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Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats Q5
Cristina Juan*, Lara Manyes, Guillermina Font, Ana Juan-García Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andr es Estell es s/n, 46100 Burjassot, Val encia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2014 Received in revised form 12 May 2014 Accepted 14 May 2014 Available online xxxx
Study of dietary supplementation with ENN A mycotoxin during 28 days of exposure time on Wistar rats to determinate its levels in serum, urine and feces and, to evaluate the immunologic effect in peripheral blood lymphocytes (PBL) is presented. The first method for ENN A extraction, determination and detection by LCeMS/MS in serum, urine and feces samples is reported. ENN A food dose administrated was detected in serum samples and influenced lymphocyte phenotyping. Levels in serum were founded from the second week of the experiment; reaching values of 4.76 mg/ml on the fourth week, which corresponds to 3.24 mg/ml in blood. PBL as T helper (CD4þ) were presented in greater percentages compared to control (p 0.001), while T cytotoxic (CD8þ) decreased significantly compared to control (p 0.001). ENN A treatment significantly increased CD4þ/CD3þ and CD4þ/CD8þ ratios but significantly decreased CD8þ/CD3þ ratio. CD4þ/CD8þ ratio was 2.94:1, indicating that PBL surface antigen expression and immune status in Wistar rats treated were impaired by the ENN A mycotoxin. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Mycotoxins Enniatin A In vivo subacute toxicity Biological samples Immunological effects
1. Introduction Enniatins (ENNs) are mycotoxins produced by Fusarium species, also known as fusarotoxins, but due to their recent finding they have been classified such as emerging mycotoxins. Fusarotoxins are commonly found in cereals and their products, stuff constituting an important part of human food and animal feed. There are four main mycotoxins belonging to ENNs group: ENN A, ENN A1, ENN B and ENN B1. Recent studies about ENNs occurrence in several countries, have been described as well as how frequent these contaminants are in cereals (wheat, barley, rye and oat) and cereals products (baby food and pasta), observing
* Corresponding author. Tel.: þ34 96 354 41 16; fax: þ34 96 354 49 54. E-mail address:
[email protected] (C. Juan).
concentrations from 5.3 to 284.2 mg/kg and from 1 to 1100 mg/kg, respectively (Juan et al., 2013a; 2013b). Regarding their biological activity, acting as enzyme inhibitors, antifungal and antibacterial agents, and also their toxicological effects, ENNs induce a wide range of effects in vitro and its action is mainly based on their ionophoric properties which are capable of transporting cations through the cell membrane, leading to toxic actions by an altered membrane potential (Ivanova et al., 2012; Jestoi et al., 2009). Furthermore, ENNs inhibit acylcoenzymeA: cholesterolacyl transferase (ACAT) and 30,50-cyclo-nucleotide phosphodiesterase enzymes causing mitochondrial dysfunction, and the inhibition of multidrug resistance associated protein-1(ABCG2) and Pglycoprotein (ABCB1) efflux pumps (Tonshin et al., 2010; Dornetshuber et al., 2009a). Many in vitro studies have reported cytotoxic effects of ENNs on several cell types, as reviewed by Jestoi (2008).
