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Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9)
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 85 (2004) 74–79 www.elsevier.com/locate/yjipa
Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9) Francesca Fornelli,* Fiorenza Minervini, and Antonio Logrieco Istituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche (CNR), Viale Einaudi 51, 70125 Bari, Italy Received 3 October 2003; accepted 5 January 2004
Abstract The toxicity of sixteen fungal metabolites produced by some entomopathogenic fungi or biological control fungi agents was evaluated on lepidopteran Spodoptera frugiperda (SF-9) cell line by Trypan blue dye exclusion and MTT-colorimetric assay, after 48 h of incubation. No statistical difference was found between IC50 values (50% Inhibiting Concentration) and CC50 values (50% Cytotoxicity Concentration) obtained by MTT test and Trypan blue dye exclusion for each fungal metabolite. By MTT assay, the cytotoxicity ranking was fusarenon X (IC50 0.3 lM) ¼ diacetoxyscirpenol (IC50 0.5 lM) ¼ beauvericin (IC50 2.5 lM) ¼ nivalenol (IC50 5.3 lM) ¼ enniatin (IC50 6.6 lM) P gliotoxin (IC50 7.5 lM) > zearalenone (IC50 17.5 lM) > deoxynivalenol (IC50 47.6 lM). By Trypan blue dye exclusion the cytotoxicity ranking was fusarenon X (CC50 0.4 lM) ¼ diacetoxyscirpenol (CC50 1.1 lM) beauvericin ¼ (CC50 3.0 lM) ¼ gliotoxin (CC50 4.0 lM) ¼ enniatin (CC50 6.7 lM) P nivalenol (CC50 9.5 lM) > zearalenone (CC50 18.3 lM) > deoxynivalenol (CC50 45.0 lM). The comparison with other bioassays showed that the SF-9 insect cell line could represent a further tool to screen for the toxic effects of fungal metabolites especially for beauvericin, gliotoxin, and zearalenone. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Fungal metabolites; Entomopathogenic fungi; Spodoptera frugiperda cell line (SF-9); Cytotoxicity; MTT-colorimetric assay; Trypan blue dye exclusion
1. Introduction Many studies are carried out in vivo on effects by fungal metabolites in order to evaluate the influence on biological fitness of insects such as total number of surviving adults, growth retardation, egg production, anti-feedant activity (Alverson, 2003; Davis et al., 1975; Dowd, 1992, 1999; Mule et al., 1992; Pangrahi, 1993; Vey et al., 2001). In vivo studies using insects provide useful information on toxicity toward target organism, but they are laborious and time consuming. In vitro assays are important and useful tools in toxicity assessment of various classes of environmental contaminants including fungal metabolites not only because they significantly reduce evaluation time, but also because they provide information about the mode of action of the toxicant. In vitro cytotoxicity tests are * Corresponding author. Fax +39-080-5486063. E-mail address: [email protected] (F. Fornelli).
0022-2011/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.01.002
useful and necessary for screening purposes to define dose and time-depend cytotoxicity, considered primarily as the potential of a compound to induce cell death, in different cell types (Eisenbrand et al., 2002). The interest in developing cellular systems for toxicity testing has increased in recent years as they are usually less expensive, more quantitative, more reproducible, and more rapid than in vivo studies. On the other hand, the possibility of obtaining a false positive result is of particular concern when using bioassays. False positives may also be due to compounds that are apparently non toxic to higher animals, but toxic to lower biological organisms, tissue or cells (Pangrahi, 1993).The comparison between in vitro data and the effects on insects is advisable, when this is possible. Spodoptera frugiperda could be the perfect foil. In this study, the toxic effects induced by sixteen fungal metabolites, with different chemical structures and biological activity (Cole and Cox, 1981) produced by some important agricultural fungi including
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entomopathogenic fungi or biological control fungi agents, were evaluated on in vitro an invertebrate model, the lepidopteran S. frugiperda (SF-9). The aim of this paper was to evaluate the sensitivity of the SF-9 cell line toward fungal metabolites. In the past (Gutleb et al., 2002; Quiot et al., 1985; Vey and Quiot, 1989) insect cell lines have been used to test toxins and some studies used the SF-9 cells in particular (Dumas et al., 1996; Liu et al., 1996). In those works the mode of action induced by fungal metabolites such as dextrusins, cyclosporin A, hirsutellin A was described, and no toxicological screening was carried out. In this study on SF-9 the MTT-colorimetric and Trypan blue dye exclusion assays were selected as bioassays because they are frequently used for toxicological purposes and because they are characterised by low cost and quick response. In addition the MTT-test and Trypan blue dye exclusion have two distinct and specific cellular targets in mitochondrial function and membrane permeability changes, respectively. The MTT test is an alternative colorimetric assay using the tetrazolium salt MTT to measure cell proliferation and survival. The principle of this reaction is the reduction of the yellow coloured MTT by mitochondrial enzymes to purple formazan crystals (Mosman, 1983). The metabolic activity measured as enzymatic cleavage of MTT, is directly correlated to the number of cells and allows determination of the cell proliferation rate. Spectrophotometrical analysis of the metabolic conversion of dyes (such as the MTT test) may provide a more objective, faster, and economical method and it is used extensively to evaluate the dose-related cytotoxicity induced by fungal metabolites (Hanelt et al., 1994; Morrison et al., 2001; Reubel et al., 1987; Visconti et al., 1991; Widestrand et al., 1999). The Trypan blue dye exclusion assay is a cell viability assay based on the ability of live cells to exclude the vital dye Trypan blue and its uptake is indicative of irreversible membrane damage preceding cell death. Trypan blue dye exclusion is used generally to quantify the reduction in the percentage of viable cells and the cytotoxic effect (Charoenpornsook et al., 1998; Minervini et al., 2004; Ueno et al., 1995).
