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.
Laboratorio di Tossicologia Comparata ed Ecotossicologia, Istituto Superiore di Sanita', Rome, Italy.
I
I
De Angelis
O
O
Vincentini
G
G
Brambilla
A
A
Stammati
F
F
Zucco
D008841:Actin Cytoskeleton / Q000187:drug effects; D000199:Actins / Q000187:drug effects; D000891:Anti-Infective Agents, Local / Q000378:metabolism / Q000633:toxicity*; D001692:Biological Transport; D018938:Caco-2 Cells / Q000187:drug effects / Q000648:ultrastructure; D016764:Cell Polarity; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005664:Furazolidone / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects; D008871:Microvilli / Q000187:drug effects / Q000648:ultrastructure; D009861:Onium Compounds / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
D000199:Actins; D000891:Anti-Infective Agents, Local; D004791:Enzyme Inhibitors; D009861:Onium Compounds; D005664:Furazolidone; C007517:diphenyleneiodonium