Sultan Qaboos University Med J, February 2014, Vol. 14, Iss. 1, pp. e65-71, Epub. 27TH Jan 14 Submitted 16TH May 13 Revision Req. 5TH Sep 13; Revision Recd. 3RD Oct 13 Accepted 31ST Oct 13 College of Medicine & Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates *Corresponding Author e-mail: suleiman.alhammadi@uaeu.ac.ae مسية األفالتوكسني يف اخلاليا البشرية اللمفاوية �صليمان �حلمادي, فريدة �ملرزوقي, عائ�صة �ملن�صوري, �ألن �صاهي, مرمي �ل�صم�صي, �إريك من�صه بر�ون, عبد �لقادر �صويد abstract: 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. Keywords: Aflatoxin B1; Oxygen Analyzer; Cellular Respiration; Mitochondria; Caspases; Leukocytes, Mononuclear. امللخ�ص: الهدف: �الأفالتوك�صي هو مركب طبيعي م�صبب لل�رشطان ولنق�س �ملناعة. هذه �لدر��صة تقّيم �الآثار �ل�صامة لالأفالتوك�صي على �خلاليا �لب�رشية عزلت خاليا دموية ملفاوية من �صبعة متطوعي �أ�صحاء. ر�صد �لتنف�س �للمفاوية. متت هذه �لدر��صة بي ت�رشين ثاين 2008 وحزير�ن 2012. الطريقة: �خللوي بو��صطة �ملوؤ�رش �لف�صفوري لك�صف �الأك�صجي. وك�صف ن�صاط �نزمي �لكا�صبي�س د�خل �خللية با�صتخد�م �ملادة �لف�صفورية �مل�صتقة من كومارين �لقادرة �يود�يد لربوبدمي �خللية غ�صاء نفاذية زيادة و �صريين �لفو�صفاتيديل ملادة �لتعر�س قيا�س طريق عن �خللية موت قيا�س مت �الإنزمي. ن�صاط قيا�س على با�صتخد�م تقنية �لتدفق �خللوي. النتائج: �الأفالتوك�صي )μM 2.5 و μM 25( ثبط �ال�صتهالك �خللوي لالأك�صجي. ظهرت فعالية �لكا�صبي�س د�خل �خلاليا بعد �صاعتي من �إ�صافة �الأفالتوك�صي. عدد �خلاليا �مليتة �اليجابية ملادة �النيكزن-V �رتفع بزيادة جرعة �الأفالتوك�صي وبزيادة زمن تعر�س �خلاليا لهذ� �ل�صم. عند �إ�صافة μM 50 من �الأفالتوك�صي �أ�صبحت ن�صبة معتربة من �خلاليا نخرية بعد 24 �صاعه )�نيكزن )+V+PI(. اخلال�سة: �الأفالتوك�صي مادة �صامة للخاليا �لب�رشية �للمفاوية و�صميته تتم عرب �ملوت �خللوي �ملربمج و�لنخر. مفتاح الكلمات: �الأفالتوك�صي؛ موؤ�رش ك�صف �الأك�صجي؛ �لتنف�س �خللوي؛ �مليتوكوندريا؛ كا�صبي�س؛ �لدم؛ �خلاليا �للمفاوية. The Cytotoxicity of Aflatoxin B 1 in Human Lymphocytes *Suleiman Al-Hammadi, Farida Marzouqi, Aysha Al-Mansouri, Allen Shahin, Mariam Al-Shamsi, Eric Mensah-Brown, Abdul-Kader Souid CLINICAL & BASIC RESEARCH Advances in Knowledge - Aflatoxin B1 (AFB1) is a potent immunosuppressant. - AFB1 induces apoptosis and necrosis in human lymphocytes. - AFB1 inhibits lymphocyte respiration (mitochondrial oxygen consumption). 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. Aflatoxin B1 (AFB1) is a mycotoxin commonly found in food contaminated by organisms, such as Aspergillus flavus.1,2 Exposure to AFB1 is linked to several human diseases, including hepatocellular carcinoma,3,4 mutagenesis,5 immunosuppression,6–8 impaired infant growth,9–12 and adverse birth outcomes.12,13 Synergistic interactions with hepatitis B and human immunodeficiency viruses have also been reported.14,15 The toxin is activated by hepatic cytochrome P450 3A4, and its active form (AFB1-exo-8,9- The Cytotoxicity of Aflatoxin B 1 in Human Lymphocytes e66 | SQU Medical Journal, February 2014, Volume 14, Issue 1 epoxide) rapidly reacts with cellular deoxyribonucleic acid (DNA) and proteins.16,17 Most of the information regarding its immuno- toxicity has been derived from animal studies.18–20 The exposure of young children to dietary aflatoxin and its toxic effects on human lymphocytes are also well established,14,21–24 including the inhibition of lymphocyte respiration.25 The term apoptosis describes the caspase- (or apoptosome)-mediated cytotoxic processes that cause mitochondrial dysfunction, membrane damage and DNA fragmentation.26 The apoptotic pathways that are independent of apoptosomes are also known.27 This study explores the mechanisms (biomarkers) of the toxic effects of AFB1 in human lymphocytes. 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.28 The 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 reagents and solutions used were as follows. The Pd(II) complex of meso- tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin ([Pd phosphor] at molecular weight [Mr]~1300) was purchased from Porphyrin Products (Frontier Scientific, Inc., Logan, Utah, USA). The dactinomycin was purchased from Merck & Co., Inc. (Whitehouse Station, New Jersey, USA). The benzyloxy-carbonyl-val-ala- DL-asp-fluoromethylketone (zVAD-fmk) was a Calbiochem® product (EMD Millipore, Billerica, Massachusetts, USA). The N-acetyl-asp-glu-val- asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was purchased from Alexis Biochemicals (Enzo Life Sciences, Inc., Farmingdale, New York, USA). The AFB1 (produced by Aspergillus flavus), glucose oxidase, D(+)-glucose and remaining reagents were from Sigma-Aldrich Co. (St. Louis, Missouri, USA). 