2. Material and methods 2.1. Fungal metabolites and reagents The following standard fungal metabolites were purchased from Sigma (Milan, Italy): fusarenon X (FUS X), diacetoxyscirpenol (DAS), beauvericin (BEA), nivalenol (NIV), gliotoxin (GL), zearalenone (ZEA), deoxynivalenol (DON), dipicolinic acid, fusaric acid (AF), oxalic acid, fumonisin B1 (FB1 ), moniliormin (MN), ochratoxin A (OTA), Kojic acid, enniatin (ENN) which
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contains A, A1 , B, and B1 enniatin homologs in the following ratio: 3, 21, 20, and 56%, respectively. The Fusaproliferin standard (FUP) with 90% of purity was a gift from Prof A. Ritieni (Department of Food Sciences University of Napoli, Italy). The reagents used, namely MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide), TNM-FH media, heat inactivated foetal calf serum (FCS), L -glutamine, penicillin–streptomycin–neomycin solution, DulbeccoÕs phosphate buffered saline (DPBS), and Trypan blue solution 0.4% were purchased from Sigma (Milan, Italy). 2.2. Cell culture and treatments The lepidopteran (S. frugiperda) cells obtained from the envelope of pupal ovaries SF-9 (Vaughn et al., 1977) were maintained at 27 °C in TNM-FH medium supplemented with 10% FCS (vol/vol), 1% L -glutamine 200 mM, and penicillin–streptomycin–neomycin solutions (vol/vol). Cultures in the early stationary phase (typical cell density, 1.6 106 cells/ml with cell viability approximately 80%) were split every 5–6 days with a starting density of 4 105 cells/ml. The fungal metabolite standards were dissolved in methanol with the exception of fumonisin B1 that was dissolved in phosphate buffered saline, as reported in the literature. The standard solutions were tested at concentrations ranging from 1 lM to 10 mM. Concerning the enniatin standard, the molar concentration was evaluated as the average molecular weight of the enniatin standard which contains enniatin homologs A, A1 , B, and B1 (the composition of the enniatin standard was described above in fungal metabolites and reagents). The control was prepared in the same manner without toxins and with appropriate solvent controls The final concentration of solvent in the cultures assayed was 1%. Cells were seeded in 96-well microtitration plates at the early stationary phase, when cell density ranged from 1 to 1.5 106 cells/ml and test solutions were added at a ratio of 1:100 (v/v). Plates were incubated for 48 h at 27 °C. 2.3. Bioassays 2.3.1. Trypan blue dye exclusion Trypan blue dye exclusion was added to cell cultures in a ratio of 1:1 and preparations were viewed at 100 with a standard light microscope. It is generally accepted that a viable cell will exclude an acid dye such as Trypan blue, and that its uptake is indicative of irreversible membrane damage preceding cell death. The ratio of live to dead cells (cell viability) was determined, standard curves were prepared and 50% cytotoxic concentrations (CC50 ), i.e., the concentrations of fungal metabolite that caused a 50% decrease in cell viability, were derived.
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2.3.2. MTT colorimetric assay This assay was carried out according to Visconti et al. (1991). Briefly, 5 mg/ml MTT was dissolved in PBS and 20 ll of this stock solution was added to culture wells. The incubation time with MTT was modified to 3 h at 27 °C because of the high cellular density. Supernatant was removed and 200 ll of 0.04 N HCl in isopropanol was added to each well before reading optical density at 580 nm with an ELISA Reader Multiskan MS Plus MK II Labsystem (Finland). The evaluation of the metabolic viability by MTT test is based on the ability of the mitochondria succinate–tetrazolium reductase system to convert the yellow compound MTT to a blue formazan dye. The amount of dye produced is proportional to the number of live cells. Dose–response curves were computer plotted after converting the mean data values to percentages of the control response. The 50% inhibiting concentration (IC50 ) was evaluated from the dose–response curves. The results obtained from both cytotoxicity assays were confirmed by repeating the experiment on three independent occasions and testing each fungal metabolite concentration or control in triplicate each time.
Table 1 Cytotoxity induced by some fungal metabolites on the SF-9 insect cell line using MTT test and Trypan blue dye exclusion Fungal metabolites
MTT test IC50 (lM)
Trypan blue test CC50 (lM)
Fusarenon X Diacetoxyscirpenol Beauvericin Nivalenol Enniatin Gliotoxin Zearalenone Deoxynivalenol Fusaproliferin Dipicolinic acid Fusaric acid Fumonisin B1 Moniliformin Ochratoxin A Oxalic acid Kojic acid
0.3 0.1 0.5 0.3 2.5 0.5 5.3 0.5 6.6 1.6a 7.5 3.5 17.5 3.5 47.6 2.5 >100 >100 >100 >100 >100 >100 >100 >100
0.4 0.2 1.1 0.5 3.0 1.0 9.5 0.8 6.7 1.7a 4.0 2.8 18.3 2.8 45.0 5.0 >100 >100 >100 >100 >100 >100 >100 >100
Data are expressed as mean standard deviation from three independent experiments. a Average molecular weight of enniatin standard, corresponding to 4.3 1.6 and 4.4 1.7 lg/ml by MTT test and Trypan blue dye exclusion, respectively. * Value differs significantly ðp < 0:001Þ for each fungal metabolites.
