TY - JOUR
AU - Heres-Pulido, María, Eugenia
AB - Abstract Chagas disease, caused by the protozoan Trypanosoma cruzi, has increased in the world due to migration, travelling and climate change; at present, the principal problem is that common trypanocidal agents have resulted in toxic or inconvenient side effects. We tested for genotoxicity in the standard (ST) and high bioactivation (HB) crosses of Drosophila wing somatic mutation and recombination test, four novel trypanocidal agents derived from 2, 4, 6-triaminquinazoline (TAQ): 2,4-diamino-6 nitro-1,3 diazonaftalene (S-1QN2-1), 2,4-diacetamino-6-amino 1,3 diazonaftalene (D-1), N6-(4,methoxybenzyl)quinazoline-2,4,6-triamine (GHPM) and N6-[4-(trifluoromethoxy)benzyl]quinazoline-2,4,6-triamine (GHPMF) at 1.9, 3.9, 7.9 and 15 µM, respectively. Also, high-pressure liquid chromatography (HPLC) analysis was run to determine the remanence of either drug in flare, and Oregon R(R)-flare flies emerged from treated larvae. S-1QN2-1 showed genotoxicity only in the ST cross, increasing the small, large and total spot frequencies at all concentrations and twin spots only at 1.9 µM; D-1 and GHPM showed significant increments of large spots only at 15 µM in the ST cross; GHPMF was not genotoxic at any concentration or either cross. In the mwh clones accumulated distribution frequencies analysis, associated with disrupted cell division, S-1QN2-1 caused alterations in the ST cross at all concentrations but only at 15 µM in the HB cross; D-1 caused alterations at 3.9, 7.9 and 15 µM in the ST cross and at 1.9 and 15 µM in the HB cross; GHPM caused alterations at 7.9 and 15 µM in the ST cross and also at 1.9, 3.9 and 7.9 µM in the HB cross; GHPMF caused those alterations at all concentrations in the ST cross and at 1.9, 3.9 and 7.9 µM in the HB cross. The HPLC results indicated no traces of either agent in the flare and Oregon R(R)-flare flies. We conclude that S-1QN2-1 is clearly genotoxic, D-1 and GHPM have an unclear genotoxicity and GHPMF was not genotoxic; all quinazoline derivatives disrupted cell division. GHPMF is a good candidate to be tested in other genotoxicity and cytotoxic bioassays. The differences in the genotoxic activity of these trypanocidal agents are correlated with differences in their chemical structure. Introduction Chagas disease is an endemic disease in Latin America, especially in poor and tropical regions, and presents two phases: one acute asymptomatic and another chronic with severe cardiac and gastrointestinal complications (mega syndromes) ending in sudden death by visceral collapse (1,2). In 2010, the World Health Organization reported approximately 10 million people infected with Chagas disease and more than 25 million in endemic zones at risk of acquiring it (3). Nowadays, due to migration, tourism processes and climate change, the disease has also spread to non-endemic zones, such as Canada, USA, Japan and several Europe’s cities where 68 000 to 123 000 people have been reported infected with Trypanosoma cruzi (1,4). The protozoan T. cruzi is transmitted to humans mainly by infected triatomine vectors (3). In Bolivia, Argentina and Chile, the vector is known as ‘vinchuca’, ‘chipo’ in Colombia, ‘kissing bugs’ in Mexico and ‘barbeiro’ in Brazil (5). These triatomines are haematophagous insects that leave its faeces on the injured skin of mammals (6). In North America, 40 species of triatomines vectors have been reported (3); in Mexico, there are at least seven genus: Belminus, Dipetalogaster, Eratyrus, Paratriatoma, Panstrongylus, Rhodnius and Triatoma, and the main vectors are: Triatoma barberi, Triatoma dimidiata, Triatoma gerstaeckeri, Triatoma longipennis, Triatoma mazzottii, Triatoma infestan, Triatoma mexicana, Triatoma pallidipennis, Triatoma phyllosoma y Triatoma picturata (3,7). Only two drugs have been clinically used as treatment for this illness: Nifurtimox [l-((5-nitrofurfurylidene)amino)-3-methyl-thiomorpholine-l, 1-dioxide] (NFX) commercially registered as Lampit® from Bayer and Benznidazol [N-benzyl-2-(2-nitroimidazol-1-yl)acetamide] (BNZ) from Hoffman-La Roche and registered as Rochagan® or Radanil® (8); however, both compounds are ineffective during the chronic phase (9), show toxicity (1) and produce important side effects: nausea, diarrhoea, weight loss, irritability, sleep disorders and periphery neuropathies (10); furthermore, both of them have been determined as genotoxic compounds in various assays (11–16), including the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster (17). Chaga’s therapy can be focussed on the specific inhibition of the bifunctional dihydrofolate reductase-thymidylate synthase of T. cruzi implied in the biosynthesis of purines, conversion of dUMP to dTMP and some amino acids (18). Our research group synthesised four novel molecules that eliminate all the stages of development of T. cruzi [2,4-diamino-6 nitro-1,3 diazonaftalene (S-1QN2-1) (Figure 1B); 2,4-diacetamino-6-amino 1,3 diazonaftalene (D-1)] (Figure 1C) (unpublished results); [N6-(4,methoxybenzyl)quinazoline-2,4,6-triamine (GHPM) (Figure 1D) and N6-[4-(trifluoromethoxy)benzyl]quinazoline-2,4,6-triamine (GHPMF) (Figure 1E)] (19), all of them derived from 2,4,6-triaminquinazoline (TAQ) (Figure 1A), which is a specific inhibitor of Dihydrofolate Reductase (DHFR) in T. cruzi (19). Mendoza-Martínez et al. (19) determined that human and T. cruzi DHFR consist of three regions, with Region I being the most important for the trypanocidal effect, because T. cruzi DHFR present aminoacids with aromatic rings (Phe or Tyr), while human DHFR is more hydrophilic (Asn64), which implies a greater especifity between quinazolines derivates and DHFR of T. cruzi. Figure 1. Open in new tabDownload slide Chemical structures of TAQ (A), S-1QN2-1 (B), D-1 (C), GHPM (D) and GHPMF (E). Figure 1. Open in new tabDownload slide Chemical structures of TAQ (A), S-1QN2-1 (B), D-1 (C), GHPM (D) and GHPMF (E). The trypanocidal molecules