The Cytotoxicity of Aflatoxin B 1 in Human Lymphocytes

Objectives: Aflatoxin B1 (AFB1) is a naturally occurring carcinogenic and immunosuppressive compound. This study was designed to measure its toxic effects on human peripheral blood mononuclear cells (PBMC). Methods: The study recruited 7 healthy volunteers. PBMC were isolated and cellular respiration was monitored using a phosphorescence oxygen analyser. The intracellular caspase activity was measured by the caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin. Phosphatidylserine exposure and membrane permeability to propidium iodide (PI) were measured by flow cytometry. Results: Cellular oxygen consumption was inhibited by 2.5 μM and 25 μM of AFB1. Intracellular caspase activity was noted after two hours of incubation with 100 μM of AFB1. The number of Annexin V-positive cells increased as a function of AFB1 concentration and incubation time. At 50 μM, a significant number of cells became necrotic after 24 hours (Annexin V-positive and PI-positive). Conclusion: The results show AFB1 is toxic to human lymphocytes and that its cytotoxicity is mediated by apoptosis and necrosis.


CLINICAL & BASIC RESEARCH
Advances in Knowledge -Aflatoxin B 1 (AFB 1 ) is a potent immunosuppressant.
-AFB 1 induces apoptosis and necrosis in human lymphocytes.

Application to Patient Care -The cytotoxicities of aflatoxin in humans include immunosuppression, mediated by lymphocyte apoptosis and necrosis.
-Public awareness of the potential immunotoxicity of aflatoxins is needed.
-Effective health regulations are required to minimise the exposure to aflatoxins, especially in immunocompromised hosts.

Methods
This study was carried out from November 2008 to June 2012.The PBMC were isolated from the whole blood of 7 healthy adult volunteers as previously described. 28The Institutional Review Board for the protection of human subjects of the United Arab Emirates University approved the collection of blood from the healthy volunteers.Informed consent was obtained from the participating volunteers.
The Pd phosphor (at 2.5 mg/mL and 2.0 mM), glucose oxidase (at 10 mg/mL) and sodium cyanide (at 1.0 M) solutions were prepared in distilled water (dH 2 O) and stored at -20 °C.The phosphate buffered saline (PBS) solution was made daily.
4][35][36] For the fluorescence-activated cell sorting (FACS) analysis, an aliquot of 10 6 of PBMC for each subject were cultured with 0, 5, 10, 50 or 165 µM of AFB 1 .The cells were harvested after 2, 16, and 24 hours, and analysed as previously described. 37The AFB 1 was prepared and measured as described. 38he reaction mixtures contained 1.5 x 10 6 cells in PBS, 10 mM of glucose and 68 µM of Ac-DEVD-AMC, with and without zVAD-fmk (at 20 µM).The mixtures were incubated at 37 °C in glass vials (in the dark with continuous agitation) for two hours, with and without AFB 1 or dactinomycin.The suspensions were then diluted with 1.0 mL of ice-cold PBS-glucose, sonicated for 60 secs and passed through 23-G needles.The supernatants were collected by centrifugation (12,300 x g for 10 mins) and separated on high-performance liquid chromatography (HPLC) as described. 37he released 7-amino-4-methylcoumarin (AMC) moiety (peak retention time of ~8.7 mins) was detected by fluorescence.A control reaction mixture containing PBS-glucose, 68 µM of Ac-DEVD-AMC and 5 µl of dimethyl sulfoxide (DMSO) (the vehicle for zVAD-fmk) without added cells was monitored periodically at 37 °C for the spontaneous release of AMC moieties; in these control reactions, the AMC peak areas at 0.5, 1, 2 and 3 hours were negligible.
The HPLC analysis of the released AMC moieties was done on a Waters Corporation (Milford, Massachusetts, USA) reversed-phase HPLC system (excitation wavelength of 380 nm and emission wavelength of 460 nm).Solvent A was acetonitrile (CH 3 CN) and water (H 2 O) at a ratio of 1:3 and solvent B was dH 2 O.The column, a 4.6 x 250 mm Ultrasphere ® ion pair column (Beckman Coulter, Inc., Brea, California, USA), was operated at 25 °C at 1.0 mL/min (0.5 mL/min of each pump).The run time was 15 mins and the injection volume was 20 µL. 37

