Antiaflatoxigenic Thiosemicarbazones as Crop-Protective Agents: A Cytotoxic and Genotoxic Study

Article abstract of DOI:10.1021/acs.jafc.9b01814

Aflatoxins are secondary fungal metabolites that can contaminate feed and food. They are a cause of growing concern worldwide, because they are potent carcinogenic agents. Thiosemicarbazones are molecules that possess interesting antiaflatoxigenic propert

Full text of DOI:10.1021/acs.jafc.9b01814

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Food Safety and Toxicology
Antiaflatoxigenic thiosemicarbazones as crop-
protective agents: a cytotoxic and genotoxic study
Jennifer Bartoli, SERENA MONTALBANO, Giorgio Spadola, Dominga Rogolino,
Giorgio Pelosi, Franco Bisceglie, Francesco Maria Restivo, Francesca Degola, Olga
Serra, Annamaria Buschini, Donatella Feretti, Claudia Zani, and Mauro Carcelli
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01814 • Publication Date (Web): 09 Sep 2019
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Journal of Agricultural and Food Chemistry
1
2
Antiaflatoxigenic thiosemicarbazones as crop-
protective agents: a cytotoxic and genotoxic study
3
4
5
Jennifer Bartoli,‡ Serena Montalbano,‡ Giorgio Spadola,‡ Dominga Rogolino,‡ Giorgio
Pelosi,‡ Franco Bisceglie,‡ Francesco Maria Restivo,‡ Francesca Degola,‡ Olga Serra,‡
Annamaria Buschini,‡ Donatella Feretti,± Claudia Zani± and Mauro Carcelli‡*
6
7
8
9
‡Department of Chemistry, Life Sciences and Environmental Sustainability and
CIRCMSB (Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi
Biologici), Università di Parma, Parco Area delle Scienze, 43124 Parma, Italy.
±Department of Medical and Surgical Specialities, Radiological Sciences and Public
10 Health, University of Brescia, Viale Europa 11, 25123 Brescia, Italy.
11 KEYWORDS: aflatoxin, thiosemicarbazone, copper complexes, antiaflatoxigenic
12 compounds, crop-protective agents.
13
14 ABSTRACT
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15 Aflatoxins are secondary fungal metabolites that can contaminate feed and food. They
16 are cause of growing concern worldwide, because they are potent carcinogenic agents.
17 Thiosemicarbazones are molecules that possess interesting antiaflatoxigenic properties
18 but, in order to use them as crop protective agents, their cytotoxic and genotoxic profiles
19 must first be assessed. In this paper, a group of thiosemicarbazones and a copper
20 complex are reported as compounds able to contrast aflatoxins biosynthesis, fungal
21 growth, and sclerotia biogenesis in Aspergillus flavus. The two most interesting
22 thiosemicarbazones found are non-cytotoxic on several cell lines (CRL1790, Hs27,
23 HFL1, and U937), and therefore they were submitted to additional analysis of
24 mutagenicity and genotoxicity on bacteria, plants and human cells. No mutagenic
25 activity was observed in bacteria, while a genotoxic activity was revealed by the Alkaline
26 Comet Assay on U937 cells and by the test of chromosomal aberrations in Allium cepa.
27
28
29 INTRODUCTION
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30 Mycotoxins, and especially aflatoxins (AFs), are considered the most widespread cause of food
31 contamination and spoilage worldwide.1,2 These secondary fungal metabolites possess a profile
32 of severe toxicity and carcinogenicity, and can cause teratogenic, carcinogenic, neurotoxic and
33 immunosuppressive effects.3 The most common producers of mycotoxins are fungi belonging to
34 the Aspergillus, Penicillium, Fusarium and Alternaria genera, which grow especially in hot and
35 humid climates. They can contaminate a large variety of important agricultural crops, like corn,
36 peanuts, wheat and rice, causing important economic losses and representing a risk for animal
37 and human health.4
38 Up to now, the most common way to fight fungal diffusion and consequent crop contamination
39 by mycotoxins has been the use of pesticides, but this has the consequence of generating noxious
40 long term residues in food and in the environment.5 Moreover, the problem of resistant pest
41 strains is an emerging global concern. All these considerations push towards the development of
42 novel plant-protection products, able contemporaneously to minimize a prolonged use of
43 synthetic fungicides and to block the production of AFs. We have recently found a series of
44 molecules, belonging to the thiosemicarbazone class, with very promising antifungal and anti-
45 aflatoxigenic profiles.6,7,8,9 Our investigations highlighted that modifications on the backbone of
46 the thiosemicarbazone scaffold provide a means to tune their activity against the two major
47 genera of mycotoxigenic fungi that contaminate cereals, i.e. Fusarium and Aspergillus, and that
48 metal chelation could improve their activity. By using these thiosemicarbazones and their
49 copper complexes, in fact, it is possible to obtain relevant anti-aflatoxigenicity, in concert with
50 moderate fungistatic activity. This behavior has the advantage of avoiding problems related to
51 undesired modifications of the composition of the microbiota in the environment.
