Environment and T regulatory cells in allergy.

The central role of T regulatory cells in the responses against harmless environmental antigens has been confirmed by many studies. Impaired T regulatory cell function is implicated in many pathological conditions, particularly allergic diseases. The "hygiene hypothesis" suggests that infections and infestations may play a protective role for allergy, whereas environmental pollutants favor the development of allergic diseases. Developing countries suffer from a variety of infections and are also facing an increasing diffusion of environmental pollutants. In these countries allergies increase in relation to the spreading use of xenobiotics (pesticides, herbicides, pollution, etc.) with a rate similar to those of developed countries, overcoming the protective effects of infections. We review here the main mechanisms of non-self tolerance, with particular regard to relations between T regulatory cell activity, infections and infestations such as helminthiasis, and exposure to environmental xenobiotics with relevant diffusion in developing countries.

Aflatoxin M1 absorption and cytotoxicity on human intestinal in vitro model.

Aflatoxin M1 (AFM1) is the principal hydroxylated Aflatoxin B1 (AFB1) metabolite and is detected in milk of mammals, after consumption of feed contaminated with AFB1. As it is classified as probable human carcinogen (group 2B of the IARC), most countries have regulated its maximum allowed levels in milk in order to reduce AFM1 risk (50 ng/kg the EU and 500 ng/kg in the USA). It was demonstrated that if AFB1 must be converted into its reactive epoxide to exert its effects, and the protein binding may play an important role in its cytotoxicity. Conversely, the AFM1 epoxidation in human liver microsomes is very limited and studies with human cell line (MCL5), expressing or not expressing cytochrome P450 enzymes, demonstrated a direct toxic potential of AFM1 in absence of metabolic activation. For this reason, while AFM1 is generally considered a detoxification product of AFB1 relatively to carcinogenicity and mutagenicity property, this is not always true for cytotoxicity activity. Aim of this work is to evaluate the intestinal absorption of AFM1 using a human in vitro model, the Caco-2 cell line. Either the parental Caco-2 cell line or its derived clone TC7, with higher metabolic competence, have been used. They were treated with different concentrations of AFM1, that mirror the milk contamination level (0.3-32 nM corresponding to 10-10,000 ng/kg), either in undifferentiated or in differentiated phase of growth. After 48 h of treatment in serum free medium, a dose dependent absorption of AFM1 has been detected in both cell lines, especially in differentiated cells, while, no appreciable effects on cell viability were observed, except for a general cellular suffering, revealed by LDH release, particularly evident in the undifferentiated cells. As well, no metabolites or AFM1 conjugates have been detected. The present results may be crucial for the evaluation of human risk to AFM1 exposure, in particular for children's population, due to their large use of milk and derivatives.

The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

Absorption of fumonisin B1 and aminopentol on an in vitro model of intestinal epithelium; the role of P-glycoprotein.

The aim of the present paper is to evaluate the absorption of fumonisin B1 and its principal metabolite, aminopentol on a human intestinal model, Caco-2 cells, cultured on semi-permeable inserts, that reproduces the two different intestinal compartments: luminal (apical) and serosal (basolateral) side. Following separate exposure in apical and in basolateral compartments, aminopentol passage through the cell layer (in particular from basolateral to apical direction) was shown, while it was not observed for the parent compound. The different aminopentol distribution between the two compartments of the culture system, and its variation in presence of verapamil or probenecid (P-gp and MRP inhibitors respectively), strongly suggests the involvement of P-glycoprotein in the influx/efflux mechanisms of aminopentol in the intestinal cells, reducing its oral bioavailability.

Toxicology investigations with cell culture systems: 20 years after.

