 5 ml of the solutions was always added to an equal volume of powdered Instant Medium. Negative water controls were included. The larvae were fed on this medium for the rest of their development (approximately 48 h) until pupation. Somatw mutatlon and recombmatton test The following cross of files carrying markers on the left arm of chromosome 3 was used: mwh females mated to fir3/In(3LR)TM3, ri pP sep b x 34e e s Ser males. Detailed information on the genetic markers is given by Lindsley and GreU (1968). The mutation fir 3 is described by GarclaBellido and Dapena (1974). The surviving flies were collected from the treatment vials on days 10-12 after egg laying. Flies of the trans-heterozygous (mwh fir+/mwh ÷ fir 3) genotype were stored in 70% ethanol. For classification, wings were mounted in Faure's solution (gum arabic 30 g, glycerol 20 ml, chloral hydrate 50 g and water 50 ml) and were scored under a compound microscope at 400 × magnification for the occurrence of spots. Different types of spots were recorded separately, namely single spots showing either the multiple wing hairs (mwh) or the flare (fir) phenotype, and twin spots showing adjacent mwh and fir areas. Single spots are produced either by mitotic recombination occurring between the 2 markers or by somatic gene mutation, deletion or another change at one or the other marker locus; twin spots are produced exclusively by rmtotlc recombination occurring between the proximal marker fir and the centromere.
101 In each case, the size of a spot was determined by counting the number of wing cells exhibiting the mutant phenotype. A detailed description of the wing spot test is given by Graf et al. (1984). Data evaluatton and stattsttcal analysts The evaluation of the wing spot data was performed with the computer program S M A R T (Wiirgler, unpublished). For the statistical analysis, the spots as described above were grouped into 3 different categories: (1) small single spots ( 1 - 2 cells in size), (2) large single spots (3 or more cells) and (3) twin spots. These 3 categories of spots were evaluated separately. More details on the statistical analysis are given in Frei and Wiirgler (1988). For the calculations, the K a s t e n b a u m Bowman test was used with P = 0.05. Based on the number of wings analyzed, the number of mwh clones and the number of cells scored in each wing (ca. 24,400), it is possible to calculate the clone formation frequency per cell cycle and 10 s
ca
20
deslpramlne
AMITRIPTYLINE CONC.: SO mM []TREATED:
116
TREATMENT: q.8 h wings
[--]CONTROL:
"~512 SPOT SIZE
TNIN
1
2
3-q
5-8 9-16 -32 SPOT SIZE
-6q
SPOTS
-128
-256 >512
Fxg 3 Spot size dlstnbutLons for single and twin spots obtained with 50 mM armtnptyhne
cells. N o clone size correction was made, because these were chronic treatments with stable compounds (see also Frei and Wiirgler, 1988).
o
Results and d i s c u s s i o n t 5
Imlpramine
OOJ
~
--
i
, a n nl~lq
OOl
Ol
amltrlptyline , , illiwj
I i ~lanlq 1
1 o
a , ,llHq lO
, i IH.. l lOO
concentration (raM)
Fig. 2. Dose-response relatlonslups for armtnptyhne, deslpramlne and lmlprarmne for total spots per wing.
Each of the 5 compounds tested was assayed in at least 2 independent experiments using 2 or more different concentrations. The compounds were tested simultaneously to obtain quantitative data which allow for a comparative analysis. All the data are given in Table 1. A large concurrent negative control series was obtained. The frequency of spontaneously o c c u m n g total spots per wing of 0.31 was within the usual range (Alonso Moraga and Graf, 1989; G r a f et al., 1989). Two of the 5 compounds gave clearly positive results. With desipramine the frequencies of total spots per wing were significantly increased over the control from 0.1 m M and higher, whereas with inupran'une this was the case from 1.0 m M and higher. The corresponding dose-response relationships are plotted in Fig. 2. With imipramine there was a drop in spot frequency at the highest concentration tested.