http://dx.doi.org/10.1016/j.toxicon.2014.05.005 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Juan, C., et al., Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.05.005
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Data on their in vivo toxicity are however lacking (Jestoi, 2008). Nevertheless, their possible subclinical effects are of greater importance as these may lead to reduced performance parameters in food producing animals and increased susceptibility to infectious disease. Opinions of the European Food Safety Authority (EFSA) on ENNs are currently being drafted on request by the European Commission. To evaluate their potential in vivo toxic effects, the knowledge of their toxicokinetic properties, namely absorption, distribution, metabolization and excretion (ADME) in livestock is crucial. Jestoi (2008, 2009) detected trace level residues of ENNs in Finnish eggs, poultry meat and liver samples. To date, only one paper carried out in our laboratory collects data on the tissue distribution of ENN A in Wistar rats (Manyes et al., 2014); however, its toxicokinetic properties in general have not been studied. This lack of data and studies could explain why there are no maximum guidance levels set for animal feed yet, in contrast to other mycotoxins (European Commission, 2006). It is obvious that for assessing animal exposure to ENNs and to investigate their toxicokinetics, the availability of sensitive and specific validated analytical methods is mandatory. In recent decades, many liquid chromatographyetandem mass spectrometric (LCeMS/MS) methods for the analysis of ENNs in food, feed and other matrices have been described (Jestoi, 2008; Uhlig and Ivanova, 2004; Uhlig et al., 2006; Sulyok et al., 2006; Juan et al., 2013a; 2013b). However, no methods for the analysis of these compounds in plasma, urine and feces have been reported. Their development and application in these fluids would allow evaluating and complete ADME studies in animals for ENNs mycotoxins. For evaluate toxicological effects in vivo, it is important to expose the animals to a single substance because previous in vitro studies have demonstrated synergic and antagonism effect between ENNs (Lu et al., 2013). Furthermore, the IC50 values for ENNs studied in CHO-K1, Caco-2, Hep-G2 and HT-29 cells, are different and depending on the exposed cell line. The highest values were obtained for ENN A and ENN B as reported by JuanGarcía et al. (2013a) and Lu et al. (2013). The goal of this study was to design a preliminary study of dietary supplementation with ENN A, as one of most toxicologically potential of enniatins mycotoxin group. ENN A mycotoxin (Fig. 1) was administrated during 28 days of exposure time in vivo (on Wistar rats) to determinate its levels in serum, urine and feces and to evaluate the immunologic effect, being the first time that ENN A immunotoxicity is evaluated. ENNs may have higher toxicity in hematopoietic progenitors than in mature blood cells. In this sense, none study with ENN A in vivo has been done at the moment and this study attempted to answer if ENN A doses and exposure time of Wistar rats altered peripheral blood lymphocytes (PBL). 2. Materials and methods 2.1. Chemical and reagents Ethyl acetate, acetonitrile, and methanol for LC mobile phase and organic solvents were HPLC grade from Merck
Fig. 1. Chemical structure of ENN A mycotoxin.
(Darmstadt, Germany), while acetic acid was obtained from Fluka (Milan, Italy). Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Enniatin A (ENN A) was purchased from Sigma Aldrich (Madrid, Spain). The stock solution and standard solution of ENN A were prepared in acetonitrile at concentration of 0.5 mg/ml and 100 mg/ml, respectively. These solutions were kept in safety conditions at 20 C. For working standard solution, it was prepared immediately before use by diluting the stock solution with methanol. Syringe filters PTFE (Polytetrafluoroethylene membrane, 15 mm, diameter 0.2 mm) were provided by Phenomenex® (Castel Maggiore, Italy). 2.2. Diets, animals, and experiment design The control diet was Autoclaved Harlan lab blocks (Spain). All components were purchased from Harlan Laboratories Inc. (Madison, WI, USA). After mixing the basal diet, ENN A was added at 465 mg/kg. Dose assayed was obtained based on reproducing experimentally the natural presence of ENN A in the food matrix after grown of Fusarium triticum strain during 30 days in our laboratory as described in Manyes et al. (2014). The food was mixed with 20% water to form a dough and then cut into 10e15 g/bar, which were air dried for 2 days until the bar weight showed no further change. Diets were done at the beginning of the experiment and stored in sealed plastic bags at 20 C. When making diets, disposable masks and gloves were used as protection. All items contaminated with ENN A were rinsed in 10% bleach before washing. Ten 6e7 week-old female Wistar Rats (z250 g) were purchased from Pharmacy animal facility (University of Valencia, Spain). Rats (5 control and 5 test animals) were housed in two plastic cages at 20 C and a 12 h light/dark cycle with bars of food in the cage top. The study room where cages were stored was maintained under controlled conditions of (23 ± 2) C and relative humidity (55 ± 10)%. Low dust absorptive bedding was changed twice per week.