From almost 20 years the "in vitro" model has gained a wide ground in toxicological investigation, providing advanced tools, reliable protocols, mechanistic information. These advancements have been done thanks to different approaches, addressed at improving chemical testing and validating procedures, at exploring the cellular and molecular basis of toxicity, at studying the modifications that xenobiotics undergo in the cellular environment. In this review the most advanced cellular models, the mechanisms of cell death, the techniques to monitor gene activation, following chemical exposure, is highlighted. Moreover the more recent in vitro models to approach the biotransformation issue will be presented.

Evaluation of Fumonisin B(1) and its metabolites absorption and toxicity on intestinal cells line Caco-2.

The aim of the present paper is to investigate intestinal absorption and toxicity of Fumonisin B(1) (FB(1)) and its partially (PHFB(1) and PHFB(2)) and totally hydrolyzed (HFB(1)) metabolites, using the human intestinal cell line Caco-2, a very well known in vitro model of intestinal epithelium for absorption and metabolism studies. Caco-2 cells were treated for 48 h with several toxin concentrations (in the range of 1-138 microM). At the end of exposure period, no significant variation on cell viability has been observed with all chemicals tested, either in undifferentiated cells or in differentiated ones, suggesting a poor toxicity of these mycotoxins for intestinal cells. In any case, FB(1) appears the most active in this respect. For which concerns the cellular absorption, FB(1), PHFB(1) and PHFB(2) are never detected into Caco-2 cells. On the contrary, a dose-dependent absorption of HFB(1) has been observed in differentiated cells, which express enzymatic and metabolic characteristics of mature enterocytes. Thus HFB(1), losing the tricarballylic acid chain, is more bioavailable than FB(1) on intestinal cell, supporting the hypothesis that in risk evaluation of fumonisins exposure its metabolites are also relevant.

Identification and coupling to adenylate cyclase of three different [(3)H]CGP 12177 binding sites in Caco-2 cell membranes.

In the present investigation the identification of beta -adrenoceptor (beta -ARs) subtypes in the Caco-2 cell line was performed using radiometric assays. beta -ARs were measured using increasing concentrations of the highly specific beta -AR antagonist (-)[(3)H]CGP 12177 (0.06-4 nM), whereas the beta(1)- and beta(2)-AR subtypes discriminated through selective binding assays using the highly selective unlabelled antagonists CGP 20712A and ICI 118551. Atypical beta -ARs were measured using an incubation system formed by higher concentrations (0.6-20 nM) of (-)[(3)H]CGP 12177. beta - Atypical binding site concentrations (69 +/- 5 fmol mg ml(-1)of membrane protein) were higher than beta(1)-ARs (7 +/- 1) and beta(2)-ARs (24 +/- 2), respectively. The different beta -AR subtype affinities were characterized by binding inhibition experiments and the adrenergic agonists displaced the radioligand from its specific binding sites in the following order of potency: isoproterenol > clenbuterol > dobutamine > SR 58611A; for antagonists the order of potency was: propranolol approximately = ICI118551 approximately = CGP20712A. For atypical beta -ARs the order was: SR 58611A > clenbuterol > dobutamine > isoproterenol for agonists and propranolol > CGP 20712A > ICI 118551 for antagonists. As far as in vitro functional studies are concerned, beta -AR subtypes were shown to be coupled to adenylyl cyclase as their stimulation produced cAMP in an amount significantly higher than basal values. cAMP production after stimulation with dobutamine, clenbuterol, isoproterenol, and SR 58611A was measured using a cAMP radioassay kit. The order of efficacy suggested that the stimulation of beta(2)-ARs was the most effective in inducing the activation of cell signalling mechanisms. The identification of functional beta -ARs in a cancer cell line represents the first step in the study of the possible adrenergic control of cellular activities (e.g. proliferation and/or differentiation), which could suggest the use of this cancer cell line as a model for the study of cell activity or possibly new therapeutic strategies.

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells.

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.

Apoptosis evaluation in epithelial cells exposed to different chemicals: relevance of floating cells.