Results
The cellular respiration results were as follows.The representative O 2 consumption runs are shown in Figure 1 and 2. In Figure 1, the PBMC (2.5 x 10 7 cells/ mL) were incubated with 12 µL/mL of DMSO or 25 µM of AFB 1 .At t = 154 mins, 1.0 mL of each mixture was simultaneously placed in the instruments for the O 2 measurements.The respiration rate, zeroorder rate (constant for cellular mitochondrial O 2 consumption (k) in µM O 2 min -1 ) for untreated cells was 3.6 and 1.2 for AFB 1 -treated cells (67% inhibition).In Figure 2, the PBMC (10 7 cells/mL) were incubated with 1.2 µL/mL of DMSO or 2.5 µM of AFB 1 .At t = 95 mins, 1.0 mL of each mixture was simultaneously placed in the instruments for the O 2 measurements.The k values were 1.6 and 1.3 µM of O 2 /min, respectively (19% inhibition).
The caspase activity was monitored after incubation for two hours at 37 °C with 100 µM of AFB 1 (which was added as a powder), with and without 20 µM of zVAD-fmk, using the caspase-3 substrate analogue Ac-DEVD-AMC.Caspase-3 cleaved Ac-DEVD-AMC, releasing the fluorogenic     3A], the AMC peak area (in arbitrary units) was 273,367 and abolished by zVAD-fmk.For cells treated with AFB 1 alone [Figure 3B], the AMC peak area was 32,347,746 (118-fold higher).For cells treated with AFB 1 and zVAD-fmk [Figure 3B], the AMC peak area was 3,522,589 (89% inhibition).Similar results were obtained in two additional experiments.For comparison, the cells were also treated with 20 µM of dactinomycin, which is well known to activate caspases.The AMC peak area in the presence of dactinomycin alone was 54,679,510; in the presence of dactinomycin and zVAD-fmk, the peak area was 4,561,062 (92% inhibition) [Figure 3C].
The caspase activation was monitored as a function of the time of incubation with AFB 1 .Three conditions were tested: untreated cells, cells treated with 100 µM of AFB 1 alone and cells treated with 100 µM of AFB 1 and 20 µM of zVAD-fmk.The Ac-DEVD-AMC cleavage was monitored at 15, 30, 60 and 120 mins after treatment.The AMC moiety appeared only after two hours of incubation with AFB 1 ; zVAD-fmk blocked ~94% of the AMC peak area.This profile of caspase activation was similar to that described for dactinomycin and doxorubicin in human immortalised T-lymphocytes. 28,29he induction of the lymphocyte apoptosis by AFB 1 was monitored by flow cytometry, using the cell membrane's permeability to propidium iodide (PI) and the phosphatidylserine exposure.There were 10 independent experiments performed, and the results of these are summarised in Figure 4.The PBMC were isolated from the blood of 7 healthy volunteers and exposed to 0, 5, 10 or 50 µM of AFB 1 .The FACS analysis was performed at 2, 16 and 24 hours.With the total number of mononuclear cells isolated at 2.5 x 10 6 , the number of Annexin V-positive cells increased with incubation time; this increase was statistically significant (P <0.05) after 16 hours of incubation for each concentration.Although the difference in the effect of 5 µM and 10 µM was not significant (P = 0.082), the increase in the number of apoptotic cells after treatment with 50 µM was significant [Figure 4 A and B].It is noteworthy that 50 µM of AFB 1 also produced a significant number of necrotic cells (annexin V-positive and PI-positive), which became more evident at 24 hours [Figure 4C].
Thus, the results of this study show that AFB 1 inhibits cyanide-sensitive cellular respiration.The toxin also induces apoptosis and necrosis.

