Migration of BADGE (bisphenol A diglycidyl-ether) and BFDGE (bisphenol F diglycidyl-ether) in canned seafood

Migration of potentially toxic materials used for the lining of commercial can goods remains an important issue, especially with respect to certain types of processed foods. Seafood is one type where more information is needed with respect to other ingredients used for adding value to fishery products. Most cans are internally coated with starters of resins such as bisphenol A diglycidyl-ether (BADGE) and bisphenol F diglycidyl-ether (BFDGE), both considered as toxic compounds. Several seafood products, sardines, tuna fish, mackerel, mussels, cod and mackerel eggs, were manufactured in different conditions changing covering sauce, time and temperature of storage and heat-treated for sterilization in cans. Migration kinetics of BADGE and BFDGE from varnish into canned products were evaluated by HPLC in 70 samples after 6, 12 or 18 months of storage. Results showed that there is no migration of BADGE in tuna fish, sardines, mussels or cod. However, migration of BFDGE occurs in all species, in a storage time-dependent way and content of fat, although migration of these compounds is not affected by sterilization conditions. All samples analyzed presented values lower than 9 mg BADGE/kg net product without exceeding European limits. However, concerning BFDGE migration, European legislation does not allow the use and/or the presence of BFDGE. Main migration takes place in mackerel reaching the highest values, 0.74 mg BFDGE/kg and 0.34 mg BADGE/kg net product, in red pepper sauce.     

Study on anti-androgenic effects of bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen.

We studied in vitro hormonal activity of bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE), which are used as a material of interior coating for food cans. We also examined related compounds such as 2,2-bis[4-(3-chloro-2-hydroxypropoxy)phenyl]propane (BADGE.2HCl), and bis[4-(3-chloro-2-hydroxypropoxy)phenyl]methane (BFDGE.2HCl) etc. For this purpose, we constructed two stably transfected CHO-K1 cell lines (AR-EcoScreen for androgenic activity and c-luc for cell toxicity evaluation). One stably expresses luciferase with induction of androgen. The other stably expresses luciferase without androgen induction. Also, we have determined the androgenic and anti-androgenic effects of the test chemicals by reporter gene assay with these cell lines. None of the chemicals tested by this assay exhibited androgen agonistic activity. However, BADGE.2HCl and BFDGE.2HCl had the conspicuous antagonistic activity for androgen. These compounds had a high binding affinity for androgen receptor. Furthermore, these two compounds did not show the estrogenic activity in vitro assays. On the contrary, bisphenol A and bisphenol F exhibited anti-androgenic activity in vitro in addition to the estrogenic activity. These results suggest that these chlorohydroxy compounds of BADGE and BFDGE act as androgen antagonist through the process of binding to androgen receptor.     

Interaction effects of Fusarium enniatins (A,A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells

Foodstuff is usually contaminated by more than one mycotoxin, however toxicological data are lacking as regards the effects in combinations compared to their individual effect. This study investigated the in vitro effects of enniatins (ENs) A, A₁, B and B₁, alone and in combinations, on Caco-2 cells viability by MTT assay after 24 h of exposure. Cells were treated with concentrations ranging from 0.9 to 15.0 μM, individually and in combination of two, three and four mycotoxins. Dose–response curves were generated for each mycotoxin and the isobologram method was used to determine the interactive effects of tested mixtures. Tested ENs produced significant cytotoxic effects both individually and in combination in a dose-dependent manner. IC₅₀ values obtained for all individually tested mycotoxins ranged from 1.3 to >15 μM. In ENs combination tests, synergistic effect in Caco-2 viability are observed for EN B + EN A₁, EN B₁ + EN A₁ and EN A + EN A₁ + EN B (CI = 0.33–0.52). All other combinations showed additive effect at medium and high affected fraction with exception of lower fraction affected and the EN B + EN B₁ mixture that produced antagonistic effect (CI = 1.76–10.36). The use of combination index-isobole method could help to better understand the potential interaction between co-occurring mycotoxins and may contribute to their risk assessment.     

