I. De Angelis

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.

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.

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.

Intestinal Cell Culture Models

The human population is exposed to xenobiotics through ingestion, inhalation and dermal absorption. However, the intestine represents the major site of exposure to xenobiotics from the oral ingestion of therapeutic agents and of environmental pollutants or other additives present in food and water. The intestinal tract is therefore particularly important in toxicology both as a target organ and as a site of access of xenobiotics into the organism.

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.

Physico-chemical characteristics and cyto-genotoxic potential of ZnO and TiO2 nanoparticles on human colon carcinoma cells

The aim of the present study is to investigate the role of the physico-chemical properties of ZnO and TiO2 NPs in the potential cytotoxicity, genotoxicity and oxidative DNA damage induction on Caco-2 cell line. As negative control, fine TiO2 particles were used. The characterization of particles was carried out by electron microscopy (SEM, TEM) using a Soft Imaging System. To evaluate the effects of ZnO and TiO2 NPs induced on Caco-2 viability, Neutral Red assay was performed after treatment with different particle concentrations. Our results showed a significant dose and time dependent effect after treatment with ZnO NPs. On the contrary, no effect was observed on Caco-2 cells exposed to TiO2 particles either in micro-and in nano-size. The role of surface in the cytotoxicity induced on Caco-2 was also considered. The levels of DNA 8-oxodG, as the main marker of oxidative DNA damage, were measured by high-performance liquid chromatography with electrochemical detection (HPLC/EC). A significant increase in the 8-oxodG levels was observed after 6 h exposure for both NPs. The estimation of the potential genotoxicity of the two NPs is ongoing by the cytokinesis-block micronucleus assay. Our preliminary results showed that a slight micronucleus increase in binucleated cells was detected in the dose range applied only for ZnO.

Cytotoxic effects of wheat gliadin-derived peptides.

The peptic-tryptic-cotazym (PTC) digest, obtained from bread wheat gliadin by simulating in vivo protein digestion, was more active than the PTC-digest of durum wheat gliadin in reversibly inhibiting HEp-2 cell proliferation and in increasing cellular acid phosphatase. Colony-forming ability of the cells was not affected by treatment with both bread or durum wheat gliadin peptides. The peptic-tryptic (PT) digest of bread wheat gliadin also showed agglutinating activity of HEp-2 cells.

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

SummaryCaco-2, a human intestinal cell lines able to differentiate in long-term culture, has been used to assess the cytotoxicity of the β-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 increase in the differentiated cells, and for the β-receptors’ binding characteristics, which are those of β1 and β2 subtypes. Isoxsuprine was shown to be the most toxic, followed by clenbuterol and salbutanol. 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.

In vitro study with Caco-2 cells on fumonisin B1: aminopentol intestinal passage and role of P-glycoprotein

Fumonisins are a group of mycotoxins produced by moulds of Fusarium, spt F. verticilloides, natural contaminants of cereals, mostly corn and corn-based foods. Fumonisina B1, has a species-specific toxicity in different animals and is related to oesophageal cancer in human (Diaz, 1994). Among the known FB1 metabolites, aminopentol (HFB1) is the totally hydrolyzed metabolite (Hopmans and Murphy, 1993), detectable in corn by-products, after processing at high temperature under alkaline conditions. HFB1 has a toxicity comparable with FB1 and higher absorption in vivo (Hopmans et al., 1997) and in vitro (Caloni et al., 2002) than that demonstrated for the parent compound. Culturing the intestinal cell line Caco-2 on semi-permeable insert, which separates the two different compartments (apical and basolateral side) simulates the real intestinal environment, it is thus possible to evaluate the passage and the absorption of FB1 and HFB1 into the cells. P-glycoprotein belongs to the MDR group, “multidrug resistance proteins”, and is located on the brush border of the enterocyte; it works like an ATP-mediated efflux pump (Wacher et al., 2001). HFB1, may be is a possible P-gp substrate for its positive charge and low polarity.

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 within 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.