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52 The analysis of the previous results indicates that the presence of the C=S group is essential to
53 observe antiaflatoxigenic activity (the corresponding semicarbazones are inactive).8 In addition
54 the antiaflatoxigenic activity depends deeply on the substituents on the phenyl ring and it is
55 increased by an increase in the lipophilicity of the thiosemicarbazone.9 This last observation
56 could be obviously associated to the ability of the molecule to cross the cell membrane.10,11 As a
57 prosecution of this research, we here present the study on the phenyl and cyclohexyl
58 thiosemicarbazones (1) - (4) (Figure 1).
59
H
N
NH2
S
H
N
NH2
S
N
N
H
NH2
H
N
NH2
S
N
N
N
S
(4)
(3)
60
61
Figure 1. Schematic representation of thiosemicarbazones (1) - (4)
62
63 These compounds have been tested for their ability to inhibit sclerotia biogenesis and AFs
64 biosynthesis in A. flavus. We also synthesized compound (5), the copper complex of (1), because
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65 previous results12 suggested that metal chelation can lead to improved AFs inhibition profiles
66 and that in particular copper compounds are good candidates to obtain inhibition of mold and
67 bacteria growth and of aflatoxin production.
68 In view of potential agronomical applications, it is important to assess the genotoxic potential
69 risks for the environment and the human health of the crop-protective agents. We have therefore
70 investigated the cyto- and geno-toxic effects of the most active compounds (3) and (4) on healthy
71 human cells, and in particular on human cell lines coming from tissues that can come into
72 contact with chemicals (gastrointestinal tract, pulmonary epithelium and epidermis). Finally, the
73 toxic and genotoxic effects of (3) and (4) were investigated on bacteria and plant cells.
74
75 MATERIALS AND METHODS
76 The following commercial products were used: chemicals (Sigma-Aldrich Srl, Milano, Italy);
77 Ham's Nutrient Mixture F-12 and Fetal bovine serum (FBS) (EuroClone s.p.a., Milano, Italy);
78 HFL1 (ATCC, CCL-153), CRL 1790 (ATCC, CCD 841 CoN), and Hs27 (ATCC, CRL1634)
79 (American Type Culture Collection, ATCC); U937 cells (American Tissue Culture Collection,
80 Rockville, MD); CellTiter96® AQueousOne Solution Cell Proliferation Assay (Promega
81 Corporation, Madison, WI, USA); Dulbecco's Modified Eagle's medium (DMEM) and RPMI-
82 1640 (Lonza Group Ltd, Basel, Switzerland).
83 Chemistry. The purity of the compounds was ≥ 95% as determined by elemental analysis.
84 Technical details about NMR, ATR-IR, ESI-MS, and ICP analyses were reported elsewhere.9
85 The synthesis of (1) - (4) (Figure 1) was performed by using a slightly modified literature
86 procedure.9
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87 Cyclohexanone thiosemicarbazone (1). Thiosemicarbazide (3.30 mmol) was dissolved in
88 refluxing ethanol. Cyclohexanone (1.2 eq., 3.96 mmol) and few drops of glacial acetic acid were
89 added to the solution and the mixture was reacted for 24 hours. The solvent was partially
90 evaporated and a precipitate was isolated by filtration. The solid was recrystallized from ethanol,
91 to give a yellow solid. Yield = 31%. M.p.: 170-171 °C. IR (ATR, cm-1): 3374 NH2); 3205,
92 3143 NH); 2940 CH 1582 C=N); 1073, 1035, 831 C=S). MS-EI: m/z= 171.1 (100,
1
93 M+). H-NMR (DMSO-d6, 25°C, ppm), : 10.12 (s, 1H, NH), 7.93(s, 1H, NH2), 7.50 (s, 1H,
94 NH2), 2.40 (t, 2H, Hcyclohexyl), 2.23 (t, 2H, Hcyclohexyl), 1.63-1.56 (m, 6H, Hcyclohexyl).
95 Dicyclohexylketone thiosemicarbazone (2). Thiosemicarbazide (3.30 mmol) was dissolved in
96 refluxing ethanol. Dicyclohexylketone (1.2 eq., 3.96 mmol) and some drops of glacial acetic acid
97 were added and the mixture was reacted for 50 hours. Solvent was removed by vacuum and the
98 solid residue was purified by chromatographic column (hexane:ethyl acetate 7:3) to give a
99 yellow solid. Yield = 15%. M.p.: 169-170 °C. IR (ATR, cm-1): 3430 NH2); 3276, 3230, 3140
1
100 NH); 2921 CH 1693 C=N); 1081, 843 C=S). MS-EI: m/z= 255.3 (80, M+). H-NMR
101 (DMSO-d6, 25°C, ppm), : 10.21 (s, 1H, NH), 8.03 (s, 1H, NH2), 7.41 (s, 1H, NH2), 3.03 (t, 1H,