Role of quercetin on Caco-2 cells against cytotoxic effects of alternariol and alternariol monomethyl ether

Molds of the genus Alternaria have been reported as contaminants of a variety of food and feed. Alternaria toxins such as alternariol (AOH) and its naturally occurring monomethyl ether (AME) produce cytotoxicity and oxidative stress in cell cultures.On the other hand, it has been proved that natural polyphenols have antioxidant effect. Quercetin (Quer) is a polyphenol present in berries and other commodities which exhibits these effects. The aims were to evaluate the cytotoxicity of AOH, AME and the binary combination of them, and the cytoprotective effect of Quer exposed simultaneously with AOH, AME and the mycotoxin mixture in human adenocarcinoma (Caco-2) cells. The cytotoxicity and the cytoprotective effect were determined by the MTT test after 24 and 48 h of exposure and interactions were evaluated with the isobologram analysis method. Cell viability decreased after 48 h of AOH and AME exposures, being the binary combination more cytotoxic, causing a synergism effect. No cytoprotective effect of Quer against AOH and AME was observed when they were exposed simultaneously in Caco-2 cells. The cytoprotective effect of Quer against mycotoxins (AOH, AME or other different which could present higher cytotoxic effect) depends on the concentration, the presence and the interaction between the compounds in food.     

Comparison of anticancer activity between lactoferrin nanoliposome and lactoferrin in Caco-2 cells in vitro

The anticancer activities of Lactoferrin (Lf) and Lf nanoliposomes in Caco-2 cells were observed in this study, and mitochondrial function (MTT assay), count kit-8(CCK-8), detection of intracellular reactive oxygen species (ROS) and apoptosis induction (AO/EB staining) assays were used to evaluate the anticancer activity. MTT results demonstrated that Lf nanoliposomes and Lf reduced the mitochondrial activity of cells in a manner of dose and time effect, and the viabilities of Caco-2 cell were significantly decreased in vitro following exposure to Lf nanoliposomes at the concentrations of 5 and 10 mg/mL. LDH leakage and ROS significantly increased in cells exposed to Lf nanoliposomes (⩾5 mg/mL), while Lf induced ROS only at higher doses (10 mg/mL). CCK-8 evaluation of cell proliferation and AO/EB double staining supported the anti-proliferative effects of Lf liposomes. Our findings demonstrated that the presence of Lf nanoliposome is more significant than Lf in inhibiting human tumor cells proliferation. Therefore, it can be concluded that Lf nanoliposomes are a potential therapeutic modality in the management of tumors.     

The cytotoxic effect of palytoxin on Caco-2 cells hinders their use for in vitro absorption studies

Palytoxin (PLTX), found in Palythoa zoanthids and Ostreopsis dinoflagellates, has also been detected in crabs and fish, through which it can enter into the food chain. Indeed, PLTX is considered the causative agent of several cases of human seafood poisoning resulting in systemic symptoms. Available epidemiological data on PLTX human toxicity suggest that the intestinal tract may be one of its in vivo targets and its potential site of access into the bloodstream. Hence, the purpose of this study was to investigate the suitability of the human intestinal Caco-2 cell line for evaluating PLTX oral absorption. A detailed analysis of PLTX cytotoxicity revealed a high sensitivity of Caco-2 cells: 4h toxin exposure reduced mitochondrial activity (MTT assay, EC(50) of 8.9±3.7×10(-12)M), cell density (SRB assay, EC(50) of 2.0±0.6×10(-11)M) and membrane integrity (LDH release, EC(50) of 4.5±1.4×10(-9)M and PI uptake, EC(50) of 1.0±0.8×10(-8)M). After low PLTX concentration (1.0×10(-11)M) exposure for 1-8h, followed by 24h recovery time in toxin-free medium, cell density reduction was only partially reversible. These results indicate that, due to the high susceptibility to PLTX cytotoxic effects, Caco-2 cells do not represent an appropriate and reliable model for investigating intestinal barrier permeation by this toxin.     

Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells

1. Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin.
2. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 μM) or bromosulphophthalein (100 μM). Similarly tetraethylammonium and N-′methylnicotinamide (10 mM) were without effect.
3. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4′-diisothiocyanostilbene-2-2′-disulphonic acid (DIDS, 400 μM).
4. Net secretion of ciprofloxacin was partially inhibited by 100 μM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretion in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK.
5. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na⁺, Cl⁻ or K⁺ in the bathing media.
6. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia.