were designed by means of strategies of assembly of pharmacophore fragments. The S-1QN2-1 (Figure 1B) was the first TAQ-derived trypanocidal agent to which an amino group was substituted in the sixth position of the TAQ molecule by a nitro group; this decision was made considering there are compounds containing a nitroaromatic group, such as BNZ (8), that have shown high trypanocidal effects; the S-1QN2-1 was an intermediate that lead to the final GHPM and GHPMF compounds. The objective was to use it as a ‘molecular probe’ to determine the trypanocidal activity of the base compound and subsequently synthesise another molecule of the same size but without the nitro group, since the mutagenic and clastogenic (11), genotoxic (11,20,21) and recombinogenic (17) effects of the nitro group are well documented. D-1 molecule (Figure 1C) is a derived trypanocidal agent in which the nitro group in the sixth position of S-1QN2-1 was substituted by an amino group and also two dicetoamine groups were added to the second and fourth positions of the molecule. On the other hand, GHPM (Figure 1D) is a derived molecule in which the nitro group in the sixth position of the TAQ molecule of S-1QN2-1 was substituted by an amino group and was added a methoxybenzyl group in this position. Finally, GHPMF (Figure 1E) is a GHPM-derived agent in which the methoxyl group was substituted by a trifluoromethoxyl group in the fifth position of the benzyl group of the TAQ molecule, which has shown an increase in the selectivity for Region I of T. cruzi DHFR enzyme (19). These four compounds have not been previously assessed through any genotoxicity test because they were recently synthesised by our group and are not commercially available. As it is well known, in the development process of novel drugs, it is necessary to conduct several toxicity and genotoxicity tests in different biological models in order to show if they are innocuous, even more so when toxicological studies of some TAQ-derived compounds have shown toxic activity. Davoll et al. (22) synthesised GHPM for the first time and found in Canis lupus familiaris clinical manifestations as diarrhoea, vomit, anorexia and weight loss at larger doses than 100 mg/kg; another TAQ-derived compound the 2-isopropyl-3 -butyl-8-(4-fluorophenylamine)-3H-imidazole[4,5-g]quinazoline has shown apoptotic effects in cancer cell lines (HepG2, SH-SY5Y, DU145, MCF-7 and A549) (23); the quinazoline nitrogen mustard derivatives produced apoptosis in the gastric epithelium cell line (GES-1) (24); on the other hand, 4-anilinoquinazoline-derived molecules showed cytotoxic activity in the L1210 cancer cell line and they induced programmed cell death in leukemia cells through the mitochondrial/caspase 9-/caspase 3-dependent pathway (25). In spite of this, our research group has obtained positive effects: Mendoza-Martínez et al. (19) worked with mammalian VERO cell line at 32 and 20 µM of GHPM and GHPMF, respectively; they did not find toxicity in murine models of cerebral malaria, caused by Plasmodium bergei, and concluded that GHPMF has a good antioxidant potential. There is a wide range of preclinic in vitro and in vivo genotoxicity tests that have not been conducted with these compounds since they are new synthetic molecules. As a first approach, we decided to use the Drosophila wing SMART bioassay because it shows advantages with respect to other systems (26–29). Drosophila melanogaster performs in Malpighi tubules, midgut and fat body analogues functions to liver and immunity system of vertebrates (30); also, Danielson et al. (31) demonstrated a strong homology between the CYP6 insect family and the CYP3 and CYP2 families of vertebrates so that the xenobiotic results obtained with D. melanogaster can be similar to those found in vertebrates. Moreover, between Drosophila and mammals, the global identity of nucleotides or amino acids is approximately of 40% homology; nevertheless, for functional domains, it can be from 80% to 90% (26). Drosophila melanogaster also presents 61.24% of orthologous genes coding for human diseases (32). The D. melanogaster DHFR shares many properties with the human DHFR protein: 17 residues are involved in substrate binding, and the inhibitor and the cofactor are identical in both organisms and differ to that of T. cruzi (18). The N-terminal sequence of Drosophila DHFR is more closely related to vertebrate than to prokaryotic DHFRs, and the Drosophila and vertebrate DHFRs show optimum enzyme activity at both an acidic (4.7) and basic (8.5) pH value but differ with protozoa, plants and most prokaryotic enzymes, which have only optimum function near pH 6–7 (33). The Drosophila wing SMART is a genotoxicity bioassay that can indicate the risk of cancer when patients are exposed to some drugs’ prescriptions (34,35). This assay is based on the loss of heterozygosity of mutant recessive markers flr3 (on chromosome 3L, 39) and mwh (on chromosome 3L, 0.0) (36,37) with phenotypical expression in the wings of adult organisms due to genotoxic activity of chemical compounds that induce point mutations, deletions, somatic recombination and aneuploidy (38). This test uses two crosses: the standard (ST) and the high bioactivation (HB) that differ in their expression levels of Cyp450s (39). The HB cross has a high expression of Cyp6a2 gene (40), allowing to assess the involvement of Cyp450s in the xenobiotic metabolism of promutagen compounds (41). For all the above reasons, the aim of this work was to assess the genotoxicity of trypanocidal agents S-1QN2-1, D-1, GHPM and GHPMF through the Drosophila wing SMART using promutagen N-nitrosodimethylamine (DMN) as a positive control because it is an indirect monofunctional alkylating agent that produces alkylations in guanine (7-methylguanine, 7-meG) (42) and DNA strand brakes (43). DMN genotoxicity has been demonstrated in the Drosophila wing SMART (38), as well as the participation of Cyp450s in its differential