Discussion
The phosphorescence O 2 analyzer, caspase assay and flow cytometry were used here to confirm the toxic effects of AFB 1 on human lymphocytes. 25The results show that AFB 1 impairs human lymphocyte mitochondrial function [Figure 1 and 2] and activates intracellular caspases [Figure 3].The AFB 1 also produces lymphocyte apoptosis and necrosis [Figure 4].The caspases become active in the cells within two hours of the AFB 1 addition [Figure 3].This period is similar to that needed to inhibit cellular respiration [Figure 1 and 2].
In one study, 32 µM of AFB 1 had a minimum effect on human lymphocyte proliferation following phytohaemagglutinin-P stimulation. 39However, an earlier study on human lymphocytes showed less lymphocyte proliferation in the presence of 16 µM of AFB 1 . 40The AFB 1 also inhibited concanavalin A-promoted lymphocyte proliferation (50% inhibition at 60 nM). 20Moreover, AFB 1 was shown to induce apoptosis in human lymphocytes. 41,42he concentrations of AFB 1 used here were relatively high, especially since the average human exposure in Eastern China is only ~0.5 mmol/day. 3 is important, however, to emphasise that the stability of AFB 1 in solutions is poor and the bulk of the toxin is deactivated by the rapid reaction with H 2 O. 43 Thus, the data presented here mainly point to the potential immunotoxicity of AFB 1 .

Figure 1 :
Figure 1: The AFB 1 (25 µM in DMSO) inhibited the PBMC respiration.The PBMC (2.5 x 10 7 cells/mL in PBS-glucose, 3 µM of Pd phosphor and 0.5% of fatfree albumin) were incubated at 37 °C with 12 µL/ mL of DMSO or 25 µM of AFB 1 (in DMSO).Min zero corresponded to the addition of AFB 1 .At t = 154 mins, 1.0 mL of each mixture was simultaneously placed in the phosphorescence O 2 analysers for O 2 measurements at 37 °C.The rates of respiration (k) were calculated from the best fit curves.The additions of 5.0 mM of NaCN and 50 µg/mL of glucose oxidase are shown.

Figure 2 A
Figure 2 A & B: The AFB 1 (2.5 µM in DMSO) inhibited the PBMC respiration.The PBMC (10 7 cells/mL in PBS-glucose, 3 µM of Pd phosphor and 0.5% of fat-free albumin) were incubated at 37 °C with 1.2 µL/mL of DMSO (A) or 2.5 µM of AFB 1 (B).Min zero corresponded to the addition of the AFB 1 .At t = 95 mins, 1.0 mL of each mixture was simultaneously placed in the phosphorescence O 2 analysers for O 2 measurements at 37 °C.The rates of respiration (k) were calculated from the best-fit curves.The additions of 5.0 mM of NaCN and 50 µg/mL of glucose oxidase are shown.

Figure 3 A
Figure 3 A-C: The intracellular caspase activation by AFB 1 and dactinomycin.Each mixture (final volume of 0.5 mL) contained 1.5 x 10 6 cells in PBS-glucose and 68 µM of Ac-DEVD-AMC, with and without 20 µM of zVAD-fmk.The suspensions were incubated at 37 °C for two hours without addition (A), with the addition of 100 µM of AFB 1 (B) or with the addition of 20 µM of dactinomycin (C).At the end of the incubation period, the cells were disrupted and their supernatants were separated via HPLC.The AMC moiety was monitored by fluorescence.The retention time for the Ac-DEVD-AMC was ~2.4 mins and ~8.7 mins for the AMC.PBMC = the human peripheral blood mononuclear cells; zVAD-fmk = benzyloxy-carbonyl-val-ala-DL-asp-fluoromethylketone; AMC = 7-amino-4-methylcoumarin; AFB 1 = aflatoxin B 1 ; PBS = phosphate buffered saline; Ac-DEVD-AMC = N-acetyl-asp-glu-val-asp-7-amino-4methylcoumarin; HPLC = high-performance liquid chromatography.

Figure 4 A
Figure 4 A-C: The flow cytometry analysis of the apoptosis.A: The percentages of the Annexin V-positive cells; data are representative of samples from 7 healthy individuals.B: The quantification of the FACS analysis of the apoptotic (Annexin V-positive) cells from 7 healthy donors stained with PI and Annexin V at 24 hours (horizontal lines), 16 hours (vertical lines) and two hours (no lines).C: The quantification of the FACS analysis of the necrotic (Annexin V-positive and PI-positive) cells after treatment with 50 µM of AFB 1 .PI = propidium iodide; FACS = fluorescence-activated cell sorting; AFB 1 = aflatoxin B 1 .*P <0.05; **P <0.005.