Edited by: Hani El-Nezami, RMIT-University, Australia
Reviewed by: Mosaad A. Abdel-Wahhab, National Research Centre, Egypt; Roger A. Coulombe, Utah State University, USA
*Correspondence: Francesca Caloni, Department of Health, Animal Science and Food Safety, Universitá degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy. e-mail:
This article was submitted to Frontiers in Predictive Toxicity, a specialty of Frontiers in Pharmacology.
This is an open-access article distributed under the terms of the
Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1). After it is formed, it is secreted in the milk of mammals. Despite the potential risk of human exposure to AFM1, data reported in literature on the metabolism, toxicity, and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM1 and possible damage to tight junctions (TJ) of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the
Aflatoxins, a group of mycotoxins produced primarily by
Aflatoxin B1 (AFB1), the most potent hepatocarcinogen known in mammals (Creppy,
Human exposure to AFM1 is partly from consumption of contaminated milk and dairy products and partly from endogenous production through AFB1 metabolism in the liver (Neal et al.,
Despite the potential risk of human exposure to AFM1, data reported in literature regarding the metabolism, toxicity, and absorption of this molecule, particularly in humans, are limited and out of date. In general, AFM1 and AFB1 cause almost identical effects of acute toxicity and carcinogenicity in different mammalian systems (Sinnhuber et al.,
A dose-dependent absorption of AFM1 in differentiated Caco-2 cells and significant lactate dehydrogenase release, particularly evident in undifferentiated cells, was reported previously (Caloni et al.,
The purpose of this study was to investigate AFM1 transport and possible damage to tight junctions (TJ) of Caco-2/TC7 cells, a clone derived from late passage of the human parental colorectal adenocarcinoma Caco-2 cell line. This clone was seen to express higher metabolic competence, such as hydrolase sucrose isomaltase and UDP-glucuronyltransferases (Turco et al.,
The 0.5-ng/μl AFM1 solution in methanol was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Water (H2O), acetonitrile (ACN), and methanol (MeOH) for HPLC analysis were obtained from J.T. Baker® (Deventer, The Netherlands) and 2-propanol (IPA) from Merck (Darmstadt, Germany). Dimethylsulfoxide (DMSO) was purchased from Carlo Erba (Milan, Italy). Hanks Balanced Salt Solution (HBSS),
Caco-2/TC7 clone, derived from late passage of Caco-2 wild type cells (provided by Dr. Ming Hu, Washington State University, Pullman) was routinely grown in an atmosphere of 5% carbon dioxide at 37°C in DMEM high glucose standard medium (Caloni et al.,
Experiment 1 was performed to evaluate AFM1
Experiment 2 was performed to evaluate AFM1
In Experiment 1 and Experiment 2 barrier impairment after exposure to AFM1 was assessed by measuring the trans-epithelial electrical resistance (TEER) which quantifies ion movement across the cellular barrier. TEER values were recorded in the culture medium at 37°C with chopstick electrodes (Millicell®-ERS, Millipore) and were expressed as Ω × cm2 according to the following equation:
For each filter, three separate measures were collected.
Experiment 3 was carried out to evaluate the AFM1 effects on TJ proteins. The expression of Zonula occludens-1 (ZO-1) and occludin, two TJ proteins located in different cellular compartments, was examined. In addition, considering that apoptosis might contribute to loss of intestinal barrier integrity (Sun et al.,
In detail, Caco-2/TC7 cells were seeded on filters as described previously and treated with AFM1 concentrations of 1,000 ng/kg (3.2 nM) and 10,000 ng/kg (32 nM) for 60 min. After two washes with PBS, monolayers were fixed with a solution of paraformaldehyde (4%) and sucrose (0.12 M) and permeabilized with TRITON×-100 (0.2%). For ZO-1 and occludin staining, cells were incubated overnight at 4°C with anti-ZO-1 (1:100 in PBS) and anti-Occludin (1:50 in PBS) and then labeled with the secondary fluorescent conjugated antibodies. For nuclear staining, after two washes with deionized water, 250 μl of Hoechst solution was added to the Ap compartment and incubated at 37°C for 30 min. Cells were observed using an inverted fluorescent microscope (LEICA DM IRB, Nussloch, Germany).