The recent increase in understanding of cell death has promoted new approaches in toxicological studies, mainly those dealing with in vitro systems where the evaluation of cell death has been the most widely adopted end-point in measuring the effects of chemical toxicants. The aim of this study was to investigate the possibility of improving the traditional cytotoxicity test protocols in order to produce more specific information on the type of cell death induced by exposure to toxicants. In particular, we characterized the mode of cell death in an established epithelial cell line, HEp-2 cells, which is frequently used in cytotoxicity testing owing to its easy handling and standardization of culture conditions. Reference chemicals for apoptosis and necrosis were selected as controls, together with other molecules that have been shown, in preliminary studies, to induce various morphological and structural modifications in relation to cell death. The results obtained show that: (a) the floating fraction of treated cells gives the clearest picture of the necrotic/apoptotic distribution; (b) morphological analysis is crucial for characterization of apoptosis; (c) more than one cytotoxic end-point is necessary to clearly identify the type of cell death.

Metabolism of furazolidone: alternative pathways and modes of toxicity in different cell lines.

1. The metabolism and cytotoxicity of the antimicrobial nitrofuran drug furazolidone have been studied in Caco-2, HEp-2 and V79 cell lines. Free radical production, metabolite pattern, formation of bound residues, inhibition of cellular replication and protection by the antioxidant glutathione were compared for the three cell lines. 2. All three cell lines produced the same nitro-anion radical with similar kinetics. Little further metabolic breakdown was observed in V79 cells, whereas Caco-2 and HEp-2 cells showed extensive degradation of furazolidone, but with different end patterns. 3. Under hypoxic conditions, the colony-forming ability was extensively impaired in HEp-2 cells whereas the other two cell lines were less affected, suggesting that irreversible damage to DNA occurred prevalently in HEp-2 cells. In V79 cells the absence of oxygen caused a 25-fold increase in the formation of protein-bound residues. 4. Brief exposure to furazolidone caused a 50% loss of endogenous glutathione in Caco-2 cells, but no loss could be detected in V79 and HEp-2 cells. Consistently, when glutathione was depleted by buthionine-[S,R]-sulphoximine (BSO) and diethylmaleate (DEM) treatment, the viability of V79 and HEp-2 cells was minimally affected by furazolidone, whereas that of Caco-2 cells was substantially reduced. 5. It is concluded that the cytotoxicity of furazolidone in these cell lines can be exerted by a number of different mechanisms, possibly related to different metabolic pathways. The cytotoxicity of nitrofuran drugs, therefore, cannot be ascribed to a single toxic intermediate, but in Caco-2 cells furazolidone is extensively metabolized and detoxified by GSH, in V79 is only partially activated and then bound to proteins, whereas in HEp-2, once activated, may react with DNA.

A contribution to safety assessment of veterinary drug residues: in vitro/ex vivo studies on the intestinal toxicity and transport of covalently bound residues.

1. The gastrointestinal fate of protein-bound residues of the model compound furazolidone (FZD) was investigated in vitro and ex vivo. Protein-bound residues were generated in rat liver microsomes, isolated by solvent extraction and digested with 0.5% hydrochloric acid and Pronase E. 2. During digestion, 3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was partly released from bound residues. 3. The absorption of free AOZ and digested protein-bound residues was tested in isolated perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2. Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer cultures, while no indications for passage of bound residues were obtained. 4. No acute toxicity of AOZ or digested food residues respectively was observed in gut segments and Caco-2 cells at concentrations that were substantially above maximum residue levels to be expected in food of animal origin after administration of therapeutic doses. 5. The results demonstrate that digestive processes can alter the chemical nature of drug residues and yield degradation products that may be bioavailable for the consumer. Thus, the covalent binding of xenobiotics to macromolecular tissue constituents cannot necessarily be regarded as an irreversible endpoint of residue bioavailability and toxicity.

Characterization of furazolidone apical-related effects to human polarized intestinal cells.