2.4. Statistical analysis of data All statistical analyses were performed using GraphPad Instat Software V 2.03 (Sigma, Italy). Parametric Turkey–Kramer Multiple Comparison Tests were used to compare statistical differences between the IC50 and CC50 values obtained with the different fungal metabolites tested in both in vitro assays. Differences were considered significant at p < 0:05.
ference was lower ðp < 0:05Þ. Following a comparison between ZEA and NIV, the IC50 value of NIV obtained by MTT test was lower ðp < 0:001Þ than the CC50 value obtained with Trypan blue dye assay ðp < 0:01Þ. In our experiments DON showed significantly ðp < 0:001Þ lower toxicity than other toxic metabolites in both assays. In dose–response curves obtained by Trypan blue dye exclusion (Fig. 1) a decrease in cell viability was observed when the cultures were exposed to BEA and NIV
3. Results and discussion The cytotoxic effects of the fungal metabolites are summarized in Table 1. No statistical difference was found between IC50 values and CC50 values obtained by both bioassays for each fungal metabolite. By MTT assay, no statistical difference was found for FUS X, DAS, BEA, NIV, ENN, with a range of IC50 values including between 0.3 and 6.6 lM. Gliotoxin showed a statistical reduction ðp < 0:05Þ of cytotoxicity respect to FUS X and DAS. By Trypan blue dye exclusion, no statistical difference was found for FUS X, DAS, BEA, GL with a range of CC50 values including between 0.4 and 4 lM. Enniatin and NIV resulted less cytotoxic ðp < 0:05Þ than FUS X. Concerning the fungal metabolites GL and ZEA, statistical differences were observed between the IC50 values and CC50 values obtained with the different bioassays. In particular by Trypan blue dye exclusion GL was significantly ðp < 0:001Þ more toxic than ZEA while, when the MTT test was used the statistical dif-
Fig. 1. Dose–response obtained with fusarenon X, gliotoxin, nivalenol, and deoxynivalenol after 48 h of incubation and evaluated by Trypan blue dye exclusion. Bars represent standard deviation of the mean cell viability (three replicates).
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at concentrations as high as 10 lM or above. For FUS X this effect was observed upto 0.3 lM. When the cell cultures treated with the highest concentration (100 lM) were examined by phase-contrast microscopy, features indicating damage cells were revealed. A reduction in cell viability (about 22%), evaluated by Trypan blue dye exclusion (Fig. 1), was only found with the highest concentration of DON tested (100 lM). A cell viability of 60, 62, and 64%, respectively was detected for FUP, OTA, and oxalic acid at the same concentration. For the other fungal metabolites the viability was found to be similar to the control (80% 4.8; mean standard deviation) with the highest concentration. The MTT test and Trypan blue dye exclusion assay, despite having different cellular targets, provided similar results for each metabolite. MTT test, and Trypan blue dye exclusion supplied a different order of cytotoxicity between the different fungal metabolites. By MTT assay, the cytotoxicity ranking was FUS X (IC50 0.3 lM) ¼ DAS (IC50 0.5 lM) ¼ BEA (IC50 2.5 lM) ¼ NIV (IC50 5.3 lM) ¼ ENN (IC50 6.6 lM) P GL (IC50 7.5 lM) > ZEA (IC50 17.5 lM) > DON (IC50 47.6 lM). By Trypan blue dye exclusion the cytotoxicity ranking was FUS X (CC50 0.4 lM) ¼ DAS (CC50 1.1 lM) BEA ¼ (CC50 3.0 lM) ¼ GL (CC50 4.0 lM) ¼ ENN (CC50 6.7 lM) P NIV (CC50 9.5 lM) > ZEA (CC50 18.3 lM) > DON (CC50 45.0 lM). The results obtained with this in vitro model show that the SF-9 cell line is sensitive to trichotecenes, such as FUS X, DAS, NIV, DON, and some fungal metabolites such as BEA, ENN GL, and ZEA. This is a first report about the cytotoxic effects of these fungal metabolites on the SF-9 cell line with the exception of BEA and FUP (Cal o et al., 2003; Logrieco et al., 1996). Although insect cell lines are used extensively as model to study the mechanism of action of the fungal metabolites (Dumas et al., 1994, 1996; Liu et al., 1996; Quiot et al., 1985; Vey and Quiot, 1989), few data have been reported on the effects of fungal metabolites tested (Abado-Becognee, 1999; Cal o et al., 2003; Logrieco et al., 1996; Vey et al., 1973). In particular AbadoBecognee (1999) reported the toxic effects induced by DON, FB1 , and MN on a lepidopteran Ploidia interpunctella wing cell line (IAL-PID-2) by MTT test, release of LDH, by MTT test, the values of IC50 were of 2.8, 102, and 260 lM for DON, FB1 , and MN, respectively. On the SF-9 cells an IC50 value of 48 lM was found for DON and no cytotoxicity was observed at the highest concentration (100 lM) of FB1 and MN. The CC50 values obtained by release of LDH were 4.2, 120M, and 320 lM for DON, FB1 , and MN, respectively. After comparison between results obtained by Trypan blue dye exclusion and those obtained by release of LDH, DON was 10 times more toxic in IAL-PID-2 than in the SF-9 cell line. This different cytotoxicity
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shows that the wing (IAL-PID-2) cell line is more sensitive to DON than the SF-9 cell line and this difference could be related to the different insect families or to the different origin of the tissue. Abado-Becognee (1999) reported a different toxicity induced by ZEA on the IAL-PID-2 cell line. A consistent release of LDH following cellular membrane damage with a CC50 value of 30 lM was reported and an IC50 of 10 lM was found by MTT test. On the SF-9 cell line no different cytotoxic effect of ZEA was observed in either assay (MTT test or Trypan blue dye exclusion). In both insect cell lines (IAL-PID-2 and SF-9), DON and ZEA were more toxic than FB1 and MN. The cytotoxicity obtained after exposure to BEA and FUP was in agreement with those reported in our previous papers (Cal o et al., 2003; Logrieco et al., 1996). Some authors reported in vivo effects of fungal metabolites in insects, measured as growth depression and changes in mortality, fertility, egg viability and metamorphosis between the egg, pupa, and adult stage (Dowd, 1992; Mule et al., 1992; Pangrahi, 1993; Vey et al., 2001). The choice of suitable insects for in vivo studies is an important consideration as there are considerable species and strain differences in the susceptibility of insects to fungal metabolites (Pangrahi, 1993). Grove and Hosken (1975) reported the toxicity of trichothecenes, after exposure of larvae of Aedis aegypti to 25 lg/ml: FUS X, induced low mortality (7%), NIV moderate (22%), and DAS high (74%). Dowd (1992) reported a moderate mortality (13%) on the fall armyworm (S. frugiperda) after incorporation of 25 ppm of DAS into artificial diets. Similar activity (17% of mortality at 50 ppm of DAS) was reported on larvae of Galleria mellonella L. by Mule et al. (1992). Dowd (1992) described no mortality on fall armyworm and corn earworm (Heliothis zea) at 25 ppm of DON, MN, and ZEA. At the same level, OTA caused high mortality (38 and 92% for corn earworm and fall armyworm, respectively). The comparison between in vivo studies and the results with the SF-9 cell line show a different toxicity ranking of regarding effects induced by FUS X, DAS, and OTA. Grove and Pople (1980) reported the toxicity of ENN and BEA after injection into adults of the blowfly Calliphora erytrocephala and larvae of the mosquito A. aegypti. Enniatin were less toxic than BEA in both insect species (20 lg/ml BEA and 75 lg/ml ENN caused 63 and 86% mortality, respectively). In the SF-9 cell line the reduced cytotoxicity of ENN with respect to BEA was confirmed. Concerning the fungal metabolites kojic acid and oxalic acid, a recent study (Alverson, 2003) reports their effects on Lygus hesperus. In particular 1500 ppm of kojic acid and oxalic acid in an artificial diet caused a reduction in the total number of surviving adults (30%).
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No significant differences were found between the two different acids. In the SF-9 cell line, no effect was observed with either fungal metabolites. The literature reports several studies on cytotoxicity of these fungal metabolites in mammalian cell lines (Cal o et al., 2004; Gutleb et al., 2002; Hanelt et al., 1994; Reubel et al., 1987; Visconti et al., 1991; Widestrand et al., 1999). The comparison between results obtained with the SF-9 cell line and mammalian cell lines was possible only for some fungal metabolites tested. This comparison showed that mammalian cells were more sensitive than the SF-9 cell line to effects of some fungal metabolites while, for other fungal metabolites, similar results were found. In particular for NIV, DON and FUS X the mammalian cells showed a sensitive 10 times higher than that of the SF-9 cell line (Visconti et al., 1991) and 100 times that for DAS and OTA (Reubel et al., 1987; Visconti et al., 1991). For fungal metabolites such as ZEA, GL, MN, and BEA similar results were found with both cell lines (Cal o et al., 2004; Gutleb et al., 2002; Hanelt et al., 1994). The comparison with other tests using small easy to manipulate animals such as Artemia salina showed that SF-9 cell line was 10-fold more sensitive than A. salina for FUS X (Schmidt, 1989), and 3-fold more sensitive for ZEA (Pangrahi, 1993). Using A. salina, a toxicity ranking was defined for FB1 , DON, and OTA, with the following LD50 (50% lethal dose) values: 1.6, 11.5, and 24.7 lM, respectively (Pangrahi, 1993; Schmidt, 1989). For DAS, NIV, BEA, and FUP, the data obtained with SF-9 cells was comparable with those obtained with A. salina (Logrieco et al., 1996; Pangrahi, 1993; Schmidt, 1989; Vey et al., 2001). In conclusion, the results obtained with this in vitro model show that the SF-9 cell line is sensitive to trichotecenes, such as FUS X, DAS, NIV, DON, and some fungal metabolites such as BEA, ENN, GL, and ZEA. The comparison with other bioassays showed that SF-9 insect cells could represent a further tool to screen for the toxic effects of fungal metabolites especially BEA, GL, and ZEA. Further studies are needed to evaluate the effects of these toxins on in vivo insects and S. frugiperda would be the perfect foil for this purpose.
Acknowledgments This work was supported by grants from the Italian Ministry of Scientific and Technological Research (MURST): Cofin 2000 and Plan for the Development of Research Networks, Law 488/92, Cluster C06+07, Project 1.1: Microorganisms and Microbial metabolites in Plant Protection. We thank Prof. A. Ritieni, Department of Food Sciences, University of Napoli, for the gift of fusaproliferin standard and Dr. L. Macchia, Department of Clinical Immunology and Allergology,
University of Bari, for the generous gift of the SF-9 cell line.
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 85 (2004) 74–79 www.elsevier.com/locate/yjipa
Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9) Francesca Fornelli,* Fiorenza Minervini, and Antonio Logrieco Istituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche (CNR), Viale Einaudi 51, 70125 Bari, Italy Received 3 October 2003; accepted 5 January 2004
Abstract The toxicity of sixteen fungal metabolites produced by some entomopathogenic fungi or biological control fungi agents was evaluated on lepidopteran Spodoptera frugiperda (SF-9) cell line by Trypan blue dye exclusion and MTT-colorimetric assay, after 48 h of incubation. No statistical difference was found between IC50 values (50% Inhibiting Concentration) and CC50 values (50% Cytotoxicity Concentration) obtained by MTT test and Trypan blue dye exclusion for each fungal metabolite. By MTT assay, the cytotoxicity ranking was fusarenon X (IC50 0.3 lM) ¼ diacetoxyscirpenol (IC50 0.5 lM) ¼ beauvericin (IC50 2.5 lM) ¼ nivalenol (IC50 5.3 lM) ¼ enniatin (IC50 6.6 lM) P gliotoxin (IC50 7.5 lM) > zearalenone (IC50 17.5 lM) > deoxynivalenol (IC50 47.6 lM). By Trypan blue dye exclusion the cytotoxicity ranking was fusarenon X (CC50 0.4 lM) ¼ diacetoxyscirpenol (CC50 1.1 lM) beauvericin ¼ (CC50 3.0 lM) ¼ gliotoxin (CC50 4.0 lM) ¼ enniatin (CC50 6.7 lM) P nivalenol (CC50 9.5 lM) > zearalenone (CC50 18.3 lM) > deoxynivalenol (CC50 45.0 lM). The comparison with other bioassays showed that the SF-9 insect cell line could represent a further tool to screen for the toxic effects of fungal metabolites especially for beauvericin, gliotoxin, and zearalenone. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Fungal metabolites; Entomopathogenic fungi; Spodoptera frugiperda cell line (SF-9); Cytotoxicity; MTT-colorimetric assay; Trypan blue dye exclusion
1. Introduction Many studies are carried out in vivo on effects by fungal metabolites in order to evaluate the influence on biological fitness of insects such as total number of surviving adults, growth retardation, egg production, anti-feedant activity (Alverson, 2003; Davis et al., 1975; Dowd, 1992, 1999; Mule et al., 1992; Pangrahi, 1993; Vey et al., 2001). In vivo studies using insects provide useful information on toxicity toward target organism, but they are laborious and time consuming. In vitro assays are important and useful tools in toxicity assessment of various classes of environmental contaminants including fungal metabolites not only because they significantly reduce evaluation time, but also because they provide information about the mode of action of the toxicant. In vitro cytotoxicity tests are * Corresponding author. Fax +39-080-5486063. E-mail address: [email protected] (F. Fornelli).
0022-2011/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.01.002
useful and necessary for screening purposes to define dose and time-depend cytotoxicity, considered primarily as the potential of a compound to induce cell death, in different cell types (Eisenbrand et al., 2002). The interest in developing cellular systems for toxicity testing has increased in recent years as they are usually less expensive, more quantitative, more reproducible, and more rapid than in vivo studies. On the other hand, the possibility of obtaining a false positive result is of particular concern when using bioassays. False positives may also be due to compounds that are apparently non toxic to higher animals, but toxic to lower biological organisms, tissue or cells (Pangrahi, 1993).The comparison between in vitro data and the effects on insects is advisable, when this is possible. Spodoptera frugiperda could be the perfect foil. In this study, the toxic effects induced by sixteen fungal metabolites, with different chemical structures and biological activity (Cole and Cox, 1981) produced by some important agricultural fungi including
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entomopathogenic fungi or biological control fungi agents, were evaluated on in vitro an invertebrate model, the lepidopteran S. frugiperda (SF-9). The aim of this paper was to evaluate the sensitivity of the SF-9 cell line toward fungal metabolites. In the past (Gutleb et al., 2002; Quiot et al., 1985; Vey and Quiot, 1989) insect cell lines have been used to test toxins and some studies used the SF-9 cells in particular (Dumas et al., 1996; Liu et al., 1996). In those works the mode of action induced by fungal metabolites such as dextrusins, cyclosporin A, hirsutellin A was described, and no toxicological screening was carried out. In this study on SF-9 the MTT-colorimetric and Trypan blue dye exclusion assays were selected as bioassays because they are frequently used for toxicological purposes and because they are characterised by low cost and quick response. In addition the MTT-test and Trypan blue dye exclusion have two distinct and specific cellular targets in mitochondrial function and membrane permeability changes, respectively. The MTT test is an alternative colorimetric assay using the tetrazolium salt MTT to measure cell proliferation and survival. The principle of this reaction is the reduction of the yellow coloured MTT by mitochondrial enzymes to purple formazan crystals (Mosman, 1983). The metabolic activity measured as enzymatic cleavage of MTT, is directly correlated to the number of cells and allows determination of the cell proliferation rate. Spectrophotometrical analysis of the metabolic conversion of dyes (such as the MTT test) may provide a more objective, faster, and economical method and it is used extensively to evaluate the dose-related cytotoxicity induced by fungal metabolites (Hanelt et al., 1994; Morrison et al., 2001; Reubel et al., 1987; Visconti et al., 1991; Widestrand et al., 1999). The Trypan blue dye exclusion assay is a cell viability assay based on the ability of live cells to exclude the vital dye Trypan blue and its uptake is indicative of irreversible membrane damage preceding cell death. Trypan blue dye exclusion is used generally to quantify the reduction in the percentage of viable cells and the cytotoxic effect (Charoenpornsook et al., 1998; Minervini et al., 2004; Ueno et al., 1995).
2. Material and methods 2.1. Fungal metabolites and reagents The following standard fungal metabolites were purchased from Sigma (Milan, Italy): fusarenon X (FUS X), diacetoxyscirpenol (DAS), beauvericin (BEA), nivalenol (NIV), gliotoxin (GL), zearalenone (ZEA), deoxynivalenol (DON), dipicolinic acid, fusaric acid (AF), oxalic acid, fumonisin B1 (FB1 ), moniliormin (MN), ochratoxin A (OTA), Kojic acid, enniatin (ENN) which
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contains A, A1 , B, and B1 enniatin homologs in the following ratio: 3, 21, 20, and 56%, respectively. The Fusaproliferin standard (FUP) with 90% of purity was a gift from Prof A. Ritieni (Department of Food Sciences University of Napoli, Italy). The reagents used, namely MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide), TNM-FH media, heat inactivated foetal calf serum (FCS), L -glutamine, penicillin–streptomycin–neomycin solution, DulbeccoÕs phosphate buffered saline (DPBS), and Trypan blue solution 0.4% were purchased from Sigma (Milan, Italy). 2.2. Cell culture and treatments The lepidopteran (S. frugiperda) cells obtained from the envelope of pupal ovaries SF-9 (Vaughn et al., 1977) were maintained at 27 °C in TNM-FH medium supplemented with 10% FCS (vol/vol), 1% L -glutamine 200 mM, and penicillin–streptomycin–neomycin solutions (vol/vol). Cultures in the early stationary phase (typical cell density, 1.6 106 cells/ml with cell viability approximately 80%) were split every 5–6 days with a starting density of 4 105 cells/ml. The fungal metabolite standards were dissolved in methanol with the exception of fumonisin B1 that was dissolved in phosphate buffered saline, as reported in the literature. The standard solutions were tested at concentrations ranging from 1 lM to 10 mM. Concerning the enniatin standard, the molar concentration was evaluated as the average molecular weight of the enniatin standard which contains enniatin homologs A, A1 , B, and B1 (the composition of the enniatin standard was described above in fungal metabolites and reagents). The control was prepared in the same manner without toxins and with appropriate solvent controls The final concentration of solvent in the cultures assayed was 1%. Cells were seeded in 96-well microtitration plates at the early stationary phase, when cell density ranged from 1 to 1.5 106 cells/ml and test solutions were added at a ratio of 1:100 (v/v). Plates were incubated for 48 h at 27 °C. 2.3. Bioassays 2.3.1. Trypan blue dye exclusion Trypan blue dye exclusion was added to cell cultures in a ratio of 1:1 and preparations were viewed at 100 with a standard light microscope. It is generally accepted that a viable cell will exclude an acid dye such as Trypan blue, and that its uptake is indicative of irreversible membrane damage preceding cell death. The ratio of live to dead cells (cell viability) was determined, standard curves were prepared and 50% cytotoxic concentrations (CC50 ), i.e., the concentrations of fungal metabolite that caused a 50% decrease in cell viability, were derived.
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2.3.2. MTT colorimetric assay This assay was carried out according to Visconti et al. (1991). Briefly, 5 mg/ml MTT was dissolved in PBS and 20 ll of this stock solution was added to culture wells. The incubation time with MTT was modified to 3 h at 27 °C because of the high cellular density. Supernatant was removed and 200 ll of 0.04 N HCl in isopropanol was added to each well before reading optical density at 580 nm with an ELISA Reader Multiskan MS Plus MK II Labsystem (Finland). The evaluation of the metabolic viability by MTT test is based on the ability of the mitochondria succinate–tetrazolium reductase system to convert the yellow compound MTT to a blue formazan dye. The amount of dye produced is proportional to the number of live cells. Dose–response curves were computer plotted after converting the mean data values to percentages of the control response. The 50% inhibiting concentration (IC50 ) was evaluated from the dose–response curves. The results obtained from both cytotoxicity assays were confirmed by repeating the experiment on three independent occasions and testing each fungal metabolite concentration or control in triplicate each time.
Table 1 Cytotoxity induced by some fungal metabolites on the SF-9 insect cell line using MTT test and Trypan blue dye exclusion Fungal metabolites
MTT test IC50 (lM)
Trypan blue test CC50 (lM)
Fusarenon X Diacetoxyscirpenol Beauvericin Nivalenol Enniatin Gliotoxin Zearalenone Deoxynivalenol Fusaproliferin Dipicolinic acid Fusaric acid Fumonisin B1 Moniliformin Ochratoxin A Oxalic acid Kojic acid
0.3 0.1 0.5 0.3 2.5 0.5 5.3 0.5 6.6 1.6a 7.5 3.5 17.5 3.5 47.6 2.5 >100 >100 >100 >100 >100 >100 >100 >100
0.4 0.2 1.1 0.5 3.0 1.0 9.5 0.8 6.7 1.7a 4.0 2.8 18.3 2.8 45.0 5.0 >100 >100 >100 >100 >100 >100 >100 >100
Data are expressed as mean standard deviation from three independent experiments. a Average molecular weight of enniatin standard, corresponding to 4.3 1.6 and 4.4 1.7 lg/ml by MTT test and Trypan blue dye exclusion, respectively. * Value differs significantly ðp < 0:001Þ for each fungal metabolites.
2.4. Statistical analysis of data All statistical analyses were performed using GraphPad Instat Software V 2.03 (Sigma, Italy). Parametric Turkey–Kramer Multiple Comparison Tests were used to compare statistical differences between the IC50 and CC50 values obtained with the different fungal metabolites tested in both in vitro assays. Differences were considered significant at p < 0:05.
ference was lower ðp < 0:05Þ. Following a comparison between ZEA and NIV, the IC50 value of NIV obtained by MTT test was lower ðp < 0:001Þ than the CC50 value obtained with Trypan blue dye assay ðp < 0:01Þ. In our experiments DON showed significantly ðp < 0:001Þ lower toxicity than other toxic metabolites in both assays. In dose–response curves obtained by Trypan blue dye exclusion (Fig. 1) a decrease in cell viability was observed when the cultures were exposed to BEA and NIV
3. Results and discussion The cytotoxic effects of the fungal metabolites are summarized in Table 1. No statistical difference was found between IC50 values and CC50 values obtained by both bioassays for each fungal metabolite. By MTT assay, no statistical difference was found for FUS X, DAS, BEA, NIV, ENN, with a range of IC50 values including between 0.3 and 6.6 lM. Gliotoxin showed a statistical reduction ðp < 0:05Þ of cytotoxicity respect to FUS X and DAS. By Trypan blue dye exclusion, no statistical difference was found for FUS X, DAS, BEA, GL with a range of CC50 values including between 0.4 and 4 lM. Enniatin and NIV resulted less cytotoxic ðp < 0:05Þ than FUS X. Concerning the fungal metabolites GL and ZEA, statistical differences were observed between the IC50 values and CC50 values obtained with the different bioassays. In particular by Trypan blue dye exclusion GL was significantly ðp < 0:001Þ more toxic than ZEA while, when the MTT test was used the statistical dif-
Fig. 1. Dose–response obtained with fusarenon X, gliotoxin, nivalenol, and deoxynivalenol after 48 h of incubation and evaluated by Trypan blue dye exclusion. Bars represent standard deviation of the mean cell viability (three replicates).
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at concentrations as high as 10 lM or above. For FUS X this effect was observed upto 0.3 lM. When the cell cultures treated with the highest concentration (100 lM) were examined by phase-contrast microscopy, features indicating damage cells were revealed. A reduction in cell viability (about 22%), evaluated by Trypan blue dye exclusion (Fig. 1), was only found with the highest concentration of DON tested (100 lM). A cell viability of 60, 62, and 64%, respectively was detected for FUP, OTA, and oxalic acid at the same concentration. For the other fungal metabolites the viability was found to be similar to the control (80% 4.8; mean standard deviation) with the highest concentration. The MTT test and Trypan blue dye exclusion assay, despite having different cellular targets, provided similar results for each metabolite. MTT test, and Trypan blue dye exclusion supplied a different order of cytotoxicity between the different fungal metabolites. By MTT assay, the cytotoxicity ranking was FUS X (IC50 0.3 lM) ¼ DAS (IC50 0.5 lM) ¼ BEA (IC50 2.5 lM) ¼ NIV (IC50 5.3 lM) ¼ ENN (IC50 6.6 lM) P GL (IC50 7.5 lM) > ZEA (IC50 17.5 lM) > DON (IC50 47.6 lM). By Trypan blue dye exclusion the cytotoxicity ranking was FUS X (CC50 0.4 lM) ¼ DAS (CC50 1.1 lM) BEA ¼ (CC50 3.0 lM) ¼ GL (CC50 4.0 lM) ¼ ENN (CC50 6.7 lM) P NIV (CC50 9.5 lM) > ZEA (CC50 18.3 lM) > DON (CC50 45.0 lM). The results obtained with this in vitro model show that the SF-9 cell line is sensitive to trichotecenes, such as FUS X, DAS, NIV, DON, and some fungal metabolites such as BEA, ENN GL, and ZEA. This is a first report about the cytotoxic effects of these fungal metabolites on the SF-9 cell line with the exception of BEA and FUP (Cal o et al., 2003; Logrieco et al., 1996). Although insect cell lines are used extensively as model to study the mechanism of action of the fungal metabolites (Dumas et al., 1994, 1996; Liu et al., 1996; Quiot et al., 1985; Vey and Quiot, 1989), few data have been reported on the effects of fungal metabolites tested (Abado-Becognee, 1999; Cal o et al., 2003; Logrieco et al., 1996; Vey et al., 1973). In particular AbadoBecognee (1999) reported the toxic effects induced by DON, FB1 , and MN on a lepidopteran Ploidia interpunctella wing cell line (IAL-PID-2) by MTT test, release of LDH, by MTT test, the values of IC50 were of 2.8, 102, and 260 lM for DON, FB1 , and MN, respectively. On the SF-9 cells an IC50 value of 48 lM was found for DON and no cytotoxicity was observed at the highest concentration (100 lM) of FB1 and MN. The CC50 values obtained by release of LDH were 4.2, 120M, and 320 lM for DON, FB1 , and MN, respectively. After comparison between results obtained by Trypan blue dye exclusion and those obtained by release of LDH, DON was 10 times more toxic in IAL-PID-2 than in the SF-9 cell line. This different cytotoxicity
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shows that the wing (IAL-PID-2) cell line is more sensitive to DON than the SF-9 cell line and this difference could be related to the different insect families or to the different origin of the tissue. Abado-Becognee (1999) reported a different toxicity induced by ZEA on the IAL-PID-2 cell line. A consistent release of LDH following cellular membrane damage with a CC50 value of 30 lM was reported and an IC50 of 10 lM was found by MTT test. On the SF-9 cell line no different cytotoxic effect of ZEA was observed in either assay (MTT test or Trypan blue dye exclusion). In both insect cell lines (IAL-PID-2 and SF-9), DON and ZEA were more toxic than FB1 and MN. The cytotoxicity obtained after exposure to BEA and FUP was in agreement with those reported in our previous papers (Cal o et al., 2003; Logrieco et al., 1996). Some authors reported in vivo effects of fungal metabolites in insects, measured as growth depression and changes in mortality, fertility, egg viability and metamorphosis between the egg, pupa, and adult stage (Dowd, 1992; Mule et al., 1992; Pangrahi, 1993; Vey et al., 2001). The choice of suitable insects for in vivo studies is an important consideration as there are considerable species and strain differences in the susceptibility of insects to fungal metabolites (Pangrahi, 1993). Grove and Hosken (1975) reported the toxicity of trichothecenes, after exposure of larvae of Aedis aegypti to 25 lg/ml: FUS X, induced low mortality (7%), NIV moderate (22%), and DAS high (74%). Dowd (1992) reported a moderate mortality (13%) on the fall armyworm (S. frugiperda) after incorporation of 25 ppm of DAS into artificial diets. Similar activity (17% of mortality at 50 ppm of DAS) was reported on larvae of Galleria mellonella L. by Mule et al. (1992). Dowd (1992) described no mortality on fall armyworm and corn earworm (Heliothis zea) at 25 ppm of DON, MN, and ZEA. At the same level, OTA caused high mortality (38 and 92% for corn earworm and fall armyworm, respectively). The comparison between in vivo studies and the results with the SF-9 cell line show a different toxicity ranking of regarding effects induced by FUS X, DAS, and OTA. Grove and Pople (1980) reported the toxicity of ENN and BEA after injection into adults of the blowfly Calliphora erytrocephala and larvae of the mosquito A. aegypti. Enniatin were less toxic than BEA in both insect species (20 lg/ml BEA and 75 lg/ml ENN caused 63 and 86% mortality, respectively). In the SF-9 cell line the reduced cytotoxicity of ENN with respect to BEA was confirmed. Concerning the fungal metabolites kojic acid and oxalic acid, a recent study (Alverson, 2003) reports their effects on Lygus hesperus. In particular 1500 ppm of kojic acid and oxalic acid in an artificial diet caused a reduction in the total number of surviving adults (30%).
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No significant differences were found between the two different acids. In the SF-9 cell line, no effect was observed with either fungal metabolites. The literature reports several studies on cytotoxicity of these fungal metabolites in mammalian cell lines (Cal o et al., 2004; Gutleb et al., 2002; Hanelt et al., 1994; Reubel et al., 1987; Visconti et al., 1991; Widestrand et al., 1999). The comparison between results obtained with the SF-9 cell line and mammalian cell lines was possible only for some fungal metabolites tested. This comparison showed that mammalian cells were more sensitive than the SF-9 cell line to effects of some fungal metabolites while, for other fungal metabolites, similar results were found. In particular for NIV, DON and FUS X the mammalian cells showed a sensitive 10 times higher than that of the SF-9 cell line (Visconti et al., 1991) and 100 times that for DAS and OTA (Reubel et al., 1987; Visconti et al., 1991). For fungal metabolites such as ZEA, GL, MN, and BEA similar results were found with both cell lines (Cal o et al., 2004; Gutleb et al., 2002; Hanelt et al., 1994). The comparison with other tests using small easy to manipulate animals such as Artemia salina showed that SF-9 cell line was 10-fold more sensitive than A. salina for FUS X (Schmidt, 1989), and 3-fold more sensitive for ZEA (Pangrahi, 1993). Using A. salina, a toxicity ranking was defined for FB1 , DON, and OTA, with the following LD50 (50% lethal dose) values: 1.6, 11.5, and 24.7 lM, respectively (Pangrahi, 1993; Schmidt, 1989). For DAS, NIV, BEA, and FUP, the data obtained with SF-9 cells was comparable with those obtained with A. salina (Logrieco et al., 1996; Pangrahi, 1993; Schmidt, 1989; Vey et al., 2001). In conclusion, the results obtained with this in vitro model show that the SF-9 cell line is sensitive to trichotecenes, such as FUS X, DAS, NIV, DON, and some fungal metabolites such as BEA, ENN, GL, and ZEA. The comparison with other bioassays showed that SF-9 insect cells could represent a further tool to screen for the toxic effects of fungal metabolites especially BEA, GL, and ZEA. Further studies are needed to evaluate the effects of these toxins on in vivo insects and S. frugiperda would be the perfect foil for this purpose.
Acknowledgments This work was supported by grants from the Italian Ministry of Scientific and Technological Research (MURST): Cofin 2000 and Plan for the Development of Research Networks, Law 488/92, Cluster C06+07, Project 1.1: Microorganisms and Microbial metabolites in Plant Protection. We thank Prof. A. Ritieni, Department of Food Sciences, University of Napoli, for the gift of fusaproliferin standard and Dr. L. Macchia, Department of Clinical Immunology and Allergology,
University of Bari, for the generous gift of the SF-9 cell line.
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