102
This was probably due to the pronounced toxicity of the compound at this concentration. The other three compounds were non-genotoxic. Amitriptyline and nortriptyline were tested over the same dose range as the 2 positive compounds (i.e., 0.001-100 mM) while protriptyline was tested only
at the 2 highest concentrations (i.e., 50 and 100 mM), and all the results obtained were negative. In Figs. 3-7 the spot size distributions for single and twin spots are given for the 50-raM treatment with each of the 5 compounds. When considering the 3 categories of spots
TABLE 1 SUMMARY OF RESULTS OBTAINED WITH 5 TRICYCLIC ANTIDEPRESSANTS IN THE WING SOMATIC MUTATION AND RECOMBINATION TEST IN Drosophda rnelanogaster Compound
Number
Spots per wing (Number of spots) Dlagnosls *
Spots with
Mean number
Frequency of clone formaUon × 10- 5
concentratxon (mM)
of wings
Small single spots (1-2 cells) [m = 2]
Large single spots ( > 2 cells) [m = 5]
Twin spots [m = 5]
Total spots [m = 2]
mwh
of cell dlvlslon cycles
observed
Control (water) Amztrtptyhne
442
0 24 (106)
0 05 (23)
0.02 (7)
0 31 (136)
136
2.10
1.3
0.01 01 10 10.0 50.0 100.0
113 156 146 156 156 28
0.13 0.19 0.25 017 0.24 0.18
(15)(30)(37)(26)(38)(5)-
0.02 ( 2 ) 0.03 ( 4 ) 0.06 ( 9 ) 004 ( 6 ) 0 07 (11)0.07 ( 2 ) -
003 0.04 0.00 0.03 0 02 000
018 0.26 032 024 0.33 0.25
(20)(41)(46)(37)(52)(7)-
19 40 46 35 50 7
2.47 230 1.98 1.86 1.94 1.86
07 1.1 1.3 0.9 1.3 10
-0.6 -0.2 0.0 -03 01 -02
80 80 80 74 158 140 64
017 (14)019 (15)0.32 (26)1.04 (77) + 0.90 (142)+ 2 09 (293)+ 1.78 (114)+
0.04 ( 3 ) 0.08 ( 6 ) 014(11)+ 0.24 (18) + 0.22 (34)+ 0.39 (54)+ 0.31 (20)+
0.03 (2)1 003 (2)1 0.01 ( 1 ) 0.04 (3)1 0.06 (9)+ 0.13 (18)+ 0.14 (9)+
0.24 (19)0.29 (23)0.47 (38)+ 1.32 (98) + 1 17 (185)+ 2 61 (365)+ 2 23 (143)+
18 23 38 98 182 359 143
1 89 2.17 2.05 1.80 1.91 1 79 1 88
0.9 1.2 19 54 4.7 10.5 9.2
-0.3 -0.1 0.7 4.2 3.5 9.3 7.9
74 120 80 120 40 80 24
011 0.21 024 0.34 0.68 0.93 0.67
(8)(25)(19)(41)w (27)+ (74)+ (16)+
0.01 0.01 0.01 0.05 0.13 0.11 0.17
(1)(1)(1)(6)(5)1 (9)w (4)+
001 0.02 0.00 0.04 0.03 0.04 0.00
(1)~ (2)(0)(5)1 (1)1 (3)1 (0)1
0.14 0.23 0.25 0.43 0.82 108 0.83
(10)(28)(20)(52)w (33)+ (86)+ (20)+
10 28 20 52 33 85 20
1.80 175 160 1 90 2.06 1.82 1.85
0.6 1.0 10 18 3.4 4.4 3.4
-07 -0.3 -02 0.5 2.1 3.1 2.2
80 80 80 80 80 80 106
015 0.26 0.30 0.20 0.21 0.28 0.33
(12)(21)(24)(16)(17)(22)(35)-
0.06 ( 5 ) 0.06 ( 5 ) 0.04 ( 3 ) 0.13(10)w 0.10 ( 8 ) 0.09 ( 7 ) 008 ( 9 ) -
004 0.01 0.06 0.01 0.03 005 000
(3)x (1)(5)+ (1)(2)i (4)i (0)-
0.25 0.34 0.40 0.34 0.34 0.41 0.42
(20)(27)(32)(27)(27)(33)(44)-
20 27 32 26 27 33 44
2.60 2.11 213 2.65 2.41 224 2.16
1.0 1.4 16 1.3 14 1.7 1.7
-02 01 0.4 0.1 0.1 0.4 0.4
022 (18)0.20 ( 4 ) -
0.09 ( 7 ) 0.10 (2)1
0.01 ( 1 ) 0.00 (0)1
0.32 (26)030 ( 6 ) -
26 6
2.15 2.00
1.3 1.2
0.1 0.0
(3)~ (7)+ (0)(5)1 (3)(0)1
clone
control corrected
Destpramme 0.001 001 01 1.0 10.0 50.0 100.0
Imlpramme 0001 001 01 1.0 10.0 500 100.0
Normptyhne 0.001 0.01 0.1 1.0 10.0 50.0 100.0
Protrtptyhne 50.0 100.0
80 20
* StatlsUcal diagnoses accordmg to Frel and Wiirgler (1988) multlphcatmn factor Probabthty levels" a = fl = 0.05. One-sided statisttcal tests.