102 HC-C=N), 2.25 (t, 1H, HC-C=N), 1.69-1.15 (20H, CHcyclohexyl).
103 Benzophenone thiosemicarbazone (3): Thiosemicarbazide (3.3 mmol) was dissolved in refluxing
104 ethanol. Benzophenone (1.2 eq., 3.96 mmol) and some drops of glacial acetic acid were added,
105 then the mixture was reacted for 36 hours. After concentration by vacuum, the precipitate was
106 isolated by filtration and recrystallized from ethanol to give a pale orange powder. Yield = 15%.
107 M.p.: 172-173 °C. IR (ATR, cm-1): 3430 NH2); 3276, 3236, 3141 NH); 2922 CH 1693
1
108 C=N); 1081, 844 C=S). MS-EI: m/z= 235.3 (80, M+). H-NMR (DMSO-d6, 25°C, ppm), :
109 8.66 (s, 1H, NH), 8.40 (d, 2H, NH2), 7.76-7.65 (m, 6H, CHarom), 7.42-7.34 (6H, CHarom).
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110 Valerophenone thiosemicarbazone (4): Thiosemicarbazide (3.3 mmol) was dissolved in refluxing
111 ethanol. Valerophenone (butylphenylketone) (1.2 eq., 3.96 mmol) and some drops of glacial
112 acetic acid were added to the solution, that was reacted for 40 hours. Solvent was removed by
113 vacuum; the solid was then purified by chromatography (CH2Cl2:CH3OH 9:1). Yield = 23%. IR
114 (ATR, cm-1): 3378 NH2); 3226, 3149 NH); 2957, 2929 CH 1588 C=N); 1066, 847
115 C=S). MS-EI: m/z= 267.5 (100, M+). 1H-NMR (DMSO-d6, 25°C, ppm), : 10.34 (s, 1H, NH),
116 8.24 (s, 1H, NH), 8.90-7.88 (3H, NH+CHarom), 7.38-7.39 (3H, CHarom), 2.87 (t, 2H, H2C-C=N),
117 1.37 (t, 4H, CHcyclohexyl), 0.88 (t, 3H, CHcyclohexyl).
118 Cu2(HL)(L)2 (5). HL: Cyclohexanone thiosemicarbazone. The solution obtained by mixing under
119 N2 100 mg (2 eq.) of (1) in 10 ml of degassed methanol, and 1 eq. of CuCl2·2H2O in 5 mL of the
120 same solvent was stirred at room temperature. After 4 hours, it was cooled and the precipitate
121 was filtered off and washed with cold diethyl ether. Green powder. Yield=34%. IR (ATR, cm-1):
122 3436 NH2); 3248, 3165 NH); 2932, 2856 CH 1604 C=N); 1034 C=S).1H-NMR
123 (DMSO-d6, 25°C) : 10.52 (s, 1H, NH); 7.64, 8.47 (2s, br, 1H+1H, NH2); 2.60 (s, br,
124 CHcyclohexyl); 1.58-1.69 (m, br, CHcyclohexyl). ESI-MS (CH3OH): m/z=701, [Cu2(HL)2L+H]+, 5%;
125 405, [Cu(HL)L+H]+, 100%; 234, [Cu(HL)]+, 50%. ICP: Cu found 20.3 ± 0.7%, calcd. for
126 Cu2(L)2(HL): 19.8 %.
127
128 Aspergillus flavus strains. Two strains were used: the toxigenic strain CR10 and the atoxigenic
129 strain TOφ isolated from corn kernels in the Po Valley.5
130 Effect on aflatoxin accumulation. Aflatoxin accumulation in a coconut-milk derived medium
131 (CCM) was valued by the high throughput procedure described in our previous works.9,13,14
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132 Effect on fungal growth and on sclerotia biogenesis. Once assessed the AF accumulation rate,
133 mycelia from single wells were recovered at the 6th day of incubation, slightly dried on hands
134 paper, and weighted. Biomass measures were then converted in percentage inhibition respect to
135 controls (DMSO-treated cultures).
136 A 5 µl aliquot of aflatoxigenic strain CR10 spore suspension (approximately 106 conidia/ml) was
137 point-inoculated in the center of Petri dishes (Ø = 5 cm) poured with Czapek Dox Agar (CZA)
138 medium added with 100 µM thiosemicarbazones. Control plates were added with 1 % (v/v)
139 DMSO. Plates were replicated in triplicate and incubated at 30˚C in darkness; after two weeks of
140 incubation, sclerotia were manually scraped from the colonies surface and washed with a 70%
141 ethanol solution to completely remove conidia, then dried for three days at 60 °C. Dry weight
142 was assessed. Thiosemicarbazones inhibition rate on sclerotia production was expressed as
143 percentage respect to the control. Plates were inoculated in triplicate.