After exposure to AFM1 the cells were processed and the samples were analyzed for AFM1 presence by using HPLC. All procedures were conducted in absence of artificial light. In short, 3 ml of H2O was added to each medium sample (1 ml) and then extracted by the Immunoaffinity Column (Afla M1 TM, Vicam, USA) as described by Sharman et al. (
Samples were processed as described previously and analyzed by HPLC (Series 200, Perkin-Elmer, USA) using a Waters Spherisorb 5 μm ODS 2 250 mm × 4.6 mm (Supelco, Inc., Sigma-Aldrich, St. Louis, MO, USA), MeOH-NaH2PO4 0.1 M (33:67, v:v) as a mobile phase (flow rate of 1 ml/min) and a fluorescence detector (LC 240 Perkin-Elmer, USA) set at an excitation wavelength of 365 nm and emission wavelength of 420 nm (Sharman et al.,
The apparent permeability coefficient (
where
Uptake ratio (absorption), i.e., the ratio between Ap → Bl and Bl → Ap
Two separate experiments, performed in triplicate, were carried out for each assay. Results were expressed as mean ± standard deviations (SD). Statistical evaluation was performed by two tailed Student’s
Trans-epithelial electrical resistance values in Experiment 1 were recorded before the treatment and after exposure for 6 and 24 h to different concentrations of AFM1 (from 1,000 to 10,000 ng/kg). Both the Ap and Bl sides were subjected to treatment. The mean TEER value of untreated cells was 256 ± 6 Ω × cm2.
As shown in Figure
Trans-epithelial electrical resistance values were basically unchanged after 48 h of AFM1 treatment (data not shown). Before and after the 40-min absorption studies (Experiment 2), TEER values of all inserts were determined in order to verify monolayer integrity. No significant variations were reported at any of the concentrations tested; moreover, the mean TEER value was always within the range of the acceptance criteria defined for this cell line (i.e., >200 Ω cm2).
After a 1-h treatment with AFM1 concentrations of 1,000 and 10,000 ng/kg, no loss of ZO-1 was observed. Occludin staining continuity was reported, indicating integrity of TJ (Figures
The detection limit for AFM1 in medium and cells in both experiments was 5 ng/kg and the volume injected was 50 μl. AFM1 extraction recoveries from Ap and Bl media samples for each transport study were calculated on 20 replicates, obtaining a range of 91.2–98.5%. Recoveries from Caco-2/TC7 cells extractions were about 100%.
Absorption of AFM1 was evaluated on the insert culture system. Caco-2/TC7 cells were exposed to different concentrations of AFM1 (1,000–10,000 ng/kg) in both Ap and Bl compartment
AFM1 ng/kg | Apical exposure (mean ± SD) |
Basolateral exposure (mean ± SD) |
||||
---|---|---|---|---|---|---|
Donor medium | Acceptor medium | Cells | Donor medium | Acceptor medium | Cells | |
1,000 | 731.7 ± 91.8 | 163.6 ± 9.1 | 53.1 ± 9.3 | 339.2 ± 77.8 | 451.6 ± 48.0 | 59.3 ± 7.5 |
5,000 | 4324.4 ± 297 | 149.0 ± 34.4 | 59.2 ± 8.3 | 2178.7 ± 239.2 | 2330.9 ± 111.8 | 60.2 ± 9.0 |
10,000 | 7341.7 ± 450 | 1914.5 ± 391.5 | 53.1 ± 9.2 | 3984.2 ± 497.8 | 4611.8 ± 449.1 | 60.5 ± 7.6 |
Forty-minutes transport studies were performed with AFM1 concentrations ranging from 10 to 1,000 ng/kg in both Ap and Bl compartment
AFM1 ng/kg | Time (min) | Apical exposure (mean ± SD) |
Basolateral exposure (mean ± SD) |
||||
---|---|---|---|---|---|---|---|
Donor medium | Acceptor medium | Cells | Donor medium | Acceptor medium | Cells | ||
10 | 10 | NA | 0.63 ± 0.21 | NA | NA | 0.57 ± 0.06 | NA |
20 | NA | 1.35 ± 0.21 | NA | NA | 0.93 ± 0.25 | NA | |
30 | NA | 2.47 ± 0.15 | NA | NA | 1.30 ± 0.20 | NA | |
40 | 7.23 ± 0.31 | 3.77 ± 0.35 | NA | 5.97 ± 0.59 | 2.17 ± 0.31 | NA | |
100 | 10 | NA | 4.53 ± 0.67 | NA | NA | 4.50 ± 0.26 | NA |
20 | NA | 5.53 ± 0.15 | NA | NA | 10.60 ± 1.57 | NA | |
30 | NA | 6.80 ± 0.60 | NA | NA | 17.17 ± 1.07 | NA | |
40 | 71.83 ± 2.28 | 9.33 ± 0.15 | 14.83 ± 3.12 | 65.73 ± 2.64 | 21.37 ± 1.96 | 7.30 ± 1.41 | |
1,000 | 10 | NA | 51.97 ± 2.52 | NA | NA | 34.17 ± 1.33 | NA |
20 | NA | 66.30 ± 4.52 | NA | NA | 88.27 ± 3.56 | NA | |
30 | NA | 89.47 ± 4.52 | NA | NA | 121.67 ± 6.50 | NA | |
40 | 718.93 ± 5.75 | 104.20 ± 1.95 | 48.87 ± 3.31 | 606.47 ± 24.45 | 228.73 ± 5.42 | 33.35 ± 1.20 |
A
The intestinal tract represents the first barrier to ingested chemicals or food contaminants and the evaluation of its integrity is crucial in assessing risk subsequent to food contaminant exposure.