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 (dH2O) and stored at -20 °C. The phosphate buffered saline (PBS) solution was made daily. The AFB1, dactinomycin, zVAD-fmk (at 2.14 mM) and Ac-DEVD-AMC (at 7.4 mM) solutions were prepared and stored as previously described.29–31 Oxygen detection was based on the principle described previously,32 and the phosphorescence oxygen (O2) analyser used as reported elsewhere.33–36 For the fluorescence-activated cell sorting (FACS) analysis, an aliquot of 106 of PBMC for each subject were cultured with 0, 5, 10, 50 or 165 µM of AFB1. The cells were harvested after 2, 16, and 24 hours, and analysed as previously described.37 The AFB1 was prepared and measured as described.38 The reaction mixtures contained 1.5 x 106 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 AFB1 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.37 The 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 (CH3CN) and water (H2O) at a ratio of 1:3 and solvent B was dH2O. 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 Suleiman Al-Hammadi, Farida Marzouqi, Aysha Al-Mansouri, Allen Shahin, Mariam Al-Shamsi, Eric Mensah-Brown and Abdul-Kader Souid Clinical and Basic Research | e67 representative O2 consumption runs are shown in Figure 1 and 2. In Figure 1, the PBMC (2.5 x 107 cells/ mL) were incubated with 12 µL/mL of DMSO or 25 µM of AFB1. At t = 154 mins, 1.0 mL of each mixture was simultaneously placed in the instruments for the O2 measurements. The respiration rate, zero- order rate (constant for cellular mitochondrial O2 consumption (k) in µM O2 min -1) for untreated cells was 3.6 and 1.2 for AFB1-treated cells (67% inhibition). In Figure 2, the PBMC (107 cells/mL) were incubated with 1.2 µL/mL of DMSO or 2.5 µM of AFB1. At t = 95 mins, 1.0 mL of each mixture was simultaneously placed in the instruments for the O2 measurements. The k values were 1.6 and 1.3 µM of O2/min, respectively (19% inhibition). In four separate experiments, the values of k for untreated lymphocytes were 2.2 ± 1.1 µM O2 min-1 per 107 cells. The corresponding values for lymphocytes treated with AFB1 (cells exposed to 2.5–75 µM of AFB1 for 60–154 mins) were 1.3 ± 0.6 µM O2 min -1 per 107 cells; the inhibition was dose- dependent and ranged from 19–67%. The caspase activity was monitored after incubation for two hours at 37 °C with 100 µM of AFB1 (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 Figure 1: The AFB1 (25 µM in DMSO) inhibited the PBMC respiration. The PBMC (2.5 x 107 cells/mL in PBS-glucose, 3 µM of Pd phosphor and 0.5% of fat- free albumin) were incubated at 37 °C with 12 µL/ mL of DMSO or 25 µM of AFB1 (in DMSO). Min zero corresponded to the addition of AFB1. At t = 154 mins, 1.0 mL of each mixture was simultaneously placed in the phosphorescence O2 analysers for O2 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. O2 = oxygen; DMSO = dimethyl sulfoxide; k = zero-order rate constant for cellular mitochondrial O2 consumption; R 2 = regression coefficient; NaCN = sodium cyanide; AFB1 = aflatoxin B1 ; PBMC = human peripheral blood mononuclear cells; PBS = phosphate buffered saline; Pd phosphor = Pd(II) complex of meso-tetra-(4- sulfonatophenyl)-tetrabenzoporphyrin. Figure 2 A & B: The AFB1 (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 AFB1 (B). Min zero corresponded to the addition of the AFB1. At t = 95 mins, 1.0 mL of each mixture was simultaneously placed in the phosphorescence O2 analysers for O2 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. O2 = oxygen; k = zero-order rate constant for cellular mitochondrial O2 consumption; R 2 = regression coefficient; NaCN = sodium cyanide; AFB1 = aflatoxin B1; DMSO = dimethyl sulfoxide; PBMC = the human peripheral blood mononuclear cells; PBS = phosphate buffered saline; Pd phosphor = Pd(II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The Cytotoxicity of Aflatoxin B 1 in Human Lymphocytes e68 | SQU Medical Journal, February 2014, Volume 14, Issue 1 moiety AMC. After cell disruption, the AMC was separated on HPLC and detected by fluorescence. For untreated cells [Figure 3A], the AMC peak area (in arbitrary units) was 273,367 and abolished by zVAD-fmk. For cells treated with AFB1 alone [Figure 3B], the AMC