144 One-way analysis of variance (Past 3.x software) was used for data analyses. Tukey’s test was
145 applied to the data relative to mycelial growth, aflatoxin accumulation and sclerotia production;
146 differences were regarded as significant if p< 0.05.
147 Cytotoxicity. The biological activity of the molecules with antifungal potential was assessed on
148 normal cells: human fibroblast cell line Hs27 (ATCC, CRL1634), human lung epithelial cell line
149 HFL1 (ATCC, CCL-153), and human colon epithelial cell line Crl1790 (ATCC, CCD 841 CoN).
150 These cell lines were chosen to investigate the exposition risks, because they are attributable to
151 human districts that could be involved in xenobiotic ways of interaction with the human body.7
152 We also performed toxicological assays on human histiocytic lymphoma cell line U937 (ATCC,
153 CRL-3253) cultured in RPMI-1640. Hs27 and CRL1790 were cultured in DMEM. HFL1 were
154 cultured in Ham's Nutrient Mixture F-12. All media were supplemented with 10% (v/v) FBS, 1%
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155 penicillin (100 U/ml)/streptomycin (100 μg/ml) and 1% L-glutamine (2 mM). Flasks and plates
156 were maintained at 37°C and 5% CO2 in a humidified atmosphere. Culture medium was
157 refreshed every two or three days during sub-culturing. Hs27, CRL1790 and HFL1 cells were
158 used between passage numbers 5 and 20. MTS assay (CellTiter96® AQueous One Solution Cell
159 Proliferation Assay) was performed to identify the antiproliferative effect of the compounds.
160 Briefly, 100 μL of a suspension of cells in exponential growth (5 ×104/mL in complete medium
161 without phenol red supplemented with 5% FBS) were added into 96-well plates 24h before
162 treatment. Plates were incubated at 37 °C in a humidified 5% CO2 incubator. After this recover
163 period, increasing concentrations of compounds (0.5-1.0-5.0-10.0-50.0-100.0 µM) were added to
164 the medium and cells were left exposed for 24-48-72h. Negative control was represented by 100
165 μM DMSO. After treatment period, 20 µl of MTS reagent was added to each well. At the end of
166 exposure time, the absorbance at 485 nm was measured by a microwell plate reader (TECAN
167 SpectraFluor Plus, Männedorf, Switzerland).
168 The cytotoxicity response parameters GI50 were extrapolated from concentration-response
169 curves.15
170 Genotoxicity on U937 cells. To verify primary DNA damage the alkaline version of Comet
171 assay was carried out on U937 cells as detailed elsewhere.16 U937 cells were seeded 24h before
172 treatment at a concentration of 1 x 105 cell/mL in 1 mL of complete medium. Cells were treated
173 with increased concentrations (25.0-50.0-75.0-100.0 μM) of the compounds for 1h and 24h.
174 DMSO (100 μM) and ethylmethanesulfonate (EMS) (2 mM) were used as negative and positive
175 controls, respectively. After treatment period at 37 °C, the percentage of live cells was assessed
176 by Trypan blue exclusion method. Only the treatments with a viability higher than 70% have
177 been processed in the Comet assay.9,16 Percentage of DNA in the tail region of the comet (TI, tail
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178 intensity) provided representative data on genotoxic effects. For each sample, coded and
179 evaluated blind, 100 cells were analyzed.