Please cite this article in press as: Juan, C., et al., Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.05.005
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Feed and water were provided ad libitum. Feed consumption and body weights were recorded once a week after measuring wastage in the cages, and rats observed daily for any signs of illness. The subchronic toxicity study was carried out for 28 days with ENN A treatment at 465 mg/kg of food and 5 replications. This project was approved by the University of Valencia Institutional Animal Care and Use Committee and the number of animals was justified and accepted for this study. 2.3. Collection of blood, urine and feces samples Feces and urine samples of each animal were collected weekly by housing each animal separately in a cage. Samples were kept in freezing (20 C) until its analysis in Eppendorf vials. All procedures were performed aseptically at room temperature. Blood samples for LCeMS/MS analysis were collected weekly by saphenous vein puncture. Serum preparation was done as follows: blood was allowed to clot by leaving it undisturbed at room temperature for 15e30 min. Later, the clot was removed by centrifuging at 1600 g by 10 min in a refrigerated centrifuge. The resulting supernatant was serum which was immediately transfer into a polypropylene tube by using a Pasteur pipette and stored at 20 C. A different procedure was followed when collecting blood samples for flow cytometry analysis, in order to achieve better fluorochrome labelling (Weaver et al., 2010). Before rats slaughtering, blood was collected into a 0.5 mL heparinized vial by mandibular vein puncture. Vials were maintained at room temperature for better retention of staining intensity. 2.4. Antibodies Monoclonal antibodies purchased from eBiosccience (Labclinics, S.A., Spain) were used for flow cytometric analysis of peripheral blood lymphocytes (PBL): fluorescein isothiocyanate (FITC)-conjugated anti-rat
3
CD4 (OX-35; IgG2a), phycoerythrin (PE)-conjugated anti-rat CD8a (OX-8; IgG1) and allophycocyanin (APC)conjugated anti-rat CD3 (G4.18; IgG3). Since blood samples were directly processed and no washable steps were included, Fc-receptors were not necessary to block as blood contains z10 mg/ml IgG in the serum which helps to block them. 2.5. Flow cytometry analysis For immunotoxicity assay, blood samples were run on a BD FACSCanto™ Flow Cytometer (Beckton-Dickinson, Italy) with a blue (488 nm) and red (633 nm) excitation sources. Emission signal was collected by starting with the longest wavelength (APC) and finishing with the shortest wavelength (FITC). Data was collected for 40.000 cells. A sample of unstained cells was used to calibrate the cytometer and a previous propidium iodide assay for showing appropriate viability of lymphocytes was carried out. Lymphocytes viability was (95 ± 2) %. Three different compensation tubes contained blood cells that were stained with only one of the three fluorochromes. Forward scatter in plane parallel to the laser path distinguished single from clumps cells. Side scatter in a plane perpendicular laser path measured intracytoplastic granularity. Data were collected using FACSDiva software (version 6.1.3). Positioning of quadrants on peripheral blood cells labelled with APC-CD3, PE-CD8 and FITC-CD4 were analysed by flow cytometry using gates based on the cells type clusters and from the CD3-APC population (Latif et al., 2001; Murphy et al., 2007). Differences in fluorescence of Q1 labelled mononuclear cells were identified to detect if dietary supplementation with ENN A affected number of T helper cells (CD4þ) or T cytotoxic cells (CD8aþ). The signal of CD3-APC indicated the number of PBL cells (see Fig. 2). Whole blood (50 mL) was transferred to 12 75 tubes and directly processed on the same day for flow cytometry analysis, by adding 5 mL of each antibody to each tube. Tubes were vortexed gently and placed on ice in the dark for 20 min. Lysing solution (2 mL, fresh daily by 10-
Fig. 2. Phenotyping peripheral blood lymphocytes in young adult Wistar rats: a) Position of population of peripheral blood cells stained with CD3-APC (P2). b) Position of quadrants in a dot-plot histogram from CD3-APC positive cells (P2) stained with CD8-PE and CD4-FITC; Q1: T cytotoxic lymphocytes (CD3þ/CD8þ/ CD4-); Q3: T helper lymphocytes (CD3þ/CD8-/CD4þ).