     

Insulin-Degrading Enzyme in a Human Colon Adenocarcinoma Cell Line (Caco-2)

The activity of insulin-degrading enzyme (IDE), a thiol metalloprotease degrading insulin in many insulin target cells, was determined in human colon adenocarcinoma (Caco-2) cells. Insulin-degrading activity was localized in the cytosol of Caco-2 cells, accounting for 88% of total activity. Western blots and immunoprecipitation showed that IDE was present in the cytosol of Caco-2 cells and contributed to more than 93% cytosolic insulin-degrading activity. Cytosolic insulin degradation was strongly inhibited by IDE inhibitors, including N-ethylmaleimide, 1,10-phenanthroline, p-chloromericuribenzoate, and EDTA, but was not significantly or not as extensively inhibited by strong inhibitors of proteasome, i.e., chymostatin, soybean trypsin inhibitor, leupeptin, and Dip-F. These results suggest that IDE is present in Caco-2 cells, that Caco-2 IDE has properties similar to those of its counterparts in insulin-target tissues, and that it significantly contributes to intracellular insulin degradation.     

Uptake and transport of the ACE-inhibitor ceronapril (SQ 29852) by monolayers of human intestinal absorptive (Caco-2) cells in vitro

The angiotensin-converting enzyme (ACE)-inhibitor ceronapril (SQ 29852) is shown to be a substrate of the intestinal dipeptide transporter. Uptake by Caco-2 cells, grown as confluent monolayers, follows a major saturable pathway (K_m, 0.91 ± 0.11 mM; 90% at 1 mM) together with a minor passive component (k_J, 32.3 ± 6.6 ng (10⁶ cells)⁻¹ (20 min)⁻¹. Uptake was inhibited by competition with dipeptides such as l-AIa-l-Pro (K_i, 2.96 mM) and l-Phe-Gly (K_i, 3.84 mM) but not by cephalosporins such as cephalexin. In contrast, transport was non-saturable, flux increased linearly with concentration and data were consistent with a passive transepithelial transport mechanism. Transport profiles showed a biphasic dependence upon time with an initial flux of 0.83 ± 0.02 ng insert⁻¹ min⁻¹ (k₁) and a terminal value of 1.65 ± 0.08 ng insert⁻¹ min⁻¹ ((k₂) at 100 μM. It is concluded that the basolateral efflux is retarded so that the passive paracellular transport controls the overall transepithelial transport characteristics in the Caco-2 model. Carrier-mediated uptake into intestinal enterocytres, followed by rate-limiting basolateral efflux, may explain the extended t_max in vivo following oral administration.     

Effect of chitosan malate on viability and cytoskeletal structures morphology of Caco-2 cells

The aim of this work was to evaluate the effects of the treatment with chitosan malate (CM) on viability of Caco-2 cells and on the morphology and the integrity of their cytoskeletal structures (microtubules, microfilaments). Cytotoxicity of CM, both as a solution and as microparticles obtained by spray drying, was evaluated by using the reduction of MTT reagent; microtubule and microfilaments organization of Caco-2 cells treated with CM solution was examined with immunofluorescence techniques in monolayers fixed with the glutaraldehyde-borohydride method.CM as a solution displayed a concentration-dependent cytotoxicity towards Caco-2 cells, with viability percentages of 5 ± 2%, 7 ± 3% and 31 ± 15% at 15, 10 and 5 mg/mL, respectively, while at 2.5 mg/mL or less cell viability was 90% or higher. CM microparticles also produced a remarkable cytotoxic effect (cell viability 84 ± 17%, 16 ± 8% and 5 ± 6% after treatment with 1, 5 and 10 mg CM per well, respectively), resulting more toxic than CM solution.Microtubules pattern of Caco-2 cells, which is a network regularly arranged around the nucleus, appeared deeply modified by CM treatment in a concentration-dependent way, with progressive microtubule changes in length and spatial disposition and deposition of fluorescent aggregates at the periphery of the cells. Furthermore, after treatment with 5-15 mg/mL CM, remarkable alterations of actin organization were observed, with a progressive disruption of the network of stress fibers and the appearance of actin aggregates inside the cell cytoplasm.In conclusion, viability and the cytoskeletal pattern of Caco-2 cells are modified by treatment with CM at high concentrations, which might be locally reached in vivo after application of drug-loaded chitosan microparticles onto mucosal cells.     

Alternariol induce toxicity via cell death and mitochondrial damage on Caco-2 cells

Alternariol (AOH), a mycotoxin produced by Alternaria sp, appears as food contaminant in fruit, vegetables and cereal products. Its toxicity has been demonstrated, but the mechanisms involved have not been elucidated yet. In this study, the pathways triggered by AOH and degradation products generated on Caco-2 cells were evaluated. Cells were exposed to AOH sub-cytotoxic concentrations of 15, 30 and 60 μM. Cell cycle disruption, the induction of apoptosis/necrosis and changes in mitochondrial membrane potential (Δψm) after 24 and 48 h was asses by flow cytometry. Also, AOH and its degradation products were evaluated after 24 and 48 h by high-performance liquid chromatography with tandem mass spectrometric (LC-MS/MS) to detect and quantify its levels. Cell cycle was significantly decreased at G1 phase and increased at S and G2/M phase at the time of exposure. AOH induced necrosis, apoptosis/necrosis and loss of Δψm in a dose and time-dependent manner. The concentrations of AOH quantified in the culture media exposed to AOH decreased as the exposure time was increased. In conclusion, AOH caused cytotoxic effects supported by blocking cell cycle, decreasing cell proliferation and increasing apoptosis/necrosis cells.     

Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells.

Diarrhetic Shellfish Poisoning (DSP) results from the consumption of shellfish contaminated with okadaic acid (OA) or one of the dinophysistoxins (DTX). It has been reported that this toxin induces apoptosis in several cell models, but the molecular events involved in this process have not been clarified. In this report we studied intracellular signals induced by OA in Caco-2 cells: mitochondrial membrane potential, F-actin depolymerization, caspases activation, cell proliferation and cell membrane integrity. Results indicate that caspases-8 and -9 increased their activity after 30 min of OA treatment according to their role as initiator caspases. In contrast, activation of the downstream caspase-3 is a later event in the execution phase of apoptosis. Mitochondrial membrane potential changes are detected at 30 min of OA exposure indicating that this is an early response in the apoptotic cascade. F-actin depolymerization occurs after 24h of incubation with OA and this effect is significant at low doses of the toxin. LDH is released into the culture medium, although there is not PI uptake, indicative of a significant cell death in addition to apoptosis. Moreover, OA led to a dose- and time-dependent decrease in cellular proliferation.     

Oxidative stress of alternariol in Caco-2 cells

Alternariol (AOH) is a mycotoxin produced by fungus Alternaria. It is found in a wide variety of fruits and cereals products. AOH is able to damage human health. The aim of this study was to evaluate the cytotoxicity of AOH in human colon adenocarcinoma (Caco-2) cells. Moreover, some events related to oxidative stress were evaluated: reactive oxygen species (ROS) generated by oxidation of 2′,7′-dichlorodihydrofluorescein diacetate; peroxidation of lipid (LPO) by malondialdehyde (MDA) production; and antioxidant enzymatic capability of catalase (CAT) and superoxide dismutase (SOD). Cytotoxicity of AOH (from 3.125 to 100 μM) was determined during 24, 48 and 72 h of exposure by different endpoints. AOH decreased cell viability by MTT, NR and PC assays. However, no IC₅₀ values were obtained by any of the assays tested. AOH induced a strong oxidative stress in Caco-2 cells by generation of ROS production and LPO associated with a rise in the SOD activity at all concentration tested. ROS increased 1.2-fold with respect to the control and MDA production ranged from 130% to 250% compared to control. Our results demonstrated that in spite of AOH showing cytotoxic effect on Caco-2 cells at the highest concentration tested, oxidative stress by LPO and ROS was observed at all concentrations assayed. This could cause an injury and be hazardous to health.     

An approach to As(III) and As(V) bioavailability studies with Caco-2 cells

Foods and drinking water are the main sources of human exposure to inorganic arsenic [As(III) and As(V)]. After oral ingestion, the intestinal epithelium is the first barrier to absorption of these species. A human intestinal cell line (Caco-2) was used to evaluate cell retention and transport of As(III) (15.6–156.0 μM) and/or As(V) (15.4–170.6 μM). Cell monolayer integrity, cell viability, membrane damage and effects on cell metabolism were evaluated. Only the highest concentrations assayed [As(III): 156.0 μM; As(V): 170.6 μM] produced a cytotoxic effect with different cellular targets: As(III) altered the permeability of tight junctions, and As(V) caused uncoupling of the respiratory chain. Retention and transport of As(III) was more efficient than that of As(V). After 4 h of exposure to As(III) or As(V), monolayer retention percentages varied between 0.87–2.28% and 0.14–0.39%, respectively. Transepithelial transport was greater for As(III) (5.82–7.71%) than for As(V) (not detectable—1.55%). The addition of As(III) and As(V) jointly produced a transport rate similar to that observed when they were added independently.     