biotransformation in both crosses of this bioassay (44). Materials and methods Chemicals Ethanol (EtOH) high pressure liquid chromatography (HPLC), CAS No. 64-17-5 (Productos Químicos Monterrey, Monterrey, N.L., Mexico); DMN, CAS No. 62-75-9 (Sigma Aldrich. St. Louis, Missouri, USA); Drosophila Instant Medium (DIM; Carolina Biological Supply Co., Burlington, NC, USA). Chemicals used for the HPLC: methanol HPLC (MeOH), CAS No. 67-56-1 (JT Baker, Mexico); phosphoric acid HPLC, CAS No. 7664-38-2 (JT Baker); acetonitrile (ACN) HPLC, CAS No. 75-05-8 (JT Baker). Trypanocidal agents: S-1QN2-1, D-1, GHPM and GHPMF were synthesised and verified according to Mendoza-Martínez et al. (19). Strains The D. melanogaster mutant strains: flare (flr3/In(3LR)TM3, ri ppsep bx34eesSer), Oregon R(R)-flare (ORR (1); ORR (2); flr3/In(3LR)TM3, ri ppsep bx34eesSer) and multiple wing hairs (mwh/mwh) were originally donated by Dr Ulrich Graf of the ETH, Zurich, Switzerland [for detailed information on the genetic markers see (36), (37), (45), (46)]. Drosophila wing SMART crosses Virgin female flies of the flare and Oregon R(R)-flare strains were mated to multiple wing hairs male flies to perform the ST and HB crosses, respectively. Eggs from both crosses were collected independently in a fresh baker´s yeast culture media for 8 h at 25°C and 65% of relative humidity (RH). Three days later, third instar larvae (72 ± 4 h) from each cross were recovered and used to carry out all the experiments. Drosophila wing SMART experiments Three independent experiments were conducted with five replicates per treatment. Larvae (72 ± 4 h) of the ST and HB crosses were chronically fed with 0.5 g of DIM and 2 ml of S-1QN2-1, D-1, GHPM or GHPMF at 1.9, 3.9, 7.9 and 15 µM, respectively, all of them dissolved in EtOH 2%; all vials were incubated until emergency at 25°C and 65% of RH; these concentrations were determined based on: (i) previous results from toxicity test and (ii) the therapeutic concentration of GHMPF against T. cruzi used for treatment in mice (9 µM) and which is within this range (data not shown); milliQ water was used as negative dissolvent control, EtOH 2% was used as dissolvent control and DMN (76 µM), dissolved in milliQ water, was the positive control. Trans-heterozygous (mwh flr3+/mwh+flr3) adult flies from treated third instar larvae were collected in alcohol 70%; each pair of wings was dissected and mounted in permanent slides made with Entellan®; afterwards, the wings of 55–60 flies (47) were scored under the optic microscope (400×). In each spot or clone, we count the number of mwh or flare trichomes produced by each mutant cell. Spots can be small, large, twins and their total (Table 1). Small ones have one to two trichomes; large ones have three or more trichomes and twin spots have mwh and flare trichomes because the somatic recombination between 3L chromosome takes place near the centromeres. Small spots are produced by aneuploidy or by intermediate compounds derived lately from the metabolism of the compound tested. Large spots have many cells because the mutation occurred early in some of the ~50 cells that each imaginal wing disk has at approximately 72 h of life. Ending the scoring of wings, we capture the data in the SMART software (Frei and Würgler, unpublished) which is based on the Kastenbaum–Bowman test (P < 0.05) and calculates the corresponding frequencies/fly and the mwh clone size for each treatment and cross (38). Also, we ran the Mann–Whitney-Wilcoxon U-test using the STAT Graphics version 6.0 software (P < 0.05) (48). On the other hand, the Kolmogorov–Smirnov test was performed to statistically analyse the accumulated mwh clones size distribution in each treatment against the corresponding control (P < 0.05); positive results indicate significant alteration of the cell division on imaginal wing cells, therefore, cytotoxic effects (38). In the three statistical tests, the milliQ water negative dissolvent control was used to determine the significant differences of the EtOH 2% and DMN controls, while EtOH 2% was used as a negative control to determine the significant differences of the treatments, of which EtOH 2% is the dissolvent and, thus, avoid the putative variation it could cause. Table 1. Summary of results obtained in the ST and HB crosses of the Drosophila wing SMART after scoring marker-heterozygous flies (mwh+/+flr3 or OR(R); mwh+/+flr3) wild-type wings Compound cross type Conc. (%, µM) Number of flies Spots per fly (number of spots) statistical diagnosisa Mean mwh clone size class Clone formation per 105 cells per cell divisionb Small single spots (1–2 cells) m = 2 Large single spots (>2 cells) m = 5 Twin spots m = 5 Total spots m = 2 mwh clones Observed Control corrected Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.93 (056) − 0.12 (007) − 0.00 (000) − 1.05 (063) − 63 1.59 4.3 −0.8  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4 −0.1  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 S-1QN2-1 treatments ST  EtOH 2 60 0.93 (056) 0.12 (007) 0.00 (000) 1.05 (063) 63 1.59 4.3  S-1QN2-1 1.9 60 2.05 (123) + 1.50 (090) + 0.20 (012) + 3.75 (225) + 225 2.51 15.4 11.1  S-1QN2-1 3.9 60 2.05 (123) + 0.50 (030) + 0.02 (001) − 2.57 (154) + 154 1.60 10.5 6.2  S-1QN2-1 7.9 60 1.88 (113) + 0.62 (037) + 0.05 (003) − 2.55 (153) + 153 1.91 10.5 6.2  S-1QN2-1 15.0 60 1.40 (084) + 0.20 (012) + 0.03 (002) − 1.63 (098) + 98 1.51 6.7 2.4 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  S-1QN2-1 1.9 60 0.48 (029) − 0.05 (003) − 0.02 (001) − 0.55 (033) − 33 1.52 2.3 −0.1  S-1QN2-1 3.9 60 0.53 (032) − 0.05 (003) − 0.00 (000) − 0.58 (035) − 33 1.39 2.3 −0.1  S-1QN2-1 7.9 60 0.45 (027) − 0.07 (004) − 0.03 (002) − 0.55 (033) − 33 1.97 2.3 −0.1  S-1QN2-1 15.0 58 0.33 (019) ↓ 0.09 (005) − 0.02 (001) − 0.43 (025) ↓ 25 2.0 1.8 −0.6 Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 D-1 treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  D-1 1.9 68 0.74 (050) − 0.09 (006) − 