+, positive; - , negative; w, weakly pos~tlve; 1, inconclusive m,
103
DESIPRQMINE CONC.: SO mM ~ T R E A T E D : qO wings
NORTRIPTYLINE
TREATMENT: q8 h [-]CONTROL: 362 ulngs
CONC. : SO mM
TREATMENT: L~8 h
~TREATED:
[~CONTROL: 4q2 w i n g s
80 u l n g s
O
z~ SINGLE SPOTS
0..=:. (,9 512 SPOT SIZE
o,P,, z¢;
THIN SPOTS
"7
~,=,
3-q
2
5-9 9-16 -32 -6q -128 -256 >612 SPOT SIZE
z¢;
THIN SPOTS
~--:.
2o
O¢=,
Z
Z
I--c,
1
2
3-q
5-8 9-16 -32 -61~ -128 -256 >512 SPOT SIZE
Fig. 4. Spot size distributions for single and twin spots obtained with 50 m M deslpramlne.
l
i-~!
TREATMENT: L~8 h [-]CONTROL:362 wings
5-9 9-16 -32 -6q -129 -256 >512 SPOT SIZE
Fig 6. Spot size distributions for single and twin spots obtained with 50 m M n o r t n p t y h n e
IMIPRAMINE
CONC.: SO mM m TREATED: 120 wings
3-q
2
PROTRIPTYLINE CONC.: SO mM
TREATMENT: q8 h
mTREATED:
rTCONTROL: 362 w i n g s
80 w i n g s
SPOTS
SINGLE SPOTS
=, z~
1
2
3-q
z~
5-8 9-16 -32 -6q -128 -256 >512 SPOT SIZE
1
2
3-q
5-8 9-16 -32 -6q -128 -256 >612 SPOT SIZE
THIN SPOTS
THIN SPOTS
-¢
(y) o
S~ o_ Z
~-512 SPOT SIZE
Fig. 5 Spot size d i s t n b u u o n s for single and twin spots obtaaned with 50 m M lrmpramme,
1
2
3-q
5-8 9-16 -32 SPOT SIZE
-6q -129 -256 >512
Fig. 7 Spot size distributions for single and twin spots obtamed with 50 m M protrlptyhne
104
separately, it 1s obvious that for deslpramine they are all significantly increased at the higher concentrations. From the positive result obtained for the twin spots it can be concluded that this compound is recomblnogenic. With itmpramlne the situation is less clear; only the small single and large single spots are significantly increased whereas the twin spots are inconclusive even at the highest concentration. It can be assumed that this is due mainly to the rather small numbers of wings analyzed at the higher concentrations. Because of the greater toxicity of this compound, the number of wings obtained was lower than that obtained at the same concentrations with desipramlne. For this reason the data do not allow a conclusion to be reached about possible recomblnogenic activity of imipramine. The most obvious difference between the 2 TCAs that were positive in this test and the 3 that were negative is the presence of an N atom at position 5 in the central ring of the positive compounds whereas the negative compounds all have a C atom at that position. A reasonable working hypothesis is that N at position 5 is responsible for the genotox~city of this group of compounds in the Drosophila wing spot test. This study demonstrates that the Drosophila somatic mutation and recombination test is useful for the study of structure-activity relationships in an eukaryote in vivo. Our results confirm that certain of the TCAs are genotoxic. Since there do not seem to be great differences in the clinical efficacy of different TCAs (Beaumont, 1989) it would seem advisable to consider possible genotoxicity together with the other factors used by clinicians in drug choice. Acknowledgements
We thank Dr. H. Frei for critically reading the manuscript. Thanks are also due to Doris Singer and Ottavina Lutz for skillful technical assistance.