In vitro assay of hydrolysis and chlorohydroxy derivatives of bisphenol A diglycidyl ether for estrogenic activity

Bisphenol A diglycidyl ether (BADGE) is an epoxy resin monomer. Epoxy-based solution coatings are used in many applications as additives for a variety of plastic coatings in food packaging. It is well known that unreacted BADGE can migrate from epoxy-based packing materials into foods. Not only BADGE but also its derivatives can easily migrate into foods and it is likely that we intake BADGE and its derivatives through food or drink. Recently, endocrine disrupting chemicals (EDCs) have attracted attention because they have been shown to affect reproduction in wildlife. The estrogenic activity of BADGE derivatives has not previously been investigated. Therefore, we investigated the estrogenic activity of the BADGE derivatives, dihydrolysed BADGE (BADGE-4OH) and chlorohydroxy BADGE (BADGE-2Cl), using breast cancer cell (T47D) proliferation assay and estrogen receptor (ER) () binding assay. These chemicals exhibited T47D cell proliferation at concentrations of 10⁻¹⁴–10⁻⁴ . However, these chemicals did not bind to ER () in the binding assay.     

The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells

Recent study suggested that the presence of phytochemicals in food could interact with nanoparticles (NPs) and consequently reduce the toxicity of NPs, which has been attributed to the antioxidant properties of phytochemicals. In this study, we investigated the interactions between ZnO NPs and two flavonoids baicalein (Ba) or baicalin (Bn) as well as the influence of the interactions on the toxicity of ZnO NPs to Caco-2 cells. The antioxidant properties of Ba and Bn were confirmed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, with Ba being stronger. However, the presence of Ba or Bn did not significantly affect cytotoxicity, intracellular superoxide or release of inflammatory cytokines of Caco-2 cells after ZnO NP exposure. When Ba was present, the cellular viability of Caco-2 cells after exposure to ZnO NPs was slightly increased, associated with a modest decrease of intracellular Zn ions, but these effects were not statistically different. Ba was more effective than Bn at changing the hydrodynamic sizes, Zeta potential and UV–Vis spectra of ZnO NPs, which indicated that Ba might increase the colloidal stability of NPs. Taken together, the results of the present study indicated that the anti-oxidative phytochemical Ba might only modestly protected Caco-2 cells from the exposure to ZnO NPs associated with an insignificant reduction of the accumulation of intracellular Zn ions. These results also indicated that when assessing the combined effects of NPs and phytochemicals to cells lining gastrointestinal tract, it might be necessary to evaluate the changes of colloidal stability of NPs altered by phytochemicals.     

The expression of efflux and uptake transporters are regulated by statins in Caco-2 and HepG2 cells

Aim： Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and efflux/uptake transporters. There is little information on the regulation of these proteins in human cells after statin therapy. In this study, the effects of atorvastatin and simvastatin on mRNA expression of efflux （ABCB1, ABCG2 and ABCC2） and uptake （SLCO1B1, SLCO2B1 and SLC22A1） drug transporters in Caco-2 and HepG2 cells were investigated.
Methods： Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins. Results： Differences in mRNA basal levels of the transporters were as follows： ABCC2〉ABCG2〉ABCB1〉SLCO1B1〉〉〉SLC22A1〉SLCO2B1 for HepG2 ceils, and SLCO2B1〉〉ABCC2〉ABCB1〉ABCG2〉〉〉SLC22A1 for Caco-2 cells. While for HepG2 cells, ABCC2, ABOG2 and SLCO2B1 mRNA levels were significantly up-regulated at 1, 10 and 20 pmol/L after 12 or 24 h treatment, in Caco-2 cells, only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin. Interestingly, whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells, in Caco-2 cells the statin significantly down-regulated ABCB1, ABCC2, SLC22A1, and SLCO2B1 mRNA levels after 12 or 24 h treatment.
Conclusion： These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters, and this effect depends on the cell type. Furthermore, alterations in the expression levels of drug transporters in the liver and/or intestine may con- tribute to the variability in oral disposition of statins.     

Differential toxicity and gene expression in Caco-2 cells exposed to arsenic species

Inorganic arsenic [As(V) + As(III)] and its metabolites, especially the trivalent forms [monomethylarsonous acid, MMA(III), and dimethylarsinous acid, DMA(III)], are considered the forms of arsenic with the highest degree of toxicity, linked to certain types of cancer and other pathologies. The gastrointestinal mucosa is exposed to these forms of arsenic, but it is not known what toxic effect these species may have on it. The aim of the present work was to evaluate the toxicity and some mechanisms of action of inorganic arsenic and its metabolites [monomethylarsonic acid, MMA(V), dimethylarsinic acid, DMA(V), MMA(III) and DMA(III)] in intestinal epithelial cells, using the Caco-2 human cell line as a model.