180 The “IBM SPSS Statistics 24” software was used for data analyses as already described.9
181 Mutagenicity assessment on bacteria. To assess the presence of mutagenic activity, the
182 standard plate incorporation method of Ames test was performed17 with S. typhimurium TA98
183 and TA100 strains, with and without metabolic activation (S9 mix). Molecules were dissolved in
184 DMSO and assayed at increasing doses (0.1, 1, 10, 50, 100 µM/plate, corresponding to a range
185 of doses from 0.025 to 25.5 µg/plate and 0.023 to 23.5 µg/plate for molecules 3 and 4,
186 respectively); negative and positive controls were also introduced.17 After 48 h, the revertant
187 colonies grown on the plates are counted and the mean of three replicates are computed with
188 their relative standard deviation (net revertants). The results were expressed as mutagenicity ratio
189 (RM) dividing the revertants/plate by spontaneous mutation rate (number of revertants in
190 negative controls). They were considered positive if two consecutive dose levels or the highest
191 non-toxic dose level produced a response at least twice that of the control and at least two of
192 these consecutive doses showed a dose-response relationship.18,19
193 Mutagenicity assessment on plants. Allium cepa toxicity test: 12 equal-sized young onion bulbs
194 were exposed for 96 hours in the dark to different concentrations of molecules dissolved in
195 DMSO changing the sample solution every day. Root length was used to calculate the EC50 value
196 of each compound and to identify the concentration to undergo the Allium cepa genotoxicity
197 assay being the highest dose correspondent to the EC50 value identified (the concentration that
198 gives a 50% reduction in root growth). Root macroscopic parameters (turgescence, consistency,
199 change in color, root tip shape) were used as toxicity indexes.20
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200 Allium cepa genotoxicity tests. To detect chromosome aberrations and micronuclei21, 22 six equal-
201 sized young bulbs per sample were exposed to sample solutions for 24 hours, then the roots were
202 fixed in acetic acid and ethanol (1:3) for 24 hours, and lastly stored in 70% ethanol for the
203 chromosome aberrations (CA) test.22 In the micronuclei (MN) test the bulbs, after exposure, were
204 replaced in saline solution (Rank’s solution) for 44 hours of recovery time, to cover two rounds
205 of mitosis so that damage induced in chromosomes during mitosis to be visible as micronuclei in
206 interphase cells. Then the roots were fixed in acetic acid and ethanol (1:3) for 24 hours and lastly
207 stored in 70% ethanol.21 The negative control was DMSO in Rank solution (the dose of DMSO
208 corresponding to volume of samples) and positive control was maleic hydrazide (10 mg/l, 6h
209 exposure). Five roots of each sample were used for microscopic analysis: 1000 cells/slide (5000
210 cells/sample) were scored for mitotic index (as a measure of cell division and hence of sample
211 toxicity), 200 in mitosis cells/slide (1000 cells/sample) for chromosomal aberrations and 2000 in
212 interphase cells/slide (10000 cells/sample) were scored for micronuclei frequency.
213 Statistical analysis was performed using chi-square test for mitotic index and chromosomal
214 aberrations, the analysis of variance and Dunnett’s t-test were performed for MN. All the
215 experiments were in duplicate (two independent assays).
216
217 RESULTS AND DISCUSSION
218 Chemistry. Compounds (1)-(4) (Figure 1) were synthesized by condensation between a ketone
219 and thiosemicarbazide accordingly to a modified literature procedure9 and they were
220 characterized by spectroscopic tools and mass spectrometry (Supporting Information). In their IR
221 spectra it is possible to observe the NH and NH2 stretching vibration bands (3430 - 3140 cm-1),
222 and (C=N) at about 1580 cm-1, while the C=S stretching absorptions are at about 1035-1080
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223 and 830-950 cm-1. In the H-NMR spectra recorded in d6-DMSO, the resonances of the
224 hydrazone NH and of NH2 groups (10.3-8.6 and 7.4-8.4 ppm, approximately) are clearly visible.
225 Even if these molecules could give rise to thione-thiol equilibrium, there are not evidences of the
226 presence of the thiolic form in solution.
227 In our previous investigations,7-9 we considered the possibility that copper coordination could
228 improve the biodisponibility of the metal ion, favoring adsorption into lipid membranes and thus
229 promoting a better anti-aflatoxigenic activity. It is well known that copper salts have been widely
230 applied in agriculture against plant pathogens. In order to further explore this aspect, we
231 synthesized the copper complex (5), by reacting (1) with CuCl2˙2H2O in methanol. As we have
232 already observed,9 also in this case the metal center undergoes a reduction to Cu(I). In (5) the
1
233 metal is present as diamagnetic d10 Cu(I) ion and it is in fact possible to record its H-NMR
234 spectrum in DMSO-d6 (Supporting Information). It is known from the literature that
235 thiosemicarbazones are subjected to intramolecular oxidative cyclization when some bases,23
1
236 oxidants24 or redox-active metals25 are present. However, IR and H NMR spectra of (5) do not
237 register the presence of cyclized ligand. In the IR spectra of (5) the strong bands attributable to
238 the symmetric and asymmetric stretching modes of NH and NH2 are clearly visible between
239 3500 and 3000 cm-1. The C=N stretching band shifts from 1582 cm-1 (the parent ligand) to 1604
240 in the complex, suggesting the coordination of the metal ion to the iminic nitrogen. Also the
241 stretching band relative to the C=S group is shifted upon coordination, from 1073 to 1034 cm-1.