The work of N.v.S. in Schwerzenbach was supported by a grant from the Swiss Federal Institute of Technology, Zurich. References Alonso Moraga, A , and U Graf (1989) Genotoxiclty testing of
antlparasltlC mtrofurans in the Drosophda somatic mutation and recombination test, Mutagenesls, 4, 105-110 Balbl, A , G Muscettola, N Staiano, G Martlre and F De Lorenzo (1980) Psychotropic drugs evaluation of mutagemc effect, Pharmacol Res Commun, 12, 423-431 Beaumont, G (1989) The toxacity of antidepressants, Br J Psychiatry, 154, 454-458 Flhppova, L M , I.A. Rapoport, Y L Shapiro and Y A. Aleksandrovskn (1975) Mutagenlc actlvlty of psychotrop~c preparaUons, Genetika, 11, 77-82 (in Russlan) Frel, H., and F E Whrgler (1988) Statistical methods to deode whether mutagenlclty test data from Drosophda assays indicate a positive, negative or inconclusive result, Mutauon Res., 203, 297-308 Fu, T K , and L F Jarvlk (1977) The in vitro effects of mupramme on human chromosomes, Mutation Res, 48, 89-94. Garcta-Belhdo, A., and J Dapena (1974) Inductton, detectton and characterizauon of cell dlfferentmtlon mutants in Drosophala, Mol. Gen. Genet, 128, 117-130 Graf, U., F E. Wurgler, A.J. Katz, H. Frei, H. Juon, C.B Hall and P.G Kale (1984) Somatic mutation and recombination test m Drosophda melanogaster, Environ. Mutagen., 6, 153-188 Graf, U., H. Frel, A Kagl, A J Katz and F E. Wlirgler (1989) Thirty compounds tested in the Drosoplula wing spot test, Mutation Res., 222, 359-373 Kawacha, T, T Komatsu, T Kada, M Ishldate, M Sasakl, T. Suglyama and Y Tazama (1980) Results of recent studies on the relevance of various short-term screening tests in Japan, in G.M. Wllhams, R. Kroes, H.W. Waaijers and K W. van de Poll (Eds.), The Predictive Value of Short-Term Screening Tests in Carcmogemoty Evaluation, Elsevier/ North Holland, Amsterdam, pp. 253-267. Lmdsley, D.L., and E.H Grell (1968) Genetic variations of Drosophda melanogaster, Carnegie Inst. Wash Publ. No 627. Paykel, E S, and J L. Wtute (1989) A European study of views on the use of monoamlne oxadase mhlbltors, Br J Psychiatry, 155 (Suppl. 6), 9-17 Saxena, R., and Y R Ahuja (1988) Genotoxaclty evaluation of the trlcychc antidepressants armtriptyhne and imapramme using human lymphocyte cultures, Enwron. Mol. Mutagen, 12, 421-430
99
MUTGEN 01646
Genotoxicity evaluation of five tricyclic antidepressants in the wing somatic m u t a t i o n and r e c o m b i n a t i o n test in Drosophilamelanogaster Nancy van Schaik 1,2 and Ulrich Graf 1 1 Insntute of Toxwology, Swiss Federal Insntute of Technology and Unwerstty of Zurwh, CH-8603 Schwerzenbach (Switzerland) and 2 Department of Genetics, University of the Wltwatersrand, 2050 Johannesburg (R S.A ) (Received 3 September 1990) (Accepted 18 October 1990)
Keywords. Tncychc antidepressants, Genotoxactty, Somatic cells; Drosoplula, Arnltnptyhne, Destpramme, Irmprarmne; Nortrlptyhne, Protnptyhne
Summary Five tricyclic antidepressants were tested for genotoxicity using the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Three-day-old larvae trans-heterozygous for 2 linked recessive wing hair mutants (multiple wing hairs and flare) were fed the test compounds in water mixed with a standard dry food for 48 h. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can be the consequence of either somatic mutation or mitotic recombination. Desipramine and imipramine were clearly genotoxic at concentrations above 1 mM whereas amitriptyline, nortriptyline and protriptyline were not genotoxic at concentrations up to 100 mM. This seems to implicate the nitrogen atom at position 5 in the 7-membered ring of the tricyclic molecule as being responsible for the genotoxic property of the compounds in Drosophila.