0.01 (001) − 0.84 (057) − 57 1.63 3.4 −0.3  D-1 3.9 55 0.80 (044) − 0.04 (002) − 0.02 (001) − 0.85 (047) − 47 1.45 3.5 −0.3  D-1 7.9 54 0.72 (039) − 0.07 (004) − 0.00 (000) − 0.80 (043) − 43 1.58 3.3 −0.5  D-1 15.0 61 0.88 (054) − 0.20 (012) + 0.03 (002) − 1.11 (068) − 68 1.72 4.6 0.8 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  D-1 1.9 56 0.64 (036) − 0.04 (002) − 0.00 (000) − 0.68 (038) − 37 1.59 2.7 0.3  D-1 3.9 56 0.46 (026) − 0.12 (007) − 0.02 (001) − 0.61 (034) − 34 2.12 2.5 0.1  D-1 7.9 58 0.40 (023) − 0.10 (006) − 0.02 (001) − 0.52 (030) − 30 1.83 2.1 −0.3  D-1 15.0 59 0.68 (040) − 0.08 (005) − 0.02 (001) − 0.78 (046) − 46 1.93 3.2 0.8 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPM treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPM 1.9 60 0.80 (048) − 0.10 (006) − 0.03 (002) − 0.93 (056) − 55 1.58 3.8 0.0  GHPM 3.9 60 0.82 (049) − 0.12 (007) − 0.08 (005) − 1.02 (061) − 59 1.86 4.0 0.3  GHPM 7.9 60 0.73 (044) − 0.03 (002) − 0.00 (000) − 0.77 (046) − 46 1.48 3.1 −0.6  GHPM 15.0 60 0.58 (035) − 0.20 (012) + 0.03 (002) − 0.82 (049) − 46 1.87 3.1 −0.6 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPM 1.9 60 0.65 (039) − 0.07 (004) − 0.03 (002) − 0.75 (045) − 45 1.64 3.1 0.2  GHPM 3.9 57 0.67 (038) − 0.14 (008) − 0.05 (003) − 0.86 (049) − 49 1.88 3.5 0.7  GHPM 7.9 60 0.92 (055) − 0.08 (005) − 0.02 (001) − 1.02 (061) − 60 1.52 4.1 1.3  GHPM 15.0 60 0.63 (038) − 0.05 (003) − 0.05 (003) − 0.73 (044) − 42 1.57 2.9 0.0 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPMF treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPMF 1.9 60 0.63 (038) − 0.10 (006) − 0.07 (004) − 0.80 (048) − 44 1.68 3.0 −0.8  GHPMF 3.9 60 0.67 (040) − 0.07 (004) − 0.02 (001) − 0.75 (045) − 43 1.51 2.9 −0.8  GHPMF 7.9 60 0.70 (042) − 0.07 (004) − 0.07 (004) − 0.83 (050) − 47 1.60 3.2 −0.5  GHPMF 15.0 60 0.58 (035) − 0.08 (005) − 0.07 (004) − 0.73 (044) − 43 1.72 2.9 −0.8 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPMF 1.9 60 0.75 (045) − 0.17 (010) − 0.07 (004) − 0.98 (059) − 59 1.95 4.0 1.2  GHPMF 3.9 59 0.75 (044) − 0.14 (008) − 0.03 (002) − 0.92 (054) − 54 2.28 3.8 0.9  GHPMF 7.9 57 0.53 (030) − 0.07 (004) − 0.07 (004) − 0.61 (035) − 35 1.8 2.5 −0.3  GHPMF 15.0 57 0.60 (034) − 0.09 (005) − 0.05 (003) − 0.74 (042) − 40 1.8 2.9 0.0 Compound cross type Conc. (%, µM) Number of flies Spots per fly (number of spots) statistical diagnosisa Mean mwh clone size class Clone formation per 105 cells per cell divisionb Small single spots (1–2 cells) m = 2 Large single spots (>2 cells) m = 5 Twin spots m = 5 Total spots m = 2 mwh clones Observed Control corrected Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.93 (056) − 0.12 (007) − 0.00 (000) − 1.05 (063) − 63 1.59 4.3 −0.8  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4 −0.1  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 S-1QN2-1 treatments ST  EtOH 2 60 0.93 (056) 0.12 (007) 0.00 (000) 1.05 (063) 63 1.59 4.3  S-1QN2-1 1.9 60 2.05 (123) + 1.50 (090) + 0.20 (012) + 3.75 (225) + 225 2.51 15.4 11.1  S-1QN2-1 3.9 60 2.05 (123) + 0.50 (030) + 0.02 (001) − 2.57 (154) + 154 1.60 10.5 6.2  S-1QN2-1 7.9 60 1.88 (113) + 0.62 (037) + 0.05 (003) − 2.55 (153) + 153 1.91 10.5 6.2  S-1QN2-1 15.0 60 1.40 (084) + 0.20 (012) + 0.03 (002) − 1.63 (098) + 98 1.51 6.7 2.4 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  S-1QN2-1 1.9 60 0.48 (029) − 0.05 (003) − 0.02 (001) − 0.55 (033) − 33 1.52 2.3 −0.1  S-1QN2-1 3.9 60 0.53 (032) − 0.05 (003) − 0.00 (000) − 0.58 (035) − 33 1.39 2.3 −0.1  S-1QN2-1 7.9 60 0.45 (027) − 0.07 (004) − 0.03 (002) − 0.55 (033) − 33 1.97 2.3 −0.1  S-1QN2-1 15.0 58 0.33 (019) ↓ 0.09 (005) − 0.02 (001) − 0.43 (025) ↓ 25 2.0 1.8 −0.6 Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 D-1 treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  D-1 1.9 68 0.74 (050) − 0.09 (006) − 0.01 (001) − 0.84 (057) − 57 1.63 3.4 −0.3  D-1 3.9 55 0.80 (044) − 0.04 (002) − 0.02 (001) − 0.85 (047) − 47 1.45 3.5 −0.3  D-1 7.9 54 0.72 (039) − 0.07 (004) − 0.00 (000) − 0.80 (043) − 43 1.58 3.3 −0.5  D-1 15.0 61 0.88 (054) − 0.20 (012) + 0.03 (002) − 1.11 (068) − 68 1.72 4.6 0.8 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  D-1 1.9 56 0.64 (036) − 0.04 (002) − 0.00 (000) − 0.68 (038) − 37 1.59 2.7 0.3  D-1 3.9 56 0.46 (026) − 0.12 (007) − 0.02 (001) − 0.61 (034) − 34 2.12 2.5 0.1  D-1 7.9 58 0.40 (023) − 0.10 (006) − 0.02 (001) − 0.52 (030) − 30 1.83 2.1 −0.3  D-1 15.0 59 0.68 (040) − 0.08 (005) − 0.02 (001) − 0.78 (046) − 46 1.93 3.2 0.8 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPM treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPM 1.9 60 0.80 (048) − 0.10 (006) − 0.03 (002) − 0.93 (056) − 55 1.58 3.8 0.0  GHPM 3.9 60 0.82 (049) − 0.12 (007) − 0.08 (005) − 1.02 (061) − 59 1.86 4.0 0.3  GHPM 7.9 60 0.73 (044) − 0.03 (002) − 0.00 (000) − 0.77 (046) − 46 1.48 3.1 −0.6  GHPM 15.0 60 0.58 (035) − 0.20 (012) + 0.03 (002) − 0.82 (049) − 46 1.87 3.1 −0.6 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPM 1.9 60 0.65 (039) − 0.07 (004) − 0.03 (002) − 0.75 (045) − 45 1.64 3.1 0.2  GHPM 3.9 57 0.67 (038) − 0.14 (008) − 0.05 (003) − 0.86 (049) − 49 1.88 3.5 0.7  GHPM 7.9 60 0.92 (055) − 0.08 (005) − 0.02 (001) − 1.02 (061) − 60 1.52 4.1 1.3  GHPM 15.0 60 0.63 (038) − 0.05 (003) − 0.05 (003) − 0.73 (044) − 42 1.57 2.9 0.0 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPMF treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPMF 1.9 60 0.63 (038) − 0.10 (006) − 0.07 (004) − 0.80 (048) − 44 1.68 3.0 −0.8  GHPMF 3.9 60 0.67 (040) − 0.07 (004) − 0.02 (001) − 0.75 (045) − 43 1.51 2.9 −0.8  GHPMF 7.9 60 0.70 (042) − 0.07 (004) − 0.07 (004) − 0.83 (050) − 47 1.60 3.2 −0.5  GHPMF 15.0 60 0.58 (035) − 0.08 (005) − 0.07 (004) − 0.73 (044) − 43 1.72 2.9 −0.8 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPMF 1.9 60 0.75 (045) − 0.17 (010) − 0.07 (004) − 0.98 (059) − 59 1.95 4.0 1.2  GHPMF 3.9 59 0.75 (044) − 0.14 (008) − 0.03 (002) − 0.92 (054) − 54 2.28 3.8 0.9  GHPMF 7.9 