242 In the literature, it is reported that by reacting copper(II) chloride with (+)-camphor and (–)-
243 carvone thiosemicarbazones,26 polynuclear copper(I) complexes are obtained; in (5) data analysis
244 suggests a 2:3 metal to ligand stoichiometry. In conclusion, if the monoprotic ligand (1) is
245 indicated as HL, for complex (5) it is possible to propose the formula Cu2(HL)(L)2 (Supporting
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246 information). The Cu2(HL)(L)2 stoichiometry is also supported by the ESI-MS mass (Supporting
247 Information) and ICP analysis.
248 Fungal growth, aflatoxin inhibition and sclerotia development. Figures 2 and 3 shows the
249 data concerning the effect of the treatments with (1)-(5) on AFs biosynthesis, biomass
250 accumulation and sclerotia development.
251
252
253 Figure 2: Antifungal and anti-aflatoxigenic activities of compounds (1)-(5) at 25, 50 and 100
254 μM concentration. Results referred to aflatoxin accumulation (A) and mycelium fresh weight (B)
255 in CCM medium; they are expressed as mean percentage inhibition in comparison with controls
256 (0.25%, 0.5% and 1% DMSO respectively). Error bars indicate the standard deviations of four
257 replications (p-value < 0.05).
258
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259
260 Figure 3. Effects of (3) and (4) on sclerotia biogenesis. Assessments were performed in Czapek
261 Dox Agar solid medium amended with 100 μM thiosemicarbazones or 1% DMSO as control
262 (CNT). Error bars indicate the standard deviations of three replications (p value <0.05).
263
264 Compound (1) inhibited only slightly AFs accumulation, whereas (2), (3) and (4) determined an
265 increasing AFs inhibition, ranging from 40% to 55% at 100 µM (Figure 2A). (5), the copper
266 complex of (1), inhibited AFs accumulation slightly more than the ligand alone, but it did not
267 show a dose dependent inhibition and its anti-mycotoxin effect was rather scarce. The biomass
268 production evaluation revealed that (2) induced a notable reduction of fresh weight (over 80% of
269 inhibition at the thiosemicarbazone highest concentration, Figure 2B). Poor antifungal activity in
270 conjunction with inhibition of aflatoxin production is highly desirable, since this can assure the
271 preservation of the environmental microbiota while ensuring the protection from noxious
272 secondary metabolites. Therefore (3) and (4) were chosen for further analyses due to their ability
273 to halve AFs accumulation without significantly affecting biomass production.
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274 Since AFs biosynthesis is known to share several regulatory steps with other developmental
275 processes belonging to the secondary metabolisms such as sclerotia biogenesis,27 the effect on
276 the production of these structures was assessed. Sclerotia formation was induced by culturing the
277 aflatoxigenic and sclerotigenic A. flavus CR10 strain in CZA solid medium amended with 100
278 µM of (3) and (4) and 1% DMSO as control. The presence of the thiosemicarbazones in the
279 culture medium reduced the formation of sclerotia, while, as expected, no mycelium growth
280 inhibition was observed. With respect to the control, the exposure to (3) and (4) limited the
281 sclerotia biogenesis to 30% and 86% respectively (Figure 3). These results are in line with our
282 previous observations regarding other thiosemicarbazones, effective in containing AFs
283 production, but with a slight effect on fungal growth, that severely impair sclerotia development
284 in A. flavus.7
285
286 Cytotoxicity. The cytotoxicity of the most interesting compounds (3) and (4) was screened over
287 a panel of human cell lines. Three normal healthy cell lines were chosen to represent possible
288 different routes of exposure to chemicals: epidermal contact (skin fibroblast, Hs27), inhalation
289 (lung epithelial cells, HFL1) and ingestion (colon epithelial cells, CRL1790). On the other hand,
290 the tumor cell line U937 is a model commonly used to identify cytotoxicity and genotoxic
291 activity of drugs.28 Growth inhibition (GI) was determined by MTS assay; data are obtained as a
292 mean of four independent experiments (Table 1).
293
294 Table 1. GI50 value (µM), concentration of compound that causes a 50% reduction of cell
295 proliferation, obtained after 24h treatment in human healthy cell lines CRL1790 (colon epithelial
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296 cells), Hs27 (skin fibroblast), HFL1 (lung epithelial cells), and U937 (histiocytic lymphoma
297 cells) cancer cell line.
298
CRL1790
>100.0
Hs27
>100.0
79.5
HFL1
>100.0
>100.0
U937
73.0
58.0
299
300
(3)
(4)
>100.0
301
302
303 Compound (3) showed no antiproliferative activity against colon, skin and lung cell lines and
304 presented a mild cytotoxic effect only on cancer cells (Table 1). A mild cytotoxic effect against
305 skin fibroblast and a more pronounced cytotoxicity on U937 cells (Table 1) were observed with
306 (4).
307 Since (3) and (4) present a good profile in term of cytotoxicity on normal cell lines, they were
308 considered for further analysis.