Depression is the most common form of mental disease, estimated to affect at least 1 in 10 persons at some time. Clinicians divide the depressions into several distinct disorders but the drugs of choice for the treatment of all of them are the tricyclic antidepressants (TCAs) (Paykel and White, 1989). These drugs have been used widely over the past 30 years and, although considerable information is available about side effects and toxicity at both therapeutic doses and overdose,
Correspondence: Dr. U. Graf, Institute of Toxicology, ETH and University of Zurich, P.O. Box 550, CH-8603 Schwerzenbach (Switzerland)
much less attention has been paid to their possible genotoxicity. What reports exist often give an unclear picture. For example, Balbi et al. (1980) reported that amitriptyline, desipramine, imipramine and nortriptyline were all negative in the Salmonella/microsome test for strains TA1534, TA1537, TA98, and TA100 either with or without $9, while Kawachi et al. (1980) reported that imipramine was positive in TA100 with activation as well as in the Bacillus subtths rec assay. Filippova et al. (1975) found amitriptyline to be negative m the Drosophila CIB sex-linked recessive lethal test but Saroten (a proprietary name for the same compound) positive in the same test. With human lymphocyte cultures, both amitriptyline
100
and imipramine have been reported to mcrease chromosomal aberrations at concentrations above 4 times plasma levels and at upper plasma levels, respectively, by Saxena and Ahuja (1988), whereas Fu and Jarvlk (1977) found no effect for imipramine at similar concentrations. Because of the paucity of reformation on certain TCAs and the conflicting evidence on others, we decided to test this group of compounds in the Drosophila wing somatic mutation and recombination test. In addition, the comparison of the results in this test of a number of chemically slrmlar compounds may help to identify chemical properties responsible for genotoxicity. Materials and m e t h o d s
Chemical compounds The 4 tricyclic antidepressants amitrlptyline hydrochloride (CAS No. 549-18-8), desiprarmne hydrochlorlde (CAS No. 58-28-6), imipramine hydrochlonde (CAS No. 113-52-0) and nortriptyline hydrochloride (CAS No. 894-71-3) were purchased from Sigma (St. Louis, MO, U.S.A.). Protnptyhne hydrochlonde (CAS No. 1225-55-4) was a gift
AMITRIPTYLINE
li
CH--CH2--CH2--N--(CH3)2 C
°PROTRIPTYLINE
I
CH2--CH2--OH2--NH--CH3 C
J~
NORTRIPTYLINE
CH--CH2--CH2--NH--CH3 N
IMIPRAMINE
i
CH2--CH2--CH2--N--(CH3)2 N
DESIPRAMINE
i
CH2--CH2--CH2--NH--OH3
Fig 1. Structural formulas of the 5 tncychc antidepressants *Protnptyhne has an additional double bond m the 7-membered ring between C10 and C l l
from Dr. E. Bachmann (Institute of Toxicology, ETH and Umverslty of Zurich, Schwerzenbach, Switzerland). The structural formulas of the 5 compounds are shown in Fig. 1. For larval feeding the compounds were d~ssolved in distilled water. Larval feedmg Eggs were collected for 8 h in standard culture bottles containing extra live yeast. Three days later, the larvae were removed from the bottles using a 20% sodium chlonde solution. They were then put into plastic wals containing a specml medium with the test compounds. The solutions of the various compounds were used to prepare Drosophila Instant Medium (Formula 4-24, Carolina Biological Supply Co., Burlington, NC, U.S.A.). 5 ml of the solutions was always added to an equal volume of powdered Instant Medium. Negative water controls were included. The larvae were fed on this medium for the rest of their development (approximately 48 h) until pupation. Somatw mutatlon and recombmatton test The following cross of files carrying markers on the left arm of chromosome 3 was used: mwh females mated to fir3/In(3LR)TM3, ri pP sep b x 34e e s Ser males. Detailed information on the genetic markers is given by Lindsley and GreU (1968). The mutation fir 3 is described by GarclaBellido and Dapena (1974). The surviving flies were collected from the treatment vials on days 10-12 after egg laying. Flies of the trans-heterozygous (mwh fir+/mwh ÷ fir 3) genotype were stored in 70% ethanol. For classification, wings were mounted in Faure's solution (gum arabic 30 g, glycerol 20 ml, chloral hydrate 50 g and water 50 ml) and were scored under a compound microscope at 400 × magnification for the occurrence of spots. Different types of spots were recorded separately, namely single spots showing either the multiple wing hairs (mwh) or the flare (fir) phenotype, and twin spots showing adjacent mwh and fir areas. Single spots are produced either by mitotic recombination occurring between the 2 markers or by somatic gene mutation, deletion or another change at one or the other marker locus; twin spots are produced exclusively by rmtotlc recombination occurring between the proximal marker fir and the centromere.
101 In each case, the size of a spot was determined by counting the number of wing cells exhibiting the mutant phenotype. A detailed description of the wing spot test is given by Graf et al. (1984). Data evaluatton and stattsttcal analysts The evaluation of the wing spot data was performed with the computer program S M A R T (Wiirgler, unpublished). For the statistical analysis, the spots as described above were grouped into 3 different categories: (1) small single spots ( 1 - 2 cells in size), (2) large single spots (3 or more cells) and (3) twin spots. These 3 categories of spots were evaluated separately. More details on the statistical analysis are given in Frei and Wiirgler (1988). For the calculations, the K a s t e n b a u m Bowman test was used with P = 0.05. Based on the number of wings analyzed, the number of mwh clones and the number of cells scored in each wing (ca. 24,400), it is possible to calculate the clone formation frequency per cell cycle and 10 s
ca
20
deslpramlne
AMITRIPTYLINE CONC.: SO mM []TREATED:
116
TREATMENT: q.8 h wings
[--]CONTROL:
"~512 SPOT SIZE
TNIN
1
2
3-q
5-8 9-16 -32 SPOT SIZE
-6q
SPOTS
-128
-256 >512
Fxg 3 Spot size dlstnbutLons for single and twin spots obtained with 50 mM armtnptyhne
cells. N o clone size correction was made, because these were chronic treatments with stable compounds (see also Frei and Wiirgler, 1988).