In studying the effects of furazolidone (FZ) on the human intestinal Caco-2 cell line grown on microporous membrane, we have previously demonstrated a higher toxicity when the compound was administered at the apical (AP) side than at the basolateral (BL) side. Moreover, we have also shown the production, in the intact cells, of a nitroanion radical from FZ by a cytochrome c P450 reductase. The aim of the present study was to investigate which specific cell structures and functions are involved in the observed domain-related toxicity. The relevance of alterations in integrity and selective properties of the intestinal barrier as first-pass site for ingested molecules is also discussed. We have confirmed that, as expected, the Caco-2 cells are protected from FZ injury by a specific inhibitor of the cytochrome c P450 reductase, and we have shown that this protection is more active on the apical side of the cells. In sublethal conditions, FZ causes increased permeability to 3H-mannitol and, to a different extent, to 3H-inulin. Again the effect is higher when the cells are apically exposed. We have thus examined the tight junctions morphology: a disruption of the apical perijunctional actin-bound cytoskeleton was detected by rhodamine-phalloidin staining and microtubule disorganization by antitubulin fluoresceinated antibodies. Again, the effect was more evident when the cells were apically treated with FZ. Preferential transport and accumulation of the compound by active transport mechanisms could be excluded, since transport of FZ was linear and no intracellular accumulation was detected either from the AP and or the BL sides. All together these results may suggest that the AP formation of the active metabolite and its possible reactivity with SH groups of perijunctional microfilaments could be responsible of the higher FZ apical toxicity. This study shows that polarized differentiated cells are very interesting in vitro models to investigate specific cellular domains as targets of toxic effects and to detect subtle changes that may be induced, in absence of cell death, in specialized epithelial layers.

In vitro toxicity and formation of early conjugates in Caco-2 cell line treated with clenbuterol, salbutamol and isoxsuprine.

Caco-2, human intestinal cell line able to differentiate in long-term culture, has been used to assess the cytotoxicity of the beta-agonists clenbuterol, salbutamol and isoxsuprine, also used at high doses to obtain lean meat in food producing animals, and to investigate the eventual in vitro formation of early conjugates of these compounds. For this purpose, the cells have been characterized for the activity of UDP-glucuronyltransferase, which is present and increases in the differentiated cells, and for the beta-receptors' binding characteristics, which are those of beta 1 and beta 2 subtypes. Isoxsuprine was shown to be the most toxic, followed by clenbuterol and salbutamol. Conjugates have been observed after incubation of the cells both with the lowest isoxsuprine and the highest salbutamol concentrations. No conjugates were detected in the case of clenbuterol.

N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone activation by the human intestinal cell line Caco-2 monitored through noninvasive electron spin resonance spectroscopy.

The pathways participating in the metabolism of the nitrofuran antimicrobial drug N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone (furazolidone) in intact cells were investigated in the human intestinal cell line Caco-2. One-electron reduction of furazolidone led to the formation of a free radical intermediate that could be monitored in dense cell suspensions by noninvasive electron spin resonance spectroscopy. The effects of enzyme inhibitors on the kinetics of radical production and decay were used to estimate the relative contribution of different enzymes to the reductive activation of the drug. Although many enzymes are known to reduce nitrofurans in vitro (e.g., xanthine oxidase, aldehyde oxidase, DT-diaphorase, mitochondrial redox chain components), their contributions were insignificant in living Caco-2 cells. The first reducing equivalent required for the formation of the nitroanion derivative of furazolidone appeared to be provided essentially by the microsomal cytochrome P450 reductase. This was confirmed through studies of the NADPH-dependent radical formation by microsomes. Differentiated Caco-2 cells, an established enterocyte model, showed only modestly increased radical formation and the same enzyme-specificity pattern as undifferentiated cells. Consistently, only a small increase in P450 reductase activity was found in differentiated cells, in contrast to the 10-fold increase seen in typical differentiation marker enzymes. With the electron spin resonance method that we describe, it is possible to distinguish between sites of bioactivation of redox active drugs in intact cells.