Please cite this article in press as: Juan, C., et al., Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.05.005
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fold dilution of FACS Lysing) was added smoothly to lysate and fix the cells. If cells were not able to run the same day, they were kept at 4 C until flow cytometric analysis. 2.6. ENN A extraction in biological samples The optimized extraction procedure for feces was carried out following Juan et al. (2013b) method, with several modifications as follows: (0.1 ± 0.03) g of sample was transferred into 1.5 mL Eppendorf Safe-Lock Microcentrifuge Tube, 3 mL of each mixture of solvents studied was added and shaking for 30 min. The mixed was centrifuged at 3500 rpm for 5 min at 4 C and 1.5 mL of supernatant phase was diluted with 2 mL of CH3CN:CH3OH (60:40). Following, 0.5 mL of the mixed was diluted with 0.5 mL of CH3CN:CH3OH (60:40) and centrifuged at 3500 rpm for 5 min at 4 C. Finally 0.3 mL of supernatant was evaporated to dryness at 45 C in an N2 stream with a TurboVap-LV (Zymark, Allschwil, Switzerland) and then redissolved in 0.3 mL of CH3OH/H2O mixture (70:30, v/v) by vortexing vigorously before the injection into the LCeMS/ MS system for analysis. On the other hand for urine and serum samples a different extraction procedure was developed. It consisted as follows: 0.2 ± 0.05 mL of sample was transferred into 1.5 mL Eppendorf Safe-Lock Microcentrifuge Tube and 0.5 mL of ethyl acetate was added, shake during 5 min, and centrifuged (3500 rpm, 5 min, 4 C). Next, the supernatant was transferred to another tube. This was repeated three times and the final volume of the transferred supernatant (1.5 mL) was evaporated to dryness using a TurboVap-LV (N2 stream). The dry residue was reconstituted in 100 mL of CH3OH/H2O mixture (70:30, v/v) and vortexing vigorously (15 s), before the sample was transferred into an autosampler vial for its injection into the LCeMS/MS. The injection volume was 20 mL. Once the different extraction solvents were studied; the method was optimized and validated. It was validated for accuracy, precision (repeatability and reproducibility) specificity and sensitivity. For that, it was validated as a quantitative confirmatory method. 2.7. LCeMS/MS analysis LC analysis of ENN A was carried out with a triple quadrupole mass spectrometry detector (QqQ), equipped with an LC Alliance 2695 system (Waters, Milford, MA) that included an autosampler and a quaternary pump. Chromatography conditions were attained on a Phenomenex (Madrid, Spain) Gemini-NX C18 (150 2 mm I.D., 3 mm particle size) analytical column, preceded by a security guard cartridge C18 (4 2 mm I.D.), using a gradient that started at 90% of A (10 mM ammonium formate in acetonitrile) and 10% of B (10 mM ammonium formate in methanol), increased linearly to 50% B in 10 min. After, it was decreased linearly to 10% of B in 3 min and it was maintained for 7 min. Flow rate was maintained at 0.2 ml min1. The QqQ mass spectrometer Quattro LC from Micromass (Manchester, UK) equipped with pneumatically assisted
electrospray probe, a Z-spray interface and Mass Lynx NT software Ver. 4.1 was used for MS/MS analyses. Analysis was performed in positive ion modes. The electrospray ionization (ESI) source values were as follows: capillary voltage 3.50 kV, cone 50 V, collision energy 25 V and positive ionization mode; extractor, 5 V; RF lens 0.5 V; source temperature, 100 C; desolvation temperature, 300 C; desolvation gas (nitrogen 99.99% purity) flow, 800 L h1. The mass transitions used for the analysis according to the optimized parameters analyte-dependent were as described previously by Juan et al. (2013b): 681.9 > 210.22 and 681.9 > 228.26 (m/z) (Table 1 and Fig. 3A). 2.8. LCeMS/MS method validation The optimized method was validated for accuracy, precision (repeatability and reproducibility) specificity and sensitivity, as a quantitative confirmatory method as described by Juan et al. (2012). Sensitivity was evaluated by limit of detection (LD) and limit of quantification (LQ) values. The LD and LQ were calculated based on signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively, and assessed for both, standards solutions and spiked samples. Spiked samples were treated identical to the samples from the feeding study. Intraday precision was assessed by calculating the relative standard deviation (RSDr), calculated from results generated under repeatability conditions of six determinations per concentration in a single day. Interday precision was calculated by the relative standard deviation (RSDR) calculated from results generated under reproducibility conditions by triplicate determination per concentration on three days. Recovery experiments were conducted at three different calibration levels for each matrix, one at limits of quantification (LQ), other 2 times LQ and the other 10 times LQ, added before the corresponding extraction procedure. 2.9. Statistical analysis Statistical analysis of data were carried out using IBM SPSS Statistic version 19.0 (SPSS, Chicago, Il, USA) statistical software package. Data were expressed as mean ± SD of four independent experiments. The statistical analysis of the results was performed by student's T-test for paired samples. Difference between groups were analysed statistically with ANOVA followed by the Turkey HDS post hoc test for multiple comparisons. The level of p 0.05 was considered statistically significant.
Table 1 Optimized parameters conditions for LCeMS/MS (ESI) detection of ENN A. Analyte
tRa [min]
Precursor ion [m/z]
Product ionsb [m/z]
Cone [eV]
CEc
Dwell time [ms]
ENN A
6.50
681.9 [M þ H]þ
210.20/228.20
40.0
35.0
0.100
a b c
Retention time. Values are given in the order quantifier ion/qualifier ion. Collision energy.