The disruption of the intestinal barrier allows increased penetration of normally excluded luminal substances that could promote intestinal disorders (Pinton et al.,
Although epidemiological evidence is still required, it is believed that food-associated exposure to certain mycotoxins could lead to the induction and/or persistence of human chronic intestinal inflammatory diseases (Maresca and Fantini,
AFM1, present in milk and dairy products, is of great importance because of the high consumption of these products by humans, especially children. Human exposure to AFM1 through milk and dairy products has been shown in several studies (Sassahara et al.,
The intake of AFM1 from milk is calculated to be 6.8 ng/person/day in the European diet but it is interesting to note that if all milk consumed were contaminated with AFM1 at the proposed maximum EU levels of 50 ng/kg, the intake of AFM1 from milk in the European regional diet would be 15 ng/person per day [Joint FAO/WHO Expert Committee on Food Additives (JECFA),
The toxicological effects of AFM1 are much less investigated than the ones caused by AFB1 and limited data are reported in literature regarding its absorption and metabolism, particularly in humans.
A previous study (Caloni et al.,
AFM1 absorption was previously evaluated in Caco-2 cells cultured in monolayer (Caloni et al.,
In the present paper, we investigated the absorption profile of AFM1 and possible damage to TJ of Caco-2/TC7 cells cultured on microporous filter supports for 21 days. The Caco-2/TC7 cell line is as suitable as the parental Caco-2 line as an intestinal model for studying absorption. Furthermore, due to its clonal origin, the TC7 cell line shows a less heterogenic cellular population, which can result in better reproducibility of results (Chantret et al.,
In epithelial tissue the initial toxic effect of several substances seems to be directed at the molecules involved in the junctional complexes (tight and adherens junctions); for this reason changes in the permeability of epithelial barriers can be considered as early indicators of adverse effects after chemical exposure (Sambuy,
In this study, the effects of AFM1 on TEER were initially studied. The TEER quantifies ion movement across a monolayer and is considered to be a good indicator of the integrity of epithelial barrier. A slight (15–20%) but significant (
Modulation of barrier properties is often mirrored by changes in specific TJ protein components, since TJ dynamic structures respond quickly to several physiological and pathological stimuli. We therefore examined whether the AFM1-induced reduction of TEER could be due to changes in the expression of certain TJ proteins. We focused our attention on the expression of two TJ proteins located in different cellular compartments: ZO-1 interacting in the cytoplasm with actin cytoskeleton and occludin interacting throughout its extracellular domain with neighboring cells (McLaughlin et al.,
The AFM1 absorption profile was evaluated on the insert culture system. In this condition the cells, after about 3 weeks of culture, were able to polarize and fully differentiate according to the enterocytic pathway, with apical microvilli and a differentiated basolateral surface, similar to the cellular surface in contact with sub-epithelial tissue. In both treatments, a very low concentration of mycotoxin was detected in the cells, indicating that AFM1 was poorly absorbed by these cells. Under these experimental conditions, AFM1 passage through the Caco-2/TC7 layer was observed at all tested concentrations after both Ap and Bl exposure and the
In particular, its passage was greater in the Bl-Ap direction than in the Ap-Bl one. The presence of asymmetric passage through Caco-2 monolayer usually suggests involvement of transporter pathways. This cell line expressed most of the known intestinal transporters overseeing influx/efflux carrier mediated processes, in a pattern similar to that reported for the small intestine (Sun et al.,
In conclusion, our results pointed out that AFM1: (i) was poorly absorbed in Caco-2/TC7 cells under the present experimental conditions, (ii) passed across the monolayer in both directions (from Ap to Bl and from Bl to Ap), even if to a different extent, (iii) did not cause viability impairment or barrier damage. Further studies need to be conducted in order to better understand the AFM1 transport mechanism.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.