Established cell lines for safety assessment of food contaminants: differing furazolidone toxicity to V 79, HEp-2 and Caco-2 cells.

In vitro models, preferentially derived from human tissues, may be valuable tools to study the biotransformation and toxicity of compounds that may be present as residues in food products. Such residues may represent a risk to human health, and therefore call for increased testing. Three established cell lines were used to study the toxic effect of furazolidone (FZ), a widely used veterinary drug: HEp-2 cells, derived from a human larynx carcinoma, previously used in toxicity screening of several compounds; Caco-2 cells, derived from a human colon adenocarcinoma, able to differentiate partially in culture, and V 79, a fibroblast cell line derived from Chinese hamster lung, widely used to assess direct toxicants. Various toxicity parameters were used, primarily dealing with cell death and cell proliferation. In all cell lines FZ at a concentration of 5 micrograms/ml caused a marked decrease in cell viability and especially in cell proliferation. Inhibition of DNA synthesis has also been observed, even if at higher concentrations. However, only in V 79 cells was the decrease in cell number accompanied by a marked increase in lactate dehydrogenase leakage due to membrane damage. Moreover, the surviving V 79 cells, after removal of FZ, fully recovered from the effect of the drug, as shown by their full capacity to attach to dishes and to form colonies. Surviving cells of the other two cell lines showed much poorer colony-forming ability. Exposure of Caco-2 cells and, to a lesser extent, HEp-2 cells, caused a marked increase in oxygen consumption, that possibly was due to redox cycling of the initially formed radical nitro anion. Biotransformation of the drug by all three cell lines was accompanied by the formation of protein-bound metabolites, HEp-2 being the most active cells. The toxic effects recorded show that cell lines provide a sensitive system in toxicity assessment. Moreover, it may be suggested that a battery of cell lines, including some of human origin, as well as a battery of endpoints, may be of help in addressing further specific mechanistic investigations.

Functional alterations induced by the food contaminant furazolidone on the human tumoral intestinal cell line Caco-2.

Caco-2 cells, which are derived from a human colon carcinoma and are able to differentiate in culture, have been used to study the effect of furazolidone (FZ), a chemical belonging to the nitrofuran family which is frequently used for the prevention of animal infections. Its potentially toxic residues could remain in some food products of animal origin and affect human health. Toxicity has been measured by different parameters, either in undifferentiated cells (day 7 of culture), or on differentiated cells (day 21 of culture). Our results indicate that FZ may seriously affect the proliferating portion of the intestinal mucosa, while the differentiated cells appear to be more resistant. However, the slight effect recorded on the aspecific and specific functions of the differentiated cells may suggest that the specialized portion of the intestine can also be compromised by the drug. Caco 2 cells seem a good model for a deeper investigation of the mechanism involved in the toxic action of FZ.

Choice and standardization of test protocols in cytotoxicology: A multicentre approach.

A major problem that interferes with the introduction of in vitro tests for toxicological risk assessment is that of defining reliable standardized protocols. This issue was approached in the present study with an interlaboratory comparison of three cytotoxicity assays detecting chemical toxicity as impairment of cell viability in confluent cultures, reduction of colony forming ability, and inhibition of cell proliferation over 3 days of treatment. The study was performed using V79 cells, which are unable to activate indirectly-acting xenobiotics, and six chemicals with different mechanisms of action: two antioxidants (butylated hydroxyanisole and butylated hydroxytoluene), an inhibitor of protein synthesis (cycloheximide), an alkylating agent requiring metabolic activation (cyclophosphamide), an uncoupler of oxidative phosphorylation (dinitrophenol), and a genotoxic metal salt (potassium dichromate). The three tests produced the same rank of relative toxic potency for the tested chemicals, based on LC(50) values. The cell viability test appeared to be the most suited for the screening of unknown chemicals, given its simplicity and better reproducibility.