57 0.53 (030) − 0.07 (004) − 0.07 (004) − 0.61 (035) − 35 1.8 2.5 −0.3  GHPMF 15.0 57 0.60 (034) − 0.09 (005) − 0.05 (003) − 0.74 (042) − 40 1.8 2.9 0.0 Trypanocidal agents S-1QN2-1, D-1, GHPM and GHPMF (1.9, 3.9, 7.9 and 15.0 µM), DMN (76 µM), EtOH [2%] and milliQ water. aStatistical diagnoses according to Frei and Würgler (47). m: minimal risk multiplication factor for the assessment of negative results. For the final statistical diagnosis of all positive (+), negative (−) and significant reduction (↓) outcomes; the non-parametric Mann–Whitney and Wilcoxon U-test with significance levels α and β = 0.05 was used in order to exclude false-positive or negative diagnoses (38). One-side binomial tests, significance levels α and β: significative results: + (α ≤ 0.05); no significant results: − (β ≤ 0.05). bClone frequencies per fly divided by the number of cells examined per fly (48 800) gives an estimate of formation frequencies per cell and per cell division in chronic exposure experiments (48). Open in new tab Table 1. Summary of results obtained in the ST and HB crosses of the Drosophila wing SMART after scoring marker-heterozygous flies (mwh+/+flr3 or OR(R); mwh+/+flr3) wild-type wings Compound cross type Conc. (%, µM) Number of flies Spots per fly (number of spots) statistical diagnosisa Mean mwh clone size class Clone formation per 105 cells per cell divisionb Small single spots (1–2 cells) m = 2 Large single spots (>2 cells) m = 5 Twin spots m = 5 Total spots m = 2 mwh clones Observed Control corrected Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.93 (056) − 0.12 (007) − 0.00 (000) − 1.05 (063) − 63 1.59 4.3 −0.8  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4 −0.1  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 S-1QN2-1 treatments ST  EtOH 2 60 0.93 (056) 0.12 (007) 0.00 (000) 1.05 (063) 63 1.59 4.3  S-1QN2-1 1.9 60 2.05 (123) + 1.50 (090) + 0.20 (012) + 3.75 (225) + 225 2.51 15.4 11.1  S-1QN2-1 3.9 60 2.05 (123) + 0.50 (030) + 0.02 (001) − 2.57 (154) + 154 1.60 10.5 6.2  S-1QN2-1 7.9 60 1.88 (113) + 0.62 (037) + 0.05 (003) − 2.55 (153) + 153 1.91 10.5 6.2  S-1QN2-1 15.0 60 1.40 (084) + 0.20 (012) + 0.03 (002) − 1.63 (098) + 98 1.51 6.7 2.4 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  S-1QN2-1 1.9 60 0.48 (029) − 0.05 (003) − 0.02 (001) − 0.55 (033) − 33 1.52 2.3 −0.1  S-1QN2-1 3.9 60 0.53 (032) − 0.05 (003) − 0.00 (000) − 0.58 (035) − 33 1.39 2.3 −0.1  S-1QN2-1 7.9 60 0.45 (027) − 0.07 (004) − 0.03 (002) − 0.55 (033) − 33 1.97 2.3 −0.1  S-1QN2-1 15.0 58 0.33 (019) ↓ 0.09 (005) − 0.02 (001) − 0.43 (025) ↓ 25 2.0 1.8 −0.6 Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 D-1 treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  D-1 1.9 68 0.74 (050) − 0.09 (006) − 0.01 (001) − 0.84 (057) − 57 1.63 3.4 −0.3  D-1 3.9 55 0.80 (044) − 0.04 (002) − 0.02 (001) − 0.85 (047) − 47 1.45 3.5 −0.3  D-1 7.9 54 0.72 (039) − 0.07 (004) − 0.00 (000) − 0.80 (043) − 43 1.58 3.3 −0.5  D-1 15.0 61 0.88 (054) − 0.20 (012) + 0.03 (002) − 1.11 (068) − 68 1.72 4.6 0.8 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  D-1 1.9 56 0.64 (036) − 0.04 (002) − 0.00 (000) − 0.68 (038) − 37 1.59 2.7 0.3  D-1 3.9 56 0.46 (026) − 0.12 (007) − 0.02 (001) − 0.61 (034) − 34 2.12 2.5 0.1  D-1 7.9 58 0.40 (023) − 0.10 (006) − 0.02 (001) − 0.52 (030) − 30 1.83 2.1 −0.3  D-1 15.0 59 0.68 (040) − 0.08 (005) − 0.02 (001) − 0.78 (046) − 46 1.93 3.2 0.8 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPM treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPM 1.9 60 0.80 (048) − 0.10 (006) − 0.03 (002) − 0.93 (056) − 55 1.58 3.8 0.0  GHPM 3.9 60 0.82 (049) − 0.12 (007) − 0.08 (005) − 1.02 (061) − 59 1.86 4.0 0.3  GHPM 7.9 60 0.73 (044) − 0.03 (002) − 0.00 (000) − 0.77 (046) − 46 1.48 3.1 −0.6  GHPM 15.0 60 0.58 (035) − 0.20 (012) + 0.03 (002) − 0.82 (049) − 46 1.87 3.1 −0.6 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPM 1.9 60 0.65 (039) − 0.07 (004) − 0.03 (002) − 0.75 (045) − 45 1.64 3.1 0.2  GHPM 3.9 57 0.67 (038) − 0.14 (008) − 0.05 (003) − 0.86 (049) − 49 1.88 3.5 0.7  GHPM 7.9 60 0.92 (055) − 0.08 (005) − 0.02 (001) − 1.02 (061) − 60 1.52 4.1 1.3  GHPM 15.0 60 0.63 (038) − 0.05 (003) − 0.05 (003) − 0.73 (044) − 42 1.57 2.9 0.0 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPMF treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPMF 1.9 60 0.63 (038) − 0.10 (006) − 0.07 (004) − 0.80 (048) − 44 1.68 3.0 −0.8  GHPMF 3.9 60 0.67 (040) − 0.07 (004) − 0.02 (001) − 0.75 (045) − 43 1.51 2.9 −0.8  GHPMF 7.9 60 0.70 (042) − 0.07 (004) − 0.07 (004) − 0.83 (050) − 47 1.60 3.2 −0.5  GHPMF 15.0 60 0.58 (035) − 0.08 (005) − 0.07 (004) − 0.73 (044) − 43 1.72 2.9 −0.8 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPMF 1.9 60 0.75 (045) − 0.17 (010) − 0.07 (004) − 0.98 (059) − 59 1.95 4.0 1.2  GHPMF 3.9 59 0.75 (044) − 0.14 (008) − 0.03 (002) − 0.92 (054) − 54 2.28 3.8 0.9  GHPMF 7.9 57 0.53 (030) − 0.07 (004) − 0.07 (004) − 0.61 (035) − 35 1.8 2.5 −0.3  GHPMF 15.0 57 0.60 (034) − 0.09 (005) − 0.05 (003) − 0.74 (042) − 40 1.8 2.9 0.0 Compound cross type Conc. (%, µM) Number of flies Spots per fly (number of spots) statistical diagnosisa Mean mwh clone size class Clone formation per 105 cells per cell divisionb Small single spots (1–2 cells) m = 2 Large single spots (>2 cells) m = 5 Twin spots m = 5 Total spots m = 2 mwh clones Observed Control corrected Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.93 (056) − 0.12 (007) − 0.00 (000) − 1.05 (063) − 63 1.59 4.3 −0.8  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4 −0.1  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 S-1QN2-1 treatments ST  EtOH 2 60 0.93 (056) 0.12 (007) 0.00 (000) 1.05 (063) 63 1.59 4.3  S-1QN2-1 1.9 60 2.05 (123) + 1.50 (090) + 0.20 (012) + 3.75 (225) + 225 2.51 15.4 11.1  S-1QN2-1 3.9 60 2.05 (123) + 0.50 (030) + 0.02 (001) − 2.57 (154) + 154 1.60 10.5 6.2  S-1QN2-1 7.9 60 1.88 (113) + 0.62 (037) + 0.05 (003) − 2.55 (153) + 153 1.91 10.5 6.2  S-1QN2-1 15.0 60 1.40 (084) + 0.20 (012) + 0.03 (002) − 1.63 (098) + 98 1.51 6.7 2.4 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  S-1QN2-1 1.9 60 0.48 (029) − 0.05 (003) − 0.02 (001) − 0.55 (033) − 33 1.52 2.3 −0.1  S-1QN2-1 3.9 60 0.53 (032) − 0.05 (003) − 0.00 (000) − 0.58 (035) − 33 1.39 2.3 −0.1  S-1QN2-1 7.9 60 0.45 (027) − 0.07 (004) − 0.03 (002) − 0.55 (033) − 33 1.97 2.3 −0.1  S-1QN2-1 15.0 58 0.33 (019) ↓ 0.09 (005) − 0.02 (001) − 0.43 (025) ↓ 25 2.0 1.8 −0.6 Negative, dissolvent and positive controls ST  Water 0 59 0.64 (038) 0.17 (010) 0.05 (003) 0.86 (051) 51 1.88 3.5  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.47 (028) 0.10 (006) 0.02 (001) 0.58 (035) 33 1.85 2.3  EtOH 2 60 0.48 (029) − 0.08 (005) − 0.02 (001) − 0.58 (035) − 35 1.69 2.4  DMN 76 59 9.58 (565) + 7.05 (416) + 3.03 (179) + 19.66 (1160) + 927 2.36 64.5 62.2 D-1 treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  D-1 1.9 68 0.74 (050) − 0.09 (006) − 0.01 (001) − 0.84 (057) − 57 1.63 3.4 −0.3  D-1 3.9 55 0.80 (044) − 0.04 (002) − 0.02 (001) − 0.85 (047) − 47 1.45 3.5 −0.3  D-1 7.9 54 0.72 (039) − 0.07 (004) − 0.00 (000) − 0.80 (043) − 43 1.58 3.3 −0.5  D-1 15.0 61 0.88 (054) − 0.20 (012) + 0.03 (002) − 1.11 (068) − 68 1.72 4.6 0.8 HB  EtOH 2 60 0.48 (029) 0.08 (005) 0.02 (001) 0.58 (035) 35 1.69 2.4  D-1 1.9 56 0.64 (036) − 0.04 (002) − 0.00 (000) − 0.68 (038) − 37 1.59 2.7 0.3  D-1 3.9 56 0.46 (026) − 0.12 (007) − 0.02 (001) − 0.61 (034) − 34 2.12 2.5 0.1  D-1 7.9 58 0.40 (023) − 0.10 (006) − 0.02 (001) − 0.52 (030) − 30 1.83 2.1 −0.3  D-1 15.0 59 0.68 (040) − 0.08 (005) − 0.02 (001) − 0.78 (046) − 46 1.93 3.2 0.8 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPM treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPM 1.9 60 0.80 (048) − 0.10 (006) − 0.03 (002) − 0.93 (056) − 55 1.58 3.8 0.0  GHPM 3.9 60 0.82 (049) − 0.12 (007) − 0.08 (005) − 1.02 (061) − 59 1.86 4.0 0.3  GHPM 7.9 60 0.73 (044) − 0.03 (002) − 0.00 (000) − 0.77 (046) − 46 1.48 3.1 −0.6  GHPM 15.0 60 0.58 (035) − 0.20 (012) + 0.03 (002) − 0.82 (049) − 46 1.87 3.1 −0.6 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPM 1.9 60 0.65 (039) − 0.07 (004) − 0.03 (002) − 0.75 (045) − 45 1.64 3.1 0.2  GHPM 3.9 57 0.67 (038) − 0.14 (008) − 0.05 (003) − 0.86 (049) − 49 1.88 3.5 0.7  GHPM 7.9 60 0.92 (055) − 0.08 (005) − 0.02 (001) − 1.02 (061) − 60 1.52 4.1 1.3  GHPM 15.0 60 0.63 (038) − 0.05 (003) − 0.05 (003) − 0.73 (044) − 42 1.57 2.9 0.0 Negative, dissolvent and positive controls ST  Water 0 60 0.65 (039) 0.17 (010) 0.05 (003) 0.87 (052) 52 1.87 3.6  EtOH 2 60 0.87 (052) − 0.07 (004) − 0.02 (001) − 0.95 (057) − 55 1.58 3.8 0.2  DMN 76 59 2.12 (125) + 0.80 (047) + 0.42 (025) + 3.34 (197) + 184 2.18 12.8 9.2 HB  Water 0 60 0.62 (037) 0.07 (004) 0.05 (003) 0.73 (044) 41 1.61 2.8  EtOH 2 58 0.66 (038) − 0.07 (004) − 0.00 (000) − 0.72 (042) − 40 1.65 2.8 0.0  DMN 76 60 5.75 (345) + 3.35 (201) + 1.10 (066) + 10.2 (612) + 522 2.35 35.7 32.9 GHPMF treatments ST  EtOH 2 60 0.87 (052) 0.07 (004) 0.02 (001) 0.95 (057) 55 1.58 3.8  GHPMF 1.9 60 0.63 (038) − 0.10 (006) − 0.07 (004) − 0.80 (048) − 44 1.68 3.0 −0.8  GHPMF 3.9 60 0.67 (040) − 0.07 (004) − 0.02 (001) − 0.75 (045) − 43 1.51 2.9 −0.8  GHPMF 7.9 60 0.70 (042) − 0.07 (004) − 0.07 (004) − 0.83 (050) − 47 1.60 3.2 −0.5  GHPMF 15.0 60 0.58 (035) − 0.08 (005) − 0.07 (004) − 0.73 (044) − 43 1.72 2.9 −0.8 HB  EtOH 2 58 0.66 (038) 0.07 (004) 0.00 (000) 0.72 (042) 40 1.65 2.8  GHPMF 1.9 60 0.75 (045) − 0.17 (010) − 0.07 (004) − 0.98 (059) − 59 1.95 4.0 1.2  GHPMF 3.9 59 0.75 (044) − 0.14 (008) − 0.03 (002) − 0.92 (054) − 54 2.28 3.8 0.9  GHPMF 7.9 57 0.53 (030) − 0.07 (004) − 0.07 (004) − 0.61 (035) − 35 1.8 2.5 −0.3  GHPMF 15.0 57 0.60 (034) − 0.09 (005) − 0.05 (003) − 0.74 (042) − 40 1.8 2.9 0.0 Trypanocidal agents S-1QN2-1, D-1, GHPM and GHPMF (1.9, 3.9, 7.9 and 15.0 µM), DMN (76 µM), EtOH [2%] and milliQ water. aStatistical diagnoses according to Frei and Würgler (47). m: minimal risk multiplication factor for the assessment of negative results. For the final statistical diagnosis of all positive (+), negative (−) and significant reduction (↓) outcomes; the non-parametric Mann–Whitney and Wilcoxon U-test with significance levels α and β = 0.05 was used in order to exclude false-positive or negative diagnoses (38). One-side binomial tests, significance levels α and β: significative results: + (α ≤ 0.05); no significant results: − (β ≤ 0.05). bClone frequencies per fly divided by the number of cells examined per fly (48 800) gives an estimate of formation frequencies per cell and per cell division in chronic exposure experiments (48). Open in new tab HPLC analysis of flare and Oregon R(R)-flare flies treated with trypanocidal agents An HPLC was run to determine the remnants of the trypanocidal agents in adult flies of the flare, and Oregon R(R)-flare strains emerged from larvae (72 ± 4 h) chronically fed with them. In this test, we used flies from those strains and not from the crosses because we only wanted to evaluate the possible influence of the Cyp450s in the xenobiotic metabolism of the trypanocidal agents, taking into account that the flare strain has basal activity of Cyp450s and the Oregon R(R)-flare strain has high levels of these enzymes (39–41). A group of 150 emerged adult flies per treatment was recovered from each strain and used to prepare extracts homogenising the flies in 500-µl MeOH (°HPLC) and centrifuging (Eppendorff 5415C) them at 14 000 rpm for 10 min. The standards used were synthesised and verified according to (19): S-1QN2-1 (48 µM), D-1 (38 µM), GHPM (40 μM), GHPMF (28 μM) and MeOH 99% (°HPLC). A 20-μl aliquot of each standard and each extract were analysed using an HPLC Hewlett–Packard series 1100 USA and an Alltech Allsphere ODS-1 column (250 × 4.6 mm, 5 μm particles size). The mobile phase consisted of a mixture of MeOH:ACN:phosphoric acid 0.005% in 50:25:25 proportion. The flux index was 1 ml/min for 18 min. Analyzes were carried out with an HP Agillent 