Please cite this article in press as: Juan, C., et al., Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.05.005
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5
Fig. 3. LCeMS/MS chromatograms from the both ENN A's identifier ions (a.1, a.2, b.1 and b.2): (A) Standard solution (0.5 mg/ml) and; (B) Positive serum (4.2 mg/ ml) from a treated rat.
3. Results and discussion 3.1. Body weight and food intake in Wistar rats All 10 female Wistar rats grew normally with no gross signs of illness. There was no significant difference in initial body weight (b.w.) and final body weight within weeks. The initial body weights were (251.12 ± 10.11) g (mean ± S.E.M.). After 28 days treatment, the body weights were (263.48 ± 21.52) g. There was no significant difference in food intake of animals feed ENN A compared with controls; food intake in rats fed with or without 465 mg/kg ENN A for 28 days was 11.8 ± 0.05 g per day. 3.2. ENN A method determination in serum, urine and feces Different extraction protocols were optimized for each biological studied sample (feces, urine and serum) with the aim of obtaining satisfactory recoveries and eliminating or reducing matrix interferences and also with less steps and economic cost, by reaching the lowest detection limits for this study. As described in materials and methods (Section 2.6) these protocols consisted in liquideliquid or
liquidesolid extraction methods with different combinations of solvents (ethyl acetate, acetonitrile, methanol or water). Each of these sample preparation protocols had its strengths and weaknesses. In this study for ENN A determination, a liquid extraction method and a sensitive LCeMS/MS method for quantitation of ENN A in rat serum, urine and feces were developed. The use of a simple and practical sample preparation procedure is desirable in order to reduce the time and cost of analysis; particularly, if the samples can be obtained during toxicokinetic studies (quantitation) since these trials generate a large number of samples. When using a sensitive and specific analytical technique such as LCeMS/MS, clean-up steps of raw extracts should be kept at minimum (De Baere et al., 2012; Devreese et al., 2012). To achieve a good compromise between simplicity of extraction and acceptable sample clean-up, a generic extraction procedure was developed. It consisted in a combination of protein precipitation with liquid extraction by using organic solvents, such as ethyl acetate, methanol and acetonitrile. In these methods, a mix of CH3CN:CH3OH (60:40) for feces, and ethyl acetate for urine and serum matrices, were
Please cite this article in press as: Juan, C., et al., Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.05.005
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used for combining precipitation of proteins and extraction of ENN A as it showed the highest extraction recoveries (R, %) and the lowest signal suppression/enhancement (SSE), reported in Table 2. Acetonitrileemethanol mix resulted in giving clear supernatants in feces after centrifugation compared to methanol or ethyl acetate when used individually. Whereas when ethyl acetate was used in urine and serum samples, greater efficiency and clear supernatants were achieved. To our knowledge, there is none study where optimization of ENN A Fusarium mycotoxin extraction from biological fluids has been performed; however, there are few in vitro studies where toxicological evaluation effects has been performed by using either ENN A solely (Prosperini et al., 2013a, 2013b; Juan-García et al., 2013a, 2013b) or mixed with other ENNs by different cell lines treatment (Dornetshuber et al., 2007, 2009b). In fact, when searching for mycotoxin on which biological fluid extractions have been studied, Aspergillus mycotoxins as aflatoxins and ochratoxin A, are the most common mycotoxins studied (Korn et al., 2011; Song et al., 2013; Ediage et al., 2012; Lattanzio et al., 2011), especially in urine and serum, either from human or animal origin. Regarding feces, there are few study where the optimization extraction for this matrix has been carried out, although for other mycotoxins as: Deoxynivalenol (DON), Deoxynivalenol-3-glucoside (D3G), Nivalenol (NIV) and, Fumonisin B1 (FB1) among others (Cavret and Lecoeur, 2006; Lattanzio et al., 2011; Nagl et al., 2012). 3.3. LCeMS/MS method validation Linearity was evaluated using the standard calibration curves that were constructed by plotting the peak area versus the ENN A concentration (calibration levels 5, 15, 31, 62, 125, 250 and 500 ng/mL). The calibration curves were 1/ x weighted in order to reduce the risk of leverage. Samples and standard solutions were analysed using MassLynx NT software Ver. 4.1. by plotting the peak area versus the ENN A concentration and linear regressions curves obtained for each sample type. The correlation coefficient (r2) obtained was