o
Results and d i s c u s s i o n t 5
Imlpramine
OOJ
~
--
i
, a n nl~lq
OOl
Ol
amltrlptyline , , illiwj
I i ~lanlq 1
1 o
a , ,llHq lO
, i IH.. l lOO
concentration (raM)
Fig. 2. Dose-response relatlonslups for armtnptyhne, deslpramlne and lmlprarmne for total spots per wing.
Each of the 5 compounds tested was assayed in at least 2 independent experiments using 2 or more different concentrations. The compounds were tested simultaneously to obtain quantitative data which allow for a comparative analysis. All the data are given in Table 1. A large concurrent negative control series was obtained. The frequency of spontaneously o c c u m n g total spots per wing of 0.31 was within the usual range (Alonso Moraga and Graf, 1989; G r a f et al., 1989). Two of the 5 compounds gave clearly positive results. With desipramine the frequencies of total spots per wing were significantly increased over the control from 0.1 m M and higher, whereas with inupran'une this was the case from 1.0 m M and higher. The corresponding dose-response relationships are plotted in Fig. 2. With imipramine there was a drop in spot frequency at the highest concentration tested.
102
This was probably due to the pronounced toxicity of the compound at this concentration. The other three compounds were non-genotoxic. Amitriptyline and nortriptyline were tested over the same dose range as the 2 positive compounds (i.e., 0.001-100 mM) while protriptyline was tested only
at the 2 highest concentrations (i.e., 50 and 100 mM), and all the results obtained were negative. In Figs. 3-7 the spot size distributions for single and twin spots are given for the 50-raM treatment with each of the 5 compounds. When considering the 3 categories of spots
TABLE 1 SUMMARY OF RESULTS OBTAINED WITH 5 TRICYCLIC ANTIDEPRESSANTS IN THE WING SOMATIC MUTATION AND RECOMBINATION TEST IN Drosophda rnelanogaster Compound
Number
Spots per wing (Number of spots) Dlagnosls *
Spots with
Mean number
Frequency of clone formaUon × 10- 5
concentratxon (mM)
of wings
Small single spots (1-2 cells) [m = 2]
Large single spots ( > 2 cells) [m = 5]
Twin spots [m = 5]
Total spots [m = 2]
mwh
of cell dlvlslon cycles
observed
Control (water) Amztrtptyhne
442
0 24 (106)
0 05 (23)
0.02 (7)
0 31 (136)
136
2.10
1.3
0.01 01 10 10.0 50.0 100.0
113 156 146 156 156 28
0.13 0.19 0.25 017 0.24 0.18
(15)(30)(37)(26)(38)(5)-
0.02 ( 2 ) 0.03 ( 4 ) 0.06 ( 9 ) 004 ( 6 ) 0 07 (11)0.07 ( 2 ) -
003 0.04 0.00 0.03 0 02 000
018 0.26 032 024 0.33 0.25
(20)(41)(46)(37)(52)(7)-
19 40 46 35 50 7
2.47 230 1.98 1.86 1.94 1.86
07 1.1 1.3 0.9 1.3 10
-0.6 -0.2 0.0 -03 01 -02
80 80 80 74 158 140 64
017 (14)019 (15)0.32 (26)1.04 (77) + 0.90 (142)+ 2 09 (293)+ 1.78 (114)+
0.04 ( 3 ) 0.08 ( 6 ) 014(11)+ 0.24 (18) + 0.22 (34)+ 0.39 (54)+ 0.31 (20)+
0.03 (2)1 003 (2)1 0.01 ( 1 ) 0.04 (3)1 0.06 (9)+ 0.13 (18)+ 0.14 (9)+
0.24 (19)0.29 (23)0.47 (38)+ 1.32 (98) + 1 17 (185)+ 2 61 (365)+ 2 23 (143)+
18 23 38 98 182 359 143
1 89 2.17 2.05 1.80 1.91 1 79 1 88
0.9 1.2 19 54 4.7 10.5 9.2
-0.3 -0.1 0.7 4.2 3.5 9.3 7.9
74 120 80 120 40 80 24
011 0.21 024 0.34 0.68 0.93 0.67
(8)(25)(19)(41)w (27)+ (74)+ (16)+
0.01 0.01 0.01 0.05 0.13 0.11 0.17
(1)(1)(1)(6)(5)1 (9)w (4)+
001 0.02 0.00 0.04 0.03 0.04 0.00
(1)~ (2)(0)(5)1 (1)1 (3)1 (0)1
0.14 0.23 0.25 0.43 0.82 108 0.83
(10)(28)(20)(52)w (33)+ (86)+ (20)+
10 28 20 52 33 85 20
1.80 175 160 1 90 2.06 1.82 1.85
0.6 1.0 10 18 3.4 4.4 3.4
-07 -0.3 -02 0.5 2.1 3.1 2.2
80 80 80 80 80 80 106
015 0.26 0.30 0.20 0.21 0.28 0.33
(12)(21)(24)(16)(17)(22)(35)-
0.06 ( 5 ) 0.06 ( 5 ) 0.04 ( 3 ) 0.13(10)w 0.10 ( 8 ) 0.09 ( 7 ) 008 ( 9 ) -
004 0.01 0.06 0.01 0.03 005 000
(3)x (1)(5)+ (1)(2)i (4)i (0)-
0.25 0.34 0.40 0.34 0.34 0.41 0.42
(20)(27)(32)(27)(27)(33)(44)-
20 27 32 26 27 33 44
2.60 2.11 213 2.65 2.41 224 2.16
1.0 1.4 16 1.3 14 1.7 1.7
-02 01 0.4 0.1 0.1 0.4 0.4
022 (18)0.20 ( 4 ) -
0.09 ( 7 ) 0.10 (2)1
0.01 ( 1 ) 0.00 (0)1
0.32 (26)030 ( 6 ) -
26 6
2.15 2.00
1.3 1.2
0.1 0.0
(3)~ (7)+ (0)(5)1 (3)(0)1
clone
control corrected
Destpramme 0.001 001 01 1.0 10.0 50.0 100.0
Imlpramme 0001 001 01 1.0 10.0 500 100.0
Normptyhne 0.001 0.01 0.1 1.0 10.0 50.0 100.0
Protrtptyhne 50.0 100.0
80 20
* StatlsUcal diagnoses accordmg to Frel and Wiirgler (1988) multlphcatmn factor Probabthty levels" a = fl = 0.05. One-sided statisttcal tests.
+, positive; - , negative; w, weakly pos~tlve; 1, inconclusive m,
103
DESIPRQMINE CONC.: SO mM ~ T R E A T E D : qO wings
NORTRIPTYLINE
TREATMENT: q8 h [-]CONTROL: 362 ulngs
CONC. : SO mM
TREATMENT: L~8 h
~TREATED:
[~CONTROL: 4q2 w i n g s
80 u l n g s
O
z~ SINGLE SPOTS
0..=:. (,9 512 SPOT SIZE
o,P,, z¢;
THIN SPOTS
"7
~,=,
3-q
2
5-9 9-16 -32 -6q -128 -256 >612 SPOT SIZE
z¢;
THIN SPOTS
~--:.
2o
O¢=,
Z
Z
I--c,
1
2
3-q
5-8 9-16 -32 -61~ -128 -256 >512 SPOT SIZE
Fig. 4. Spot size distributions for single and twin spots obtained with 50 m M deslpramlne.
l
i-~!
