Pergamon 0887-2333@4)002124 Toxic. in Vifro Vol. 9, No. 3, 251-255, pp. 1995 Copyright 0 1995 ElsevicrScienceLtd Printed zyxwvutsrqponmlkjihgfedcbaZY in Great Britain. All rights reserved 0887-2333/95 89.50 + 0.00 Cytotoxic Effect of Prolamin-derived Peptides on In V&o Cultures of Cell Line Caco-2: Implications for Coeliac Disease C. GIOVANNINI, L. MAIURI* and M. DE VINCENZIt Department of Metabolism and Pathological Biochemistry, Istituto Superiore di SanitP, Viale Regina Elena 299, 00161 Rome and *University Federico II of Naples, Via S. Panzini 5, 80131 Naples, Italy (Accepted 29 November 1994) Abstract-The cytotoxic effects of various prolamin-derived peptides’on Caco-2 cells were investigated by measuring the alterations of several parameters at different stages of cell differentiation. The PT digest of bread wheat was active in inhibiting cell proliferation (by about 50%). whereas the other digests from durum wheat, maize and bovine serum albumin (BSA) did not affect the proliferating activity of cells. Compared with the control, colony-forming ability was inhibited by 20% by treatment with cereals that are toxic in coeliac disease (bread wheat, rye, oats and barley). BSA and maize peptides are devoid of this zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in vifro effect. However, the decrease in alkaline phosphatase activity during Caco-2 cell differentiation was observed in the presence of bread wheat. This could be due to slowing down of the enterocytic differentiation of cells that are susceptible to interaction with toxic peptides. Therefore, long-term cultures of Caco-2 cells constitute a useful in vitro model to assess the ability of cereal proteins to damage the coeliac small intestine. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA INTRODUCTION The mechanism by which wheat gliadins produce damage to the coeliac small intestine is still unknown. Most investigators favour the hypothesis that dereg- ulated immune responses to gliadin underlie the abnormality to coeliac disease (CD) patients (Strober, 1979). However, non-immunomediated cytotoxic activity of gliadin peptides on the small intestine and other cells might also be involved in the pathogenesis of mucosal damage, and evidence exists to support this hypothesis. Gliadin peptides reversibly inhibited development and morphogenesis of the very immature small intestine of 17-day-old rat foetuses, whereas they had no effect on in vitro cultured differentiated jejunum from 21