309
310 Genotoxicological assessment. The Alkaline Comet Assay was performed on U937 cells to
311 identify the genotoxic potential of (3) and (4). The test is able to measure DNA damage and is
312 usually carried out at pH>13 to detect, in addition to single and double strand breaks, alkali-
313 labile sites such as adducts, apurinic and apyrimidinic sites, oxidation of the nitrogenous bases,
314 etc. The percentage of DNA in the tail of the comet (TI%) provided a representative data on
315 genotoxic effects of the molecules. Cells were treated with 25.0-50.0-75.0-100.0 µM of the
316 molecules for 1 and 24h. Against U937 cells, compound (3) induced DNA strand breaks in a
317 dose-dependent manner in both the treatment times (1 and 24h) (Figure 4A). Compound (4)
318 after 1h of treatment induced DNA damage at the highest concentrations (75.0 and 100.0 µM),
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319 while after 24h it was genotoxic already at 25.0 µM (Figure 4B); it caused, as expected, a
320 reduction in cell viability and showed a toxic effect at the highest concentrations.
321
322
323 Figure 4. Genotoxic activity of compounds (3) (A) and (4) (B) by using Alkaline Comet Assay
324 on U937 cells treated for 1 h and 24 h. The significance of the difference between the control
325 (DMSO) and the exposure group is indicated by: *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA,
326 Bonferroni’s post-hoc test). TOX: viability <70%
327
328 The results of the Ames test showed that (3) and (4) had no mutagenic activity both in
329 Salmonella typhimurium strains TA98 and in TA100, with and without metabolic activation
330 (Table 2).
331
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332 Table 2. Results of the Ames test expressed as revertants/plate and as mutagenicity ratio (MR).
TA 98
TA98+S9
TA 100
TA100+S9
Doses
(µM/plate)
Samples
mean ± SD MR mean ± SD MR mean ± SD MR mean ± SD MR
0.1
1
14.5 ± 0.7
18.5 ± 2.1
11.5 ± 0.7
19.0 ± 2.8
16.5 ± 0.7
12.0 ± 4.2
17.5 ± 12.0
15.5 ± 4.9
14.5 ± 0.7
17.5 ± 3.5
1.0 22.3 ± 4.11
1.2 30.0 ± 1.41
0.8 20.5 ± 0.71
1.2 22.0 ± 0.0
1.1 20.5 ± 6.36
0.8 15.5 ± 6.36
1.1 26.0 ± 0.71
1.0 16.0 ± 2.12
1.0 22.0 ± 6.36
1.1 22.0 ± 3.54
1.0
1.3
0.9
0.9
0.9
0.7
1.1
0.7
0.9
0.9
94.5 ± 19.1
92 ± 7.1
1.2
1.1
1.2
1.2
1.2
0.9
1.0
0.9
1.0
0.9
108.0 ± 0.0
99.5 ± 10.6
83.5 ± 6.4
86.5 ± 14.8
79.0 ± 17.0
86.0 ± 17.0
86.5 ± 3.5
101.5 ± 16.3
92.5 ± 10.7
75.5 ± 3.5
1,2
1,1
0,9
0,9
0,9
0,9
0,9
1,1
1,0
0,8
10
50
100
0.1
1
96.5 ± 20.5
100.5 ± 7.8
95.5 ± 7.8
75.5 ± 12.0
77.5 ± 12.0
74 ± 5.7
(3)
10
50
100
(4)
82 ± 1.4
75 ± 9.9
Negative controls
15.25 ± 4.11
23.3 ± 10.6
81.5 ± 4.65
91.3 ± 11.32
333 Negative control: DMSO, 100 µM/plate. Positive control –S9: TA98 10 µg/plate 2-nitrofluorene;
334 TA100 10 µg/plate sodium azide; positive control +S9: TA98, TA100 20 µg/plate 2-
335 aminofluorene. Positive control results: TA 98±S9 > 1,000 revertants/plate; TA100±S9 > 1,000
336 revertants/plate.
337
338 Unfortunately, in preliminary tests, solutions of (4) exposed to the roots of Allium cepa showed a
339 high toxicity already to the lowest doses, with a decrease in the number of cell division (too low
340 mitotic index); for this reason, it was not possible to carry out a genotoxicity test. On the
341 contrary, for (3) it was evaluated, by a root toxicity test, the dose corresponding to the EC50,
342 which was used as the maximum dose for the further genotoxicity evaluations. The molecule had
343 EC50 = 50 μM, with a slight toxic activity at the highest tested dose (100 μM). The test of
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344 chromosomal aberrations in Allium cepa showed a clear genotoxic activity for (3) already at the
345 lowest dose tested, with a statistically significant increase of chromosomal aberrations compared
346 to the negative control, without a reduction in the number of cell divisions and mitotic index
347 (Table 3).
348
349 Table 3. Allium cepa chromosomal aberrations test on compound (3): mitotic index and total
350 chromosomal aberrations in metaphase root cells.