1100 series diode array detector (Agillent Technologies, USA). Results Drosophila wing SMART In both crosses, the mutant clone frequencies for EtOH 2% did not differ with respect to those of milliQ water negative control (Table 1), nor there were differences between them. In both crosses, DMN produced significant differences in all types of mutant clone frequencies. As expected, when comparing between crosses, we found 3-fold higher frequencies in the HB cross (Table 1). A genotoxic effect of S-1QN2-1 was observed in the ST cross, which showed significant increases in small, large and total clone frequencies at all concentrations and a significant increase of twin clone frequencies at 1.9 µM. On the other hand, we did not find differences in the clone frequencies between treatments in the HB cross compared against EtOH 2%, except at 15 μM, with significant reduction in small and total clone frequencies. In addition, we found differences between crosses in small, large and total clone frequencies at all concentrations (Table 1). In the ST cross, D-1 showed significant increase in the large clone frequencies at 15 μM. Moreover, we did not find differences between treatments in the HB cross compared against EtOH 2% or between crosses (Table 1). In the ST cross, GHPM (15 μM) showed significant increase in the large clone frequencies. We did not find differences between treatments in the HB cross or differences between crosses (Table 1). The GHPMF treatments did not produce significant differences in the frequencies of any spot type or between crosses (Table 1). In the mwh clones accumulated distribution frequencies analysis, we did find statistical differences between EtOH 2% and the milliQ water control, only in the ST cross, but not significant differences between the crosses. It showed clearly significant differences between DMN and milliQ water controls in the ST and HB crosses and, since DMN is also a positive control of bioactivation by Cyp450s enzymes (44), we found differences between crosses. In the ST cross, treated with S-1QN2-1, this distribution was altered at all concentrations (Figure 2A) more than in the HB cross, where it was altered only at 15 µM (Figure 2B); differences between both crosses were observed at all concentrations. The mwh clones accumulated distribution frequency was altered in the D-1 treatment at 3.9, 7.9 and 15 µM in the ST cross (Figure 2C) more than in the HB cross at 1.9 and 15 µM (Figure 2D), and we obtained differences between both crosses at all concentrations. With GHPM at 7.9 and 15 µM, this parameter was altered in the ST cross (Figure 2E); in the HB cross, alterations were observed at 1.9, 3.9 and 7.9 µM (Figure 2F) and the analysis between crosses pointed out differences only at 7.9 µM. Finally, all used concentrations of GHPMF modified the mwh clones accumulated distribution frequencies in the ST cross (Figure 2G), while, in the HB cross, they were altered only at 1.9, 3.9 and 7.9 μM (Figure 2H) and differences between both crosses were only observed at 1.9 and 7.9 µM. Figure 2. Open in new tabDownload slide Size distribution of mwh clones. EtOH 2% against S-1NQN2-1: (A) standard cross, (B) high bioactivation cross; D-1: (C) standard cross, (D) high bioactivation cross. GHPM: (E) standard cross, (F) high bioactivation cross; and GHPMF: (G) standard cross, (H) high bioactivation cross. The Kolmogorov–Smirnov test was performed to determine statistically significant differences in the mwh clones accumulated distribution frequency. Significance level α = 0.05 (*; Graf et al. 1984). Figure 2. Open in new tabDownload slide Size distribution of mwh clones. EtOH 2% against S-1NQN2-1: (A) standard cross, (B) high bioactivation cross; D-1: (C) standard cross, (D) high bioactivation cross. GHPM: (E) standard cross, (F) high bioactivation cross; and GHPMF: (G) standard cross, (H) high bioactivation cross. The Kolmogorov–Smirnov test was performed to determine statistically significant differences in the mwh clones accumulated distribution frequency. Significance level α = 0.05 (*; Graf et al. 1984). HPLC Comparison of standards S-1QN2-1 (48 µM), D-1 (38 µM), GHPM (40 µM) and GHPMF (28 µM) against the extracts of S-1QN2-1, D-1, GHPM and GHPMF treatments (1.9, 3.9, 7.9 and 15 µM) yielded chromatograms where there were not remnants of these compounds, although there were some differences of abundance and presence of other undetermined compounds (data not shown). This data and the SMART results lead us to conclude that the trypanocidal agents were absorbed, metabolised and even possibly excreted. Discussion It is clear that the investigation on chemical compounds designed to cure or control the Chagas’ disease has recently taken more importance (49). As mentioned before, the main problems with trypanocidal agents are that they present adverse effects in the host (5,9–17). It is worldwide known that one undesirable effect in novel drugs could be genotoxicity, and this can be studied with short-time assays using different biological models. We decided to test for genotoxic potentials of four TAQ-derived trypanocidal agents using the well-known in vivo assay Drosophila wing SMART (34,35,50). Trypanocidal agents 2,4-diamino-6 nitro-1,3 diazonaftalene The increase in the ST cross frequencies indicate clear genotoxicity of S-1QN2-1, in contrast with the other three compounds (D-1, GHPM y GHPMF) that resulted not genotoxic. These results were as expected because S-1QN2-1 was the TAQ-derived trypanocidal agent with a nitro group. It has been proved in bacteria that the enzymatic reduction of the nitro group by nitro reductases to amines or nitroso and hydroxylamine intermediates can react with biomolecules and exert toxic (51) and mutagenic effects (49); also, hydroxylamine produces oxidative stress in DNA through superoxide anions or hydroxyl ions (52). It has been proved the mutagenicity of NFX and BNZ in the Ames test, comet and the micronucleus assays (11,16). Boechat et al. (20) demonstrated genotoxicity and mutagenicity of nitroimidazoles in the same tests and Würgler et al. (21) demonstrated the genotoxicity of the nitroimidazole methotrexate. Although nitrosation has been proved in D. melanogaster(53), Moraga and Graf (17) propose that, beside this bioactivation, other metabolic pathways could be implicated in genotoxicity of nitro compounds. Boechat et al. (20) assessed the effect of different groups in several positions of the molecule and found that the effects did not depend on the presence of the nitro group but on the susbtituents and their positions. It is possible that there exist other pathways in D. melanogaster for the metabolism of nitro compounds (17) as it occurs in mammals by the action of xanthine oxidase, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-cytochrome C reductase and aldehyde oxidase (54), all present in D. melanogaster (55–57); in support of this, the genotoxicity of nitro compounds has been reported in D. melanogaster (21,58–60). The lack of genotoxicity in the HB cross and the decrease in the small and total spot frequencies at 15 µM could be explained according to Sun et al. (61), who propose that in the insecticide-resistant D. melanogaster strains, like Oregon-R(R)-flare, with high levels of Cyp450s and glutathione S-transferase (GST), occurs a detoxification of the reactive oxygen species generated by quinazoline derivates. Likewise, the alterations in the mwh clones accumulated distribution frequency promoted by the compounds indicates a disruption in the cell division of the wing imaginal disc cells (38) perhaps due to the oxidative stress as has been reported for nitro compounds (25). 2,4-diacetamino-6-amino 1,3 diazonaftalene D-1 increased the large spot frequency only at 15 µM in the ST cross. This is an unclear genotoxic effect and it could be insufficient to determine a genotoxic activity of this compound (38). The alterations in the mwh clones accumulated distribution frequency indicate an effect on the cell division and it agrees with the antiproliferative properties of quinazolines reported previously, such as the inhibition of cell cycle checkpoints, the inhibition of cell cycle progression (23), the apoptosis induction (24), the oxidative stress cytotoxicity (25) or an aneuploidy effect (38). N6-(4,methoxybenzyl)quinazoline-2,4,6-triamine GHPM has a trypanocidal effect and a successful molecular interaction with DHFR and pteridine reductase (PTR) of T. cruzi (19). It increased the large spot frequency only at 15 µM in the ST cross; therefore, the data are insufficient to affirm its genotoxicity. The alterations in the mwh clones accumulated distribution frequency indicate a disrupting effect on cell division (38). This is in agreement with adverse effects reported by Davoll et al. (22), Yadav et al. (62) and toxic effects in mice at an oral dose of 99 mg/kg (~334.6 µM; unpublished data). N6-[4-(trifluoromethoxy)benzyl]quinazoline-2,4,6-triamine This quinazoline derivate has a trifluoromethoxy group that reduces its ability to bind to the human DHFR enzyme but, at the same time, increases selectivity by interaction with T. cruzi’s Region 1 DHFR with thymidylate synthetase-dihydrofolate reductase activity (19). In addition, it has shown trypanocidal effect at 9 μM in two T. cruzi strains (NINOA and INC-5), Leishmania mexicana and Plasmodium berghei, and did not exhibit cytotoxicity in the VERO cell line (19). As expected, GHPMF did not show genotoxicity at any of the concentrations studied or the crosses of the Drosophila wing SMART. GHPMF produced alteration in the mwh clones accumulated distribution frequencies at all concentrations in the ST cross and at 1.9, 3.9 and 7.9 μM in the HB cross; these effects are in agreement with some previous studies of TAQ molecules as those used in antineoplastic therapy (23,24). We propose that this compound is a good candidate to be tested in in vivo and in vitro genotoxicity assays, such as the chromosomal aberration test or the micronucleus test, in order to finally be assayed in clinical protocols. HPLC analysis The HPLC analysis showed that S-1QN2-1, D-1, GHPM and GHPMF were not present in the samples; for that reason, we assume that these compounds were transported, metabolised and/or excreted completely; since there were no differences between the strains treated with D-1, GHPM and GHPMF, there is not enough evidence of the participation of the Cyp450s enzymes in the xenobiotic metabolism of these trypanocidal agents. Conclusions We propose that the nitro group in the sixth position of S-1QN2-1 was responsible for its genotoxicity, and the inconclusive results for D-1 and GHPM were due to the substitution of the nitro group by other radicals; we propose that our GHPMF genotoxic negative results were obtained because of the trifluormethoxyl substitution in the fifth position of the benzyl group of the TAQ molecule; based on these experimental results and the fact that this agent eliminates T. cruzi efficiently in all its stages of development (19), we concluded that it is a good candidate to be used as a trypanocidal agent, albeit further studies with other preclinical tests and models are needed to support this proposal. Acknowledgements The authors thank Dr Rosario Rodríguez-Arnáiz of the Faculty of Sciences, UNAM, for her valuable suggestions. Funding This work was supported by a UNAM DGAPA PAPIIT grant (#IG200814 to P.I.E. and H-P.M.E.) and a UNAM-Iztacala DIP grant (#9/1/1 to H-P.M.E.). 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TI - Genotoxicity assessment of four novel quinazoline-derived trypanocidal agents in the Drosophila wing somatic mutation and recombination test
JF - Mutagenesis
DO - 10.1093/mutage/gez042
DA - 2003-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/genotoxicity-assessment-of-four-novel-quinazoline-derived-trypanocidal-0KJYITaC1h
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -