T-2 toxin immunotoxicity on human B and T lymphoid cell lines

Toxicology 210 (2005) 81–91

T-2 toxin immunotoxicity on human B and T lymphoid cell lines Fiorenza Minervini a, ∗ , Francesca Fornelli a , Giacomo Lucivero b , Ciro Romano b , Angelo Visconti a b

a Istituto di Scienze delle Produzioni Alimentari (ISPA), CNR, Via Amendola 122/O, 70124 Bari, Italy Divisione di Medicina Interna e Immunoallergologia, Dipartimento di Gerontologia, Geriatria e Malattie del Metabolismo, Seconda Universit`a degli Studi di Napoli, Piazza Miraglia, 3 Napoli, Italy

Received 1 October 2004; accepted 10 January 2005 Available online 17 February 2005

Abstract T-2 toxin belongs to a group of mycotoxins synthesized by Fusarium fungi that are widely encountered as natural contaminants in cereals. Human lymphoid cell lines of T (MOLT-4) or B (IM-9) lineage were used to characterize the cytotoxic effects mediated by T-2 at different concentrations (0.1 pg/ml to 1 ␮g/ml). After 24 h, membrane damage was observed by Trypan blue dye exclusion in IM-9 cells with a 50% cytotoxic concentration (CC50 ) of 0.2 ng/ml, whereas CC50 for MOLT-4 cells was 0.6 ␮g/ml (gmicro). At a T-2 concentration of 0.01 ␮g/ml, apoptosis was seen in MOLT-4 cells by Annexin V binding as early as after 4 h. T-2 toxin determined sustained (48 h) immunosuppression on both cell lines, as evaluated by BrdU and MTT assays. Cytotoxicity appeared to be due to early apoptosis in MOLT-4 cells, as indicated by increased Annexin V binding and activation of caspase-3, and to direct cell membrane damage in IM-9 cells. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: MOLT-4; IM-9; Apoptosis; Flow cytometry; Cytotoxicity; T-2 toxin

1. Introduction Mycotoxins represent a diverse group of secondary fungal metabolites, which vary widely in their chemistry and toxicology. Several species of Fusarium fungi ∗ Corresponding author. Tel.: +39 080 5929360; fax: +39 080 5929374. E-mail address: [email protected] (F. Minervini).

are capable of producing mycotoxins, including trichothecenes. Fusarium sporotrichioides and F. poae are contaminants of certain agricultural commodities and are also species of economic importance capable of producing the potent trichothecene T-2 toxin. Alimentary toxic aleukia was reported in the former USSR during the period 1931–1947 and was attributed to the presence of toxic Fusarium species, specifically F. poae and F. sporotrichioides, in mouldy over-wintered

0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.01.007

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grain. The main pathological findings were leukopenia and necrotic lesions of the oral cavity, oesophagus, and stomach. The disease was lethal in a high proportion of cases but the presence of T-2 toxin was not identified (Canady et al., 2001). Acute exposure to trichothecenes results in severe damage to actively dividing cells in tissues such as bone marrow, lymph nodes, spleen, thymus, and intestinal mucosa. The capacity of trichothecene mycotoxins to be potent protein synthesis inhibitors and to interact with the cell membrane apparently contributes to their potential to modulate immune functions (Canady et al., 2001). Among trichothecenes, T-2 is the most cytotoxic toxin. Lymphocytes are more sensitive to T-2 than other cultured cell lines and this corresponds well with data from in vivo experiments showing that trichothecenes act as immunosuppressive agents (Gutleb et al., 2002). Specifically, T-2 effects on human lymphocytes included blunting of mitogeninduced blast transformation, inhibition of antibodydependent cellular cytotoxicity, and suppression of natural killer activity (Berek et al., 2001). The aim of this work was therefore to characterize the immunotoxic effects induced by T-2 toxin in human T (MOLT-4) and B (IM-9) lymphoid cell lines by means of different bioassays.

BD PharMingen. CaspACE assay system colorimetric (G-7351) was purchased from Promega. 2.2. Cell lines and culture procedures MOLT-4 (BS TCL 85), a T lymphoblastic human cell line, and IM-9 (BS TCL 98), an EBVtransformed B lymphoblastoid human cell line, were purchased from the Cell Bank of Istituto Zooprofilattico Sperimentale (Brescia, Italy) and maintained in RPMI 1640 medium supplemented with 10% FBS, 200 mM l-glutamine (1%), and penicillin–streptomycin–neomycin solution (1%) at 37 ◦ C in ambient air with 5% CO2 . The starting densities were 4 × 105 and 5 × 105 cells/ml for MOLT-4 and IM-9 cell lines, respectively. Culture medium was renewed every 2–3 days. T-2 toxin was solubilized in a mixture of ethanol, dimethyl-sulfoxide and RPMI 1640, as described by Visconti et al. (1991) at a final concentration of 50 ␮g/ml. Mycotoxin serial dilutions (1:10) were prepared at concentrations ranging from 0.2 pg/ml to 2 ␮g/ml (gmicro) and added to cell cultures at a ratio of 1:1 (v/v). Control solutions were prepared in the same manner but without toxin. The final DMSO and ethanol concentration of 0.1% had no significant effect on the growth and viability of both cell lines.

2. Materials and methods 2.3. Viability assessment 2.1. Mycotoxin and reagents T-2 toxin (T-4887), Trypan blue solution 0.4% (T-8154), MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide) (M-2128), dimethylsulfoxide (DMSO) (D-5879), RPMI 1640 medium (R-5886), heat-inactivated foetal bovine serum (FBS) (F-6178), l-glutamine (G-7513), penicillin– streptomycin–neomycin solution (P-9032), ascorbic acid (A-4403), BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid tetrakis (acetoxymethylester) (A-1076), camptothecin (C-9911), propidium iodide solution (PI) (P-4864), Triton X-100 (T-8787), ribonuclease A (R-4875) were purchased from Sigma. Cell proliferation ELISA BrdU colorimetric assay (1647229) was purchased from Roche. Caspase-3&7 FLICA apoptosis Detection kit (ICT-93-T025) was purchased from Vinci-Biochem. Annexin V binding assay (556547) was obtained from

MOLT-4 and IM-9 cells were seeded in a 96 multiwell plate at a density of 4 × 105 and 5 × 105 cells/ml, respectively, and different concentrations of T-2 toxin ranging from 0.2 pg/ml to 2 ␮g/ml were added at a ratio of 1:1 (v/v). Cell viability was assessed by Trypan blue dye exclusion in time-course experiments (2–48 h). Briefly, an aliquot (50 ␮l) of cell suspension was incubated with an equal volume of Trypan blue for 2 min. Cells were transferred to the chamber of a hemocytometer and counted by light microscopy. Dead cells were defined as those stained with the dye, its uptake being indicative of irreversible membrane damage. Dose–response curves were prepared and the 50% cytotoxic concentration (CC50 ), i.e., the concentration of mycotoxin that caused a 50% decrease in cell viability, was derived by linear extrapolation. Six independent experiments were carried out and each mycotoxin concentration or control was tested in triplicate each time.

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2.4. MTT colorimetric assay Quantification of viable cells was performed using the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) to a blue coloured product (formazan), which is indicative of mitochondrial succinate-dehydrogenase activity in viable cells. This assay was carried out according to Visconti et al. (1991). Briefly, 4.0 × 104 MOLT-4 cells/100 ␮l or 5.0 × 104 IM-9 cells/100 ␮l were seeded in a 96-well plate and supplemented with 100 ␮l of different concentrations of T-2 toxin, as described in Section 2.2. At two different incubation time points (24 and 48 h), 20 ␮l of 5 mg/ml MTT dissolved in PBS were added to culture wells and plates were incubated for 4 h at 37 ◦ C. Supernatants were removed and 200 ␮l of 0.04N HCl in isopropanol was added to each well prior to optical density measurement at 580 nm with an ELISA-Reader Multiskan MS Plus. Dose–response curves were plotted after converting the mean values into percentages of control response. Mycotoxin doses resulting in 50% inhibition of live cells (ID50 ) were derived from plotted data by linear extrapolation. 2.5. BrdU uptake colorimetric assay 5-Bromo-2 -deoxyuridine (BrdU) uptake colorimetric assay was used to determine cell proliferation through BrdU incorporation into cellular DNA. BrdU is a thymidine analogue, which is incorporated into DNA during the S-phase of the cell cycle and can be detected by immunoassay. The assay was performed according to the manufacturer’s instructions, as in Minervini et al. (2004). After exposure of cell cultures to different concentrations of T-2 toxin for 48 h, cells were incubated for 4 h at 37 ◦ C with 10 ␮M BrdU and then fixed. DNA was denatured in order to improve the accessibility of the incorporated BrdU for detection by the antibody and a peroxidase-conjugated mouse monoclonal anti-BrdU-POD antibody was added. Immune complex formation was revealed by measuring the absorbance of the substrate reaction (tetramethylbenzydine) at 450 nm with an ELISA-Reader. Absorbance values directly correlated with the amount of DNA synthesis and thereby with the number of proliferating cells in the respective microcultures. Mean absorbance values at each toxin concentration were compared to mean control values and were expressed

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as percent of control response. Dose–response curves were computer-plotted and the mycotoxin concentration resulting in 50% inhibition of proliferation (IC50 ) were derived from the plotted data by linear extrapolation. 2.6. Flow cytometry 2.6.1. Cell cycle analysis This assay was performed using a FACSCalibur flow cytometer (Beckton-Dickinson, Milan, Italy) equipped with an argon ion laser. Cells (1 × 106 ), treated with different T-2 toxin concentrations or induced to undergo apoptosis with camptothecin (12 ␮M) were suspended in 1 ml PI solution (PI 50 ␮g/ml in 0.1% sodium citrate plus 0.1% Triton X-100 plus 0.1% RNase at 0.5 mg/ml) in polypropylene tubes. PI fluorescence was obtained using linear (FL2 584–642 nm) amplification. At least 20,000 events were recorded for each sample. A timecourse analysis was carried out between 4 and 48 h. Cell cycle analysis was calculated by rectangular fitting (MODIFIT, Becton-Dickinson, Milan, Italy), using 1024 channels which produced histograms with a single G0 /G1 peak at channel 200 when DNA is diploid, an S-peak between channels 200 and 400 when DNA is replicating, a G2 /M peak at channel 400 when DNA is tetraploid and a debris peak between channels 100 and 200 when DNA is hypodiploid or damaged. Histograms were considered reliable when the coefficient of variation (CV) of the G0 /G1 diploid peak was lower than 5%. Assays were repeated at least on three separate experiments. 2.6.2. Annexin V-FITC apoptosis Annexin V, an impermeable plasma protein, which specifically binds to surface phosphatidylserine (PS), is commonly used to detect cells undergoing apoptosis because the translocation of PS from the inner leaflet of the cell membrane to the outer leaflet occurs in the earliest stages of apoptosis. Staining with Annexin V-FITC is typically used in conjunction with a vital dye, such as propidium iodide (PI), in order to distinguish necrotic cells from early apoptotic cells. Approximately 1.0 × 106 cells/well were plated in a 24-well plate and medium containing different T-2 toxin concentrations were added at a 1:1 ratio. The culture control treated with 12 ␮M camptothecin (a topoisomerase I inhibitor) was used as positive control of apoptosis.

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To determine the optimal time-course for apoptosis induction, cells were harvested after 4 and 6 h of incubation, washed in PBS, and centrifuged at 200 × g for 5 min. Cellular suspensions containing 1.0 × 105 cell/ 100 ␮l were stained with Annexin V-FITC and PI, gently mixed, and incubated for 15 min at room temperature (15–25 ◦ C) in the dark. Samples were then subjected to flow cytometry on a FACSCalibur and analyses were carried out by Cell Quest software. Assays were repeated on two separate experiments. 2.7. Caspase-3 activation assay 2.7.1. Caspase-3&7 FLICA kit Cells (1.0 × 106 cells/ml) were incubated with different T-2 toxin concentrations or 12 ␮M camptothecin for 5 h at 37 ◦ C prior to flow cytometry analysis. Briefly, FLICA reagent (FAM·DEVD-FMK) was added to cells prior to incubation for 1 h at 37 ◦ C in 5% CO2 . After thorough washings, cells were centrifuged (350 × g for 5 min at room temperature) twice. Two sets of cell samples were used for single and dual colour staining, respectively, for flow cytometry analysis. For singlecolour analysis, fluorescein was measured on the log FL1 channel (x-axis) whereas cell number was set on the y-axis. In this histogram, two cell populations, represented by two peaks, appeared. The caspase negative cells occurred within the first log decade of the FL1 axis (M1), whereas the caspase-positive cell population appeared as a separate peak (M2) with increased fluorescence intensity. For dual colour analysis, cells were stained with both PI and the FLICA reagent. Live cells, dead cells, caspase-negative, and caspasepositive cells could be detected by measuring fluorescein on the FL1 channel and red fluorescence (PI) on the FL2 channel. Assays were repeated on two separate experiments. 2.7.2. Colorimetric determination A caspase-3 colorimetric assay kit was used to assess caspase-3 activation in MOLT and IM-9 cell lines exposed to T-2 toxin. As positive control for apoptosis, camptothecin at a concentration of 12 ␮M (gmicro) was used. Z-VAD-FMK inhibitor at a final concentration of 50 ␮M was used for inhibition of apoptosis. The assay was performed according to the manufacturer’s instructions (Promega). Briefly, after a 16 h incubation, cells (1.0 × 106 /ml) were disrupted by in-

cubation in ice-cold lysing buffer for 10 min and then centrifuged at 15,000 × g for 20 min. Supernatants (cell extracts containing caspase-3) were retrieved and 50 ␮l aliquots (100–200 ␮g total protein) along with AcDEVD substrate labelled with the chromophore pnitrianiline (pNA) were added in a 96-well flat bottomed microplate. In the presence of active caspase-3, cleavage and release of pNA from the substrate occurs. Free pNA produces a yellow color that can be detected by a photometer at 405 nm. Additional controls, some free from cell lysates and others lacking substrate, were included. The results were expressed as caspase-3 specific activity (IU/mg protein). 2.8. Statistical analysis of data All statistical analyses were performed using the GraphPad Instat Software version 2.03 (Sigma, Italy). Statistical differences between the mean CC50 , IC50 , and ID50 values and the percentage values of cell cycle analysis were calculated by analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparisons test. Bonferroni multiple comparison test was used to compare the results obtained with the FLICA kit.

3. Results 3.1. Trypan blue A decrease in cellular viability was found in both cell lines. In time-course (2–48 h) analysis, a reduced viability (mean ± S.E. = 58% ± 4.5) was observed on IM-9 line after 8 h with 1 ␮g/ml T-2 toxin. A further increase in the proportion of membrane-damaged cells was found after 12 h incubation. MOLT-4 showed a membrane damage (41% ± 0.3 cell viability) only after 24 h incubation at the highest T-2 toxin concentration. After 24 h, the sensitivity to cytotoxicity was further different in the two cell lines, with IM-9 cells showing a significantly (p < 0.001) lower CC50 values than MOLT-4 cells (Table 1). After 48 h, cytotoxicity significantly increased in T-2-treated MOLT-4 cells. No significant difference was seen in IM-9 cells at the same time point with respect to 24 h. Experiments carried out with ascorbic acid (0.005 mM), known antioxidant substance, showed

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Table 1 Cytotoxicity (CC50 ), cell metabolism (ID50 ) and inhibition of cell proliferation (IC50 ) induced by T-2 toxin on MOLT-4 and IM-9 human cell lines Cell line

Trypan blue dye exclusion CC50 (␮g/ml) 24 h

MOLT-4 IM-9

0.6 ± 0.03 (n = 6)*** 0.0002 ± 0.0001 (n = 7)

MTT assay ID50 (␮g/ml)

BrdU assay IC50 (␮g/ml)

48 h

48 h

48 h

0.006 ± 0.0006 (n = 6) 0.0002 ± 0.0001 (n = 7)

0.001 ± 0.0004 (n = 6)* 0.0006 ± 0.00006 (n = 6)

0.003 ± 0.0009 (n = 6) 0.00002 ± 0.000011 (n = 6)

Statistical analysis evaluated by ANOVA parametric Tukey–Kramer multiple comparison test; comparison between results obtained on both cell lines and by different bioassays. Values are mean ± S.E. ∗ p < 0.05 after comparison between Trypan blue and MTT test at 48 h carried out on MOLT-7 cells. ∗∗∗ p < 0.001 after comparison between Trypan blue dye exclusion at 24 and 48 h carried out on MOLT-4 cells, p < 0.001 after comparison between both cell line of Trypan blue dye exclusion carried out at 24 h.

a significantly (p < 0.01) decreased cytotoxicity (80% ±3 versus 58% ± 4.5) on IM-9 cells after 8 h with 1 ␮g/ml T-2 toxin. As reported in Fig. 1, a further significant (p < 0.05) inhibition of cytotoxicity was observed up to 12 h. MOLT-4 cells shown after 24 h incubation a slight protective effect (60% ± 0.2 versus 41% ± 0.3) observed upon supplementation of ascorbic acid (data not shown). 3.2. MTT bioassay A reduction in mitochondrial activity was found in both cell lines following exposure to T-2 toxin, as reported in Table 1. No significant difference was observed in ID50 values between MOLT-4 and IM-9 cells.

Significant difference (p < 0.05) was observed between CC50 and ID50 values found on MOLT-4 line. 3.3. BrdU assay Inhibition of cell proliferation was observed in both cell lines after 48 h exposure to T-2 toxin. As reported in Table 1, no significant difference was found in IC50 values between both cell lines. No differences in cytotoxicity were found in both cell lines among bioassays (Trypan blue dye exclusion, MTT test). 3.4. Cell cycle perturbation Analysis of DNA content by flow cytometry provides a means of looking at cell cycle perturbation, and

Fig. 1. Cytotoxicity induced by T-2 toxin ± ascorbic acid on IM-9 cell line. Dose–response obtained and evaluated by Trypan blue dye exclusion. Data are mean ± S.E. of three separate experiments significant differences are shown as: *** p < 0.001, ** p < 0.01 and * p < 0.05 after comparison between each concentration and control by Tukey–Kramer multiple comparisons test.

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Table 2 Cell cycle analysis of MOLT-4 and IM-9 cell lines treated with T-2 toxin T-2 toxin concentration ␮g/ml (n)

G0 /G1 phase (%)

MOLT-4 cell line After 24 h of incubation Control (5) 1 (5) 0.1 (5) 0.01 (5) 0.001 (5)

62.7 69.4 55.3 54 74

± ± ± ± ±

2 6.7 3.2 1.8 6.3

30.1 29.9 39.1 38.3 21.3

± ± ± ± ±

2.2 5 3.9 2.4 5.4

7.2 4 5.5 8.8 5.4

± ± ± ± ±

0.3 0.4 1.5 2.1 1.0

15.5 81.7 63.7 53 21.2

± ± ± ± ±

2.3 5.3*** 16.5*** 12** 5.3

After 48 h of incubation Control (5) 1 (5) 0.1 (5) 0.01 (5) 0.001 (5)

57.7 80.5 59 56.8 64.8

± ± ± ± ±

1.8 7.4 1.3 1.8 5.1

33.8 17.4 34 35.5 28.3

± ± ± ± ±

1.7 6.8 3.6 2 4.5

7.8 4 6.6 5.9 6.8

± ± ± ± ±

0.4 1.6 3 2 1

13.9 63.7 62.9 45.5 23

± ± ± ± ±

3.1 9.8*** 15.5*** 19.3*** 6.3

IM-9 cell line After 12 h of incubation Control (3) 1 (3) 0.1 (3) 0.01 (3) 0.001 (3)

60.8 51.9 50.9 51.4 51.7

± ± ± ± ±

3.5 4.5 3.5 3.8 3.9

27.5 41.7 44.0 43.7 31.9

± ± ± ± ±

3.6 7.3 6 6.0 2.9

11.7 5.7 5.1 7.9 16.3

± ± ± ± ±

0.08 2.2 2.8 1.1 1.1

34.0 67.7 60.4 59.5 54.9

± ± ± ± ±

8.1 5.7*** 1.3* 5.6* 8.4

After 24 h of incubation Control (3) 1 (3) 0.1 (3) 0.01 (3) 0.001 (3) 0.0001 (3)

69.1 – – 59.2 67.9 67.1

± 1.1

21.2 – – 28.5 20.8 21.8

± 0.5

10.2 – – 11.9 11.3 11.1

± 0.8

After 48 h of incubation Control (3) 1 (3) 0.1 (3) 0.01 (3) 0.001 (3) 0.0001 (3)

67.9 – – 42.1 64.6 68

±3

22.8 – – 17.9 23.6 23.7

± 2.7

± 1.6* ± 2.1 ± 0.7

± 21.1 ± 2.3 ± 2.9

S-phase (%)

± 1.1 ± 1.5 ± 0.4

G2 /M phase (%)

± 0.8 ± 0.7 ± 0.4

9.6 ± 0.8 – –

± 9.7 ± 2.3 ± 2.1

6.7 ± 3.8 11.9 ± 0.6 9.8 ± 0.4

Debris (%)

16.2 ± 100*** 100*** 86.1 ± 20.3 ± 17.8 ±

4.7

15.6 ± 100*** 100*** 78.1 ± 23.4 ± 13.1 ±

5.6

2.5*** 2.9 1

11.6*** 7.3 3.8

One-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison test. Values are mean ± S.E. ∗ p < 0.05. ∗∗∗ p < 0.001.

detects accumulation or depletion of cells in particular phases. Indeed, time-course analysis carried out in MOLT-4 and IM-9 cells showed cell cycle perturbation following exposure to T-2 toxin. No modification was recorded in MOLT-4 cells following treatment with T-2 after incubation for 4–12 h (data not shown). As reported in Table 2, a significant increase of debris was observed between 24 and 48 h after incubation of

MOLT-4 cells with T-2 toxin. Significant changes in cell cycle, characterized by debris increase, were observed in IM-9 cells at early incubation time (12 h) regardless of the T-2 toxin compound used. At 24 and 48 h, 100% debris was found up to 0.01 ␮g/ml T-2 toxin. The apoptosis positive (camptothecin) control showed no cell cycle perturbation at all incubation time points on both cell lines (data not shown).

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Fig. 2. Early apoptosis induction on MOLT-4 and IM-9 cell lines after 6 h of exposure to T-2 toxin evaluated by Annexin V binding. Representative two color FACS analysis of early apoptosis induction on MOLT-4 and IM-9 cell lines. Both lines were treated with different concentrations of T-2 toxin and, after 6 h, were collected for Annexin V-FITC staining. MOLT-4: (a) control; (b) apoptosis positive control (camptothecin); (c) T-2 toxin at 0.01 ␮g/ml IM-9; (d) control; (e) apoptosis positive control (camptothecin); (f) T-2 toxin at 1 ␮g/ml. Apoptotic cells are indicated by arrows.

3.5. Detection of apoptosis by annexin V/PI staining Annexin V-FITC binding (FL1-H) and PI staining (FL2-H) by flow cytometry were used as criteria for distinguishing cycling, early apoptotic, late apoptotic, or necrotic cells. With regard to MOLT-4 cells, the early apoptotic cell population increased dose-dependently (0.01–1 ␮g/ml of T-2 toxin) from 3- to 7-fold with respect to control cells after 4 h incubation. At the same time point, the camptothecin-treated positive control showed a 14-fold increase in Annexin V-FITC+ cells. A further increase in Annexin V-FITC+ cells was recorded after 6 h following T-2 exposure (Fig. 2). With regard to IM-9 cells, a 4 h exposure to different concentrations of T-2 toxin did not yield changes in the fraction of early apoptotic cells. High mortality, as determined by detection of Annexin V-FITC+ /PI+ cells, was found both in control (34.7 ± 16%) and treated cell cultures (from 22 to 28%) after a 4 h incubation. In the positive control for apoptosis, a 2-fold increase in the apoptotic population was seen with respect to

untreated cells after 4 h of incubation. No difference was observed at 6 h with respect to the 4 h time point (Fig. 2). The treatment of MOLT-4 cells with BAPTA-AM, a chelator agent of Ca2+ , was carried out in order to evaluate a reduction of apoptosis after T-2 treatment or camptotecin, following the protocol reported by Holme et al. (2003). As shown in Fig. 3, no differences in apoptosis were seen between T-2-treated cultures with or without BAPTA-AM. Similar results were observed in camptothecin-treated cells. 3.6. Caspase-3 activation 3.6.1. FLICA kit This novel approach to detect active caspases in situ is based on the entry of fluorochrome inhibitor of caspase (FLICA) inside the cell, on the covalent link of FLICA inhibitor carboxyfluorescein-labeled DEVD-FMK to a reactive cystine residue that resides on the large subunit of the activated caspase-3 and -7 with following inhibition of further enzymatic activ-

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3.6.2. Colorimetric assay The cleavage of synthetic substrate upon incubation with lysates of apoptotic cells is another commonly used tool to detect caspase activity. The intensity of the signal is proportional to the amount of cleaved substrate, which in turn, is dependent on caspase activity and is related to the percentage of apoptotic cells in a cell population. For technical problems this determination was done only once, preventing us from repeating this assay enough times. As shown in Fig. 5, an increase in caspase activity was found in camptothecinstimulated MOLT-4 cells and in MOLT-4 cultures supplemented with up to 0.001 ␮g/ml T-2 toxin. Conversely, lower levels of caspase activity were detected in both positive control and T-2-treated IM-9 cells.

Fig. 3. Evaluation of apoptotic process induced by T-2 toxin ± BAPTA-AM (6 h of incubation) on MOLT-4 cell line and detected by Annexin V-FITC binding. Values are mean ± S.E. obtained with three separate experiments.

4. Discussion

ity (Darzynkiewicz et al., 2002). The green fluorescent signal is a direct measure of the number of active caspase enzyme that is present in the cell at the time the reagent is added. As reported in Fig. 4, on monoparametric analysis, the caspase positive population (M2) was increased in MOLT-4 cell cultures treated with camptothecin (2.6-fold with respect to control) or T-2 toxin 0.1 and 1 ␮g/ml (1.9-fold). This proportional increase was confirmed by biparametric analysis (Table 3), which allowed to distinguish the caspase negative living cells, the necrotic, membranecompromised caspase negative cells, and the apoptotic caspase-dependent cells. The camptothecin and T-2treated MOLT-4 cells showed a statistical increase of apoptotic-caspase+ cells respect to control. In contrast, no variations in caspase-positive cells between control and treated (T-2 toxin or camptothecin) cultures were recorded in IM-9 cells using monoparametric or biparametric analysis (data not shown).

Trichothecenes are agriculturally important mycotoxins of relevance to human health produced by certain fungi belonging to Fusarium, Myrothecium or Stachybotrys species. Fusarium species, found worldwide in cereals and other food type for human and animal consumption, are the most important toxigenic fungi in northern temperate regions and represent a big economical problem for the agricultural sector (Gutleb et al., 2002). About 20% of the crops grown in the European Union for foods and animal feeds contain measurable amounts of mycotoxins. T-2 toxin is the most potent trichothecene, which acts as an inhibitor of eukaryotic protein synthesis by binding to the 60S ribosomal subunit and interacting with the enzyme peptidyltransferase (Yang et al., 2000). This mycotoxin is extremely toxic to leukocytes and other rapidly dividing cells resulting in in vivo and in vitro immunosuppressing

Table 3 Caspase-3 activation induced by T-2 toxin on MOLT-4 and detected by FLICA kit T-2 toxin levels (␮g/ml) (n = 2)

Live cells (FL1− /PI− )

Control 1 0.1 0.01 Camptothecin (12 ␮M)

52.9 30.2 31.1 52.9 11.0

0.6 0.3*** 0.3*** 0.6 0.3***

are mean ± S.E. p < 0.001 evaluated Bonferroni multiple comparisons test.

a Values ∗∗∗

± ± ± ± ±

Necrotic cells (FL1− /PI+ )a 26.2 26.1 21.6 8.0 22.3

± ± ± ± ±

0.7 1.9 1.1 0.2*** 0.8

Caspase positive dead cells (FL1+ /PI+ )a 20.7 43.6 47.2 38.2 66.6

± ± ± ± ±

1.4 2*** 1.4*** 1.3*** 0.4***

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Fig. 4. Caspase-3 activation on MOLT-4 cell line evaluated by FLICA kit after 5 h of incubation with T-2 toxin. Representative monoparametric FACS analysis of activated caspase-3 determination by FLICA kit. MOLT-4 cell line was treated with different concentrations of T-2 toxin for 5 h. Fluorochrome inhibitor of caspase (FLICA) was added 1 h before and a single-color analysis was carried out. In the histogram two cell populations, represented as two peaks, appeared. The caspase negative cells occurred within the first log decade (MI), the caspase positive cell population appeared as a separate peak (M2). (a) Control; (b) apoptotic positive control with camptothecin (12 ␮M); (c) 1 ␮g/ml T-2 toxin; (d) 0.1 ␮g/ml T-2 toxin; (e) 0.01 ␮g/ml T-2; (f) 0.001 ␮g/ml T-2.

effects even at concentrations lower than those found in cereals responsible for endemics or influencing incidence of secondary diseases (Gutleb et al., 2002). Indeed, lymphocytes are more sensitive to T-2 toxin than other cell types and either DNA or protein synthesis inhibition were sensitive endpoints in cell systems when compared to general cytotoxicity (Forsell et al., 1985; Charoenpornsook et al., 1998; Widestrand et al., 1999; Gutleb et al., 2002). Because of the known immunotoxic activity of T-2 toxin, we set out to investigate its effects on two lymphoid human cell lines, MOLT-4 (T lineage) and IM-9 (B lineage). The use of different bioassays allowed us to discriminate different mechanisms of T-2-mediated toxicity in these two cell lines. On IM-9 cells, an early (8 h) membrane damage, evaluated by Trypan blue dye exclusion, was observed after T-2 treatment. This type of membrane damage was in accordance with previous data reported by Rizzo et al. (1992), who detected a hemolytic activity on rat erythrocytes as early as after a 3 h exposure to 200 ␮g/ml T-2 toxin. This cyto-

toxic effect was attributed to other properties shared by trichothecenes, such as lipophilia, free radical production, and lipid peroxide generation (Rizzo et al., 1992) and a protective role of antioxidant nutrients (selenium, ascorbic acid, lycopene) on cytotoxicity induced by

Fig. 5. Activation of caspase-3 after T-2 toxin exposure for 16 h.

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T-2 toxin was reported by several authors (Rizzo et al., 1994; Leal et al., 1998; Shokri et al., 2000; Keshavarz et al., 2001; Vil`a et al., 2002). Consistently, we observed a protective effect of ascorbic acid on T-2-mediated cytotoxicity in IM-9 cells. On MOLT-4 cells the main and earlier (4 h) effect observed after T-2-treatment was apoptosis induction as evaluated by Annexin V-FITC binding. Several authors reported the induction of apoptosis by T-2 toxin in vivo (Ihara et al., 1997; Islam et al., 1998; Gogal et al., 2000; Nagata et al., 2001) and in vitro systems (Yoshino et al., 1996, 1997; Yang et al., 2000) in different cell types. In particular, in myeloid cell models (RAW 264.7 and U937), the generation of reactive oxygen species or a ‘ribotoxic stress response’ by translation inhibitors, such as are trichothecenes, have been suggested to explain SAPK/JNK activation and consequent apoptosis (Yang et al., 2000). In HL-60 cell line, upon T-2 exposure, Nagase et al. (2001) and Holme et al. (2003) suggested the involvement of several active caspases as early events of apoptosis. Specifically, cellular insults, linked to prolonged endoplasmic reticulum stress, as following T-2 exposure, were suggested to induce apoptosis through caspase-7-mediated caspase-12 activation and receptor-mediated caspase-8 activation (Holme et al., 2003). In our study, caspase-3 activation in MOLT-4 cells following exposure to T-2 toxin supports the model proposed by Nagase et al. (2001) and Holme et al. (2003). Early induction of apoptosis and activation of caspase-3 were not seen after exposure of IM-9 cells to T-2 toxin, pointing to cytotoxicity as the major mechanism of T-2-mediated damage in this cell line. In addition, Nagase et al. (2001) reported Ca2+ involvement in the activation of several caspases and subsequent DNA degradation. The addition of BAPTA-AM, an intracellular Ca2+ chelator, was reported by Holme et al. (2003) to completely block T2-mediated apoptosis induction in HL-60, with reduced activation of caspases 7–9. These data were in agreement with those by Yoshino et al. (1996), who reported block of T-2 toxin-induced apoptosis in HL-60 cells by BAPTA-AM, but not EDTA, an extracellular Ca2+ chelator. In these studies, no differences of apoptosis were seen after treatment with T-2 toxin and BAPTAAM, suggesting involvement of a Ca2+ independent mechanism in MOLT-4 cells. These findings suggest that T-2 toxin effects may be cell type-specific, as also pointed out by Holme

et al. (2003). Consistent with this, cell cycle analysis data were found to differ in MOLT-4 and IM-9 cells following treatment with T-2 toxin. Specifically, T-2treated IM-9 cells showed a debris increase after 12 h and 100% debris after 24 h. In T-2-treated MOLT-4 cells, the only change, manifested as debris increase, was recorded after 24 h. These changes progressed up to 48 h in cultures treated with T-2 toxin. With regard to cell cycle perturbation after T-2 treatment, Yoshino et al. (1996) reported reduced percentages of each cycle phase in HL-60 cells treated with 100 ng/ml T-2 toxin for 3 h, whereas Holme et al. (2003) did not observe major changes in the relative number of HL-60 cells in G0 /G1 , S, or G2 /M phases during the first 8 h of incubation with 12.5 ng/ml T-2 toxin. These discrepancies, including our data obtained in lymphoid cells of different lineages, may be related to the different T-2 concentrations and incubation times used. T-2 toxin induced immunosuppression in the two cell lines used in this study, as evaluated by MTT test and BrdU assay, and these finding are in close agreement with other studies on human lymphocytes and lymphoid cell lines (Visconti et al., 1991; Berek et al., 2001; Meky et al., 2001). No different IC50 values were reported by Visconti et al. (1991) and Meky et al. (2001) in PHA-stimulated human lymphocytes and lymphoid cell lines (MIN-GL1 and K-562). Several studies (Babich and Borenfreund, 1991; Visconti et al., 1991; Hanelt et al., 1994; Juranic et al. (1998) showed an inhibition of proliferation in neoplastic cell lines of different histotypes. Juranic et al. (1998) has suggested a possible use of T-2 toxin as a chemiotherapic agent but this hypothesis could be questionable because the known toxic effects induced by T-2 toxin. In fact European Commission (2001) established for T-2 toxin a ‘temporary total daily intake’ (t-TDI) value of 0.06 ␮g/kg T-2 kg/bw. This t-TDI value would protect against to chronic and subchronic effects but in this study lower levels than t-TDI values were found active on human lymphoid cell lines. Further studies are thus warranted.

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