peak area was 32,347,746 (118-fold higher). For cells treated with AFB1 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 AFB1. Three conditions were tested: untreated cells, cells treated with 100 µM of AFB1 alone and cells treated with 100 µM of AFB1 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 AFB1; 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.28,29 The induction of the lymphocyte apoptosis by AFB1 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 AFB1. The FACS analysis was performed at 2, 16 and 24 hours. With the total number of mononuclear cells isolated at 2.5 x 106, 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 AFB1 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 AFB1 inhibits cyanide-sensitive cellular respiration. The toxin also induces apoptosis and necrosis. Discussion The phosphorescence O2 analyzer, caspase assay and flow cytometry were used here to confirm the toxic effects of AFB1 on human lymphocytes. 25 The results show that AFB1 impairs human lymphocyte Figure 3 A‒C: The intracellular caspase activation by AFB1 and dactinomycin. Each mixture (final volume of 0.5 mL) contained 1.5 x 106 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 AFB1 (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; AFB1 = aflatoxin B1 ; PBS = phosphate buffered saline; Ac-DEVD-AMC = N-acetyl- asp-glu-val-asp-7-amino-4- methylcoumarin; HPLC = high-performance liquid chromatography. Suleiman Al-Hammadi, Farida Marzouqi, Aysha Al-Mansouri, Allen Shahin, Mariam Al-Shamsi, Eric Mensah-Brown and Abdul-Kader Souid Clinical and Basic Research | e69 mitochondrial function [Figure 1 and 2] and activates intracellular caspases [Figure 3]. The AFB1 also produces lymphocyte apoptosis and necrosis [Figure 4]. The caspases become active in the cells within two hours of the AFB1 addition [Figure 3]. This period is similar to that needed to inhibit cellular respiration [Figure 1 and 2]. In one study, 32 µM of AFB1 had a minimum effect on human lymphocyte proliferation following phytohaemagglutinin-P stimulation.39 However, an earlier study on human lymphocytes showed less lymphocyte proliferation in the presence of 16 µM of AFB1. 40 The AFB1 also inhibited concanavalin A-promoted lymphocyte proliferation (50% inhibition at 60 nM).20 Moreover, AFB1 was shown to induce apoptosis in human lymphocytes.41,42 The concentrations of AFB1 used here were relatively high, especially since the average human exposure in Eastern China is only ~0.5 mmol/day.3 It is important, however, to emphasise that the stability of AFB1 in solutions is poor and the bulk of the toxin is deactivated by the rapid reaction with H2O. 43 Thus, the data presented here mainly point to the potential immunotoxicity of AFB1. The caspase activity reaction described was previously calibrated using recombinant human caspase-3.28 The AMC was expressed as peak areas (in arbitrary units) per number of cells. The reactions were conducted in the presence and absence of the pan-caspase inhibitor zVAD-fmk. It is of note that the substrate Ac-DEVD-AMC can be cleaved by several caspases, including caspase-3 (turnover number [kcat]/ Michaelis constant [Km] = 218,000 s-1); caspase-7 (kcat/Km = 37,000 s-1); caspase-1/interleukin-1 converting enzyme (kcat/Km = 30,000 s-1); caspase-6 (kcat/Km = 2,000 s-1), and caspase-4 (kcat/Km = 1,800 s-1). 44 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 AFB1. PI = propidium iodide; FACS = fluorescence-activated cell sorting ; AFB1 = aflatoxin B1 . *P <0.05; **P <0.005. The Cytotoxicity of Aflatoxin B 1 in Human Lymphocytes e70 | SQU Medical Journal, February 2014, Volume 14, Issue 1 Conclusion Human lymphocytes exposed in vitro to AFB1 exhibit impairments in cellular respiration, caspase activation and necrosis. The results underscore the immunosuppressive activity of aflatoxins in humans exposed to this natural fungal toxicant. References 1. Eaton DL, Gallagher EP. Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 1994; 34:135–72. 2. Williams JH, Phillips TD, Jolly PE, Stiles JK, Jolly CM, Aggarwal D. Human aflatoxicosis in developing countries: A review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 2004; 80:1106–22. 3. Wang LY, Hatch M, Chen CJ, Levin B, You SL, Lu SN, et al. Aflatoxin exposure and risk of hepatocellular carcinoma in Taiwan. Int J Cancer 1996; 67:620–5. 4. 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