TREATMENT: L~8 h [-]CONTROL:362 wings
5-9 9-16 -32 -6q -129 -256 >512 SPOT SIZE
Fig 6. Spot size distributions for single and twin spots obtained with 50 m M n o r t n p t y h n e
IMIPRAMINE
CONC.: SO mM m TREATED: 120 wings
3-q
2
PROTRIPTYLINE CONC.: SO mM
TREATMENT: q8 h
mTREATED:
rTCONTROL: 362 w i n g s
80 w i n g s
SPOTS
SINGLE SPOTS
=, z~
1
2
3-q
z~
5-8 9-16 -32 -6q -128 -256 >512 SPOT SIZE
1
2
3-q
5-8 9-16 -32 -6q -128 -256 >612 SPOT SIZE
THIN SPOTS
THIN SPOTS
-¢
(y) o
S~ o_ Z
~-512 SPOT SIZE
Fig. 5 Spot size d i s t n b u u o n s for single and twin spots obtaaned with 50 m M lrmpramme,
1
2
3-q
5-8 9-16 -32 SPOT SIZE
-6q -129 -256 >512
Fig. 7 Spot size distributions for single and twin spots obtamed with 50 m M protrlptyhne
104
separately, it 1s obvious that for deslpramine they are all significantly increased at the higher concentrations. From the positive result obtained for the twin spots it can be concluded that this compound is recomblnogenic. With itmpramlne the situation is less clear; only the small single and large single spots are significantly increased whereas the twin spots are inconclusive even at the highest concentration. It can be assumed that this is due mainly to the rather small numbers of wings analyzed at the higher concentrations. Because of the greater toxicity of this compound, the number of wings obtained was lower than that obtained at the same concentrations with desipramlne. For this reason the data do not allow a conclusion to be reached about possible recomblnogenic activity of imipramine. The most obvious difference between the 2 TCAs that were positive in this test and the 3 that were negative is the presence of an N atom at position 5 in the central ring of the positive compounds whereas the negative compounds all have a C atom at that position. A reasonable working hypothesis is that N at position 5 is responsible for the genotox~city of this group of compounds in the Drosophila wing spot test. This study demonstrates that the Drosophila somatic mutation and recombination test is useful for the study of structure-activity relationships in an eukaryote in vivo. Our results confirm that certain of the TCAs are genotoxic. Since there do not seem to be great differences in the clinical efficacy of different TCAs (Beaumont, 1989) it would seem advisable to consider possible genotoxicity together with the other factors used by clinicians in drug choice. Acknowledgements
We thank Dr. H. Frei for critically reading the manuscript. Thanks are also due to Doris Singer and Ottavina Lutz for skillful technical assistance.
The work of N.v.S. in Schwerzenbach was supported by a grant from the Swiss Federal Institute of Technology, Zurich. References Alonso Moraga, A , and U Graf (1989) Genotoxiclty testing of
antlparasltlC mtrofurans in the Drosophda somatic mutation and recombination test, Mutagenesls, 4, 105-110 Balbl, A , G Muscettola, N Staiano, G Martlre and F De Lorenzo (1980) Psychotropic drugs evaluation of mutagemc effect, Pharmacol Res Commun, 12, 423-431 Beaumont, G (1989) The toxacity of antidepressants, Br J Psychiatry, 154, 454-458 Flhppova, L M , I.A. Rapoport, Y L Shapiro and Y A. Aleksandrovskn (1975) Mutagenlc actlvlty of psychotrop~c preparaUons, Genetika, 11, 77-82 (in Russlan) Frel, H., and F E Whrgler (1988) Statistical methods to deode whether mutagenlclty test data from Drosophda assays indicate a positive, negative or inconclusive result, Mutauon Res., 203, 297-308 Fu, T K , and L F Jarvlk (1977) The in vitro effects of mupramme on human chromosomes, Mutation Res, 48, 89-94. Garcta-Belhdo, A., and J Dapena (1974) Inductton, detectton and characterizauon of cell dlfferentmtlon mutants in Drosophala, Mol. Gen. Genet, 128, 117-130 Graf, U., F E. Wurgler, A.J. Katz, H. Frei, H. Juon, C.B Hall and P.G Kale (1984) Somatic mutation and recombination test m Drosophda melanogaster, Environ. Mutagen., 6, 153-188 Graf, U., H. Frel, A Kagl, A J Katz and F E. Wlirgler (1989) Thirty compounds tested in the Drosoplula wing spot test, Mutation Res., 222, 359-373 Kawacha, T, T Komatsu, T Kada, M Ishldate, M Sasakl, T. Suglyama and Y Tazama (1980) Results of recent studies on the relevance of various short-term screening tests in Japan, in G.M. Wllhams, R. Kroes, H.W. Waaijers and K W. van de Poll (Eds.), The Predictive Value of Short-Term Screening Tests in Carcmogemoty Evaluation, Elsevier/ North Holland, Amsterdam, pp. 253-267. Lmdsley, D.L., and E.H Grell (1968) Genetic variations of Drosophda melanogaster, Carnegie Inst. Wash Publ. No 627. Paykel, E S, and J L. Wtute (1989) A European study of views on the use of monoamlne oxadase mhlbltors, Br J Psychiatry, 155 (Suppl. 6), 9-17 Saxena, R., and Y R Ahuja (1988) Genotoxaclty evaluation of the trlcychc antidepressants armtriptyhne and imapramme using human lymphocyte cultures, Enwron. Mol. Mutagen, 12, 421-430
