Simonetta Ferruzza Yula Sambuy Andrea Onetti Muda Fabio Nobili and Maria Laura Scarino

1. COPPER AND INTESTINAL TOXICITY

Copper in humans plays different roles: It is an essential nutrient, but, depending on the dose, it can act as a toxic substance. However, it remains controversial where to set the limits between essential and toxic levels of intake.

Sensitive and accurate indicators of copper toxicity at the level of the gastrointestinal tract are still missing, and those available (e.g., nausea, vomiting, abdominal pain) do not discriminate from other toxicants and respond at high levels of ingested copper (1). Setting the toxic levels is therefore difficult and most information on copper toxicity in humans derive from data collected in clinical cases of acute poisoning (2-4).

The oral absorption of xenobiotics is a critical factor for their fate into the body and is mainly controlled by their passage across the intestinal ephitelium. The barrier function of the intestinal mucosa is guaranteed by the presence and the function of the tight junctions, which are highly organized and finely regulated structures joining adjacent epithelial cells. However, tight junctions do not represent an absolute diffusion barrier and restrict diffusion in a manner that depends on the molecular weight and on the charge of the tracer; furthermore, paracellular permeabilty is regulated by different physiological and pathological stimuli (5-8).

In the present study, we investigated the effects of ionic copper and of copper chelates on the tight junctions' structure and function using a human intestinal in vitro model, the Caco-2 cell line, in order to elucidate the effect of copper on the permeability of the intestinal epithelium and the mechanisms of copper interaction with tight junctions.

In addition, such a study contributes to the establishment of a more sensitive end point of the effect of copper at the intestinal level (i.e., modification in intestinal permeability), which could help toxicologist in establishing upper limits of tolerable copper intake.

2. THE HUMAN INTESTINAL CACO-2 CELL MODEL FOR TRANSPORT AND TOXICITY STUDIES

2.1. The Model

The Caco-2 cell line was established by Fogh and co-workers in 1977 from a human colon adeno-carcinoma and was originally used for the screening of the cytotoxic effects of antitumoral drugs and

From: Handbook of Copper Pharmacology and Toxicology Edited by: E. J. Massaro © Humana Press Inc., Totowa, NJ

for the study of resistance mechanisms (9). Later, it was demonstrated that Caco-2 cells can spontaneously differentiate both morphologically and functionally, when maintained at confluency, into a polarized epithelium resembling that of the mature small intestine (10-13). Differentiated Caco-2 cells develop ultrastructural characteristics, such as functional tight junctions and microvilli on the apical (AP) surface, which are typical of the small intestinal absorptive enterocytes. In addition, during differentiation, the cells progressively express hydrolase activities associated with the AP membrane (sucrase-isomaltase, lactase, aminopeptidase N and dipeptidylpeptidase IV), normally expressed on the microvilli of the absorptive enterocyte of the small intestine. Although a transient expression of these enzyme activities is observed in the fetal human colon around the 15 th wk of gestation, these enzymes are not present in the mature colon (14). Conversely, electrical properties, ionic conductivity, and permeability characteristics of the differentiated Caco-2 cells resemble those of the colon crypt cells (15,16). In addition, transport activities of both small intestinal and colonic type have been observed in Caco-2 cells (17). Overall, some but not all small intestinal functions have been shown to be expressed in the differentiated Caco-2 cell line, leading to the conclusion that these cells, because of their tumoral origin, may not represent a single cytotype, but they exhibit some biochemical characteristics of the normal adult intestine, others of the fetal colon, and others of the normal adult colon (18).

Caco-2 cells can be grown and differentiated on permeable filter supports leading to the formation of a monolayer of differentiated cells joined by functional tight junctions separating the AP medium in the upper chamber from the basolateral (BL) medium in the lower chamber (Fig. 1). Such arrangement mimics the barrier function of the intestinal epithelium, facing the intestinal lumen on the AP side and the blood capillaries and connective tissue on the basal side. Caco-2 cells on filters have been extensively employed to study polarized uptake and transepithelial transport of nutrients and xenobiotics, including several heavy metals (19-26). In addition, in toxicology, this model has frequently been used to study the effect and the metabolism of natural and synthetic compounds (27-31).

Figure 1 shows a schematic representation of the Caco-2 cell monolayer on the polycarbonate filter insert with AP medium on the luminal side and BL medium on the mucosal side. Differentiated Caco-2 cells on the filter can also be viewed in cross-section by optical microscopy after inclusion in polyacrylate resin and staining of 3-pm sections with hematoxylin-eosin. Cells are disposed in a monolayer and they are well polarized with nuclei on the basal side and brush border on the AP side (Fig. 2).

2.2. Cell Culture

The human intestinal Caco-2 cell line was obtained from Alain Zweibaum (INSERM, Villejuif, Paris, France). Caco-2 cells were grown and maintained as previously described (21) in Dulbecco's modified minimum essential medium (DMEM) containing 25 mM glucose, 3.7 g/L NaHCO3, and supplemented with 4 mM L-glutamine, 1% nonessential amino acids (containing 8.9 mg/L L-alanine, 15 mg/L L-asparagine, 13.3 mg/L L-aspartate, 14.7 mg/L L-glycine, 11.5 mg/L L-proline, 10.5 mg/L L-serine), 1 x 105 U/L penicillin, 100 pg/L streptomycin, and 10% heat-inactivated fetal calf serum (complete culture medium). Tissue culture media and supplements were obtained from Euroclone UK Ltd (Pero, Milan, Italy). For experiments monitoring the effects of copper on tight-junction permeability, the cells were seeded on polycarbonate filter cell culture chamber inserts (Transwell, 12 mm diameter, 1.13 cm2 area, 0.45 pm pore diameter; Costar Europe, Badhoevedorp, The Netherlands). Cells were seeded at a density of 4 x 105 cells/cm2 and were allowed to differentiate for 16-19 d after confluency; the medium was regularly changed three times a week.

2.3. Assays of Tight-Junction Permeability

After seeding on the filters, Caco-2 cells start growing and, given the high seeding density, reach confluency within 2 d. After confluency, Caco-2 cells initiate a process of differentiation that lasts

Fig. 1. Schematic view of Caco-2 cells' monolayer cultured on permeable filter culture inserts. Caco-2 cells on polycarbonate filter inserts after differentiation form a monolayer of cells coupled by tight junctions separating the AP from the BL medium. This system allows free access to both compartments and permits studies of the vectorial transport of molecules in both directions and of the toxic effects to the barrier function of the intestinal epithelium.

Fig. 1. Schematic view of Caco-2 cells' monolayer cultured on permeable filter culture inserts. Caco-2 cells on polycarbonate filter inserts after differentiation form a monolayer of cells coupled by tight junctions separating the AP from the BL medium. This system allows free access to both compartments and permits studies of the vectorial transport of molecules in both directions and of the toxic effects to the barrier function of the intestinal epithelium.

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