1st experiment
2nd experiment
Mitotic Cromosomal
Cromosomal
Aberration
AC (%)
Doses
(µM)
Mitotic
index (%)
index
(%)
Aberration p values*
AC (%)
p values*
10
25
12.6
11.6
10.3
12.3
10.5
7.2
3.5
< 0.01
< 0.01
< 0.01
< 0.01
13.3
11.2
10.0
11.8
9.9
3.7
4.6
5.1
7.4
1.9
10.1
< 0.01
< 0.01
< 0.01
< 0.01
4.5
50
4.7
100
7.4
C-
1.2
C+
8.8
8.7
351 Negative control, C-: 100 µM DMSO; positive control, C+: maleic hydrazide (10-2M)
352 * Statistically significant according to 2 test
353
354 In the linear trend analysis, the sample gave a positive response, showing a clear dose-response
355 relationship which is statistically significant (p<0.05) (Figure 5).
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356
357 Figure 5: Linear trends of chromosomal aberrations in Allium cepa root cells tested with
358 compound (3)
359
360 The frequency of micronuclei in interphase cells of Allium cepa is reported in Table 4: there is
361 no increase of micronuclei compared with negative control for compound (3).
362
363 Table 4. Allium cepa micronuclei test on compound (3): mitotic index and total chromosomal
364 aberrations in metaphase root cells.
1st experiment
2nd experiment
Doses
(µM)
Mitotic index
MCN
(mean±DS)
Mitotic index
MCN
(mean±DS)
(%)
(%)
10 µM
25 µM
50 µM
11.5
11.4
9.6
0.03 ± 0.03
0.03 ± 0.03
0.08 ± 0.03
0.04 ± 0.07
11.8
11.5
10.4
12.6
0.03 ± 0.07
0.04 ± 0.04
0.07 ± 0.07
0.02 ± 0.04
C-
12.5
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C+
7.6
16.8 ± 8.4
7.2
17.2 ± 12.0
365 C-: negative control, DMSO; C+: positive control, maleic hydrazide (10-2M). MCN, micronuclei
366 frequency
367
368
369 In conclusion, we were focused on finding specific inhibitors of AFs biosynthesis, rather than
370 inhibitors of fungal growth, and benzophenone thiosemicarbazone (3) and valerophenone
371 thiosemicarbazone (4) seem interesting in this sense. Compound (3) and (4) are promising also
372 because they showed no antiproliferative activity against colon, skin and lung cell lines and were
373 characterized by the absence of mutagenic activity on bacteria. However, the Alkaline Comet
374 Assay performed on U937 cells points out that (3) and (4) produce DNA damages. (4) is toxic
375 for the root of Allium cepa, and the test of chromosomal aberrations still in Allium cepa showed a
376 clear genotoxic activity for (3), already at the lowest doses. Allium cepa micronuclei test was
377 negative instead: compound (3) caused directly DNA damage and chromosomal alterations, but
378 did not cause disturbance in the mitotic cycle and damage in mitotic spindle.
379 Compounds (3) and (4) revealed to be promising hit compounds and the next efforts will
380 be devoted to investigate the action mechanisms of the antiaflatoxigenic
381 thiosemicarbazones and, at the same time, control their genotoxic character.
382
383 ASSOCIATED CONTENT
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384 Supporting Information. The Supporting Information is available free of charge on the
385 ACS Publications website at DOI: xxx.
1
386 Figures: IR and H NMR spectra of compounds (1), (2), (3), and (4) are reported,
387 together with IR, 1H NMR and mass spectra of (5).
388
389 ACKNOWLEDGMENTS
390 This work was funded by “Fondazione Cariplo” (Project N. 2014-0555; http://aflatox.it ).
391 Authors thank ‘‘Centro Interdipartimentale Misure Giuseppe Casnati” of the University of Parma
392 for facilities.
393
394
395
396
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397 AUTHOR INFORMATION
398 Corresponding Author
399 Prof. Mauro Carcelli
400 Email: mauro.carcelli@unipr.it
401 Phone: +39 0521905427
402
403
404 Author Contributions
405 Jennifer Bartoli, Serena Montalbano, and Giorgio Spadola are considered co-first authors. The
406 manuscript was written through contributions of all authors. All authors have given approval to
407 the final version of the manuscript. DR, JB, MC, FB, GP: molecular design and chemical
408 synthesis; FMR, FD, GS: fungal growth, sclerotia development and aflatoxin inhibition
409 evaluation. AB, SM, OS: cyto- and geno-toxicological assessment on human cells. DF, CZ:
410 genotoxicological assessment on bacteria, and plants.
411
412 Funding Sources
413 This study was supported by a grant from “Fondazione Cariplo” (Project N. 2014-0555;
414
415
http://aflatox.it ).
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416 Notes
417 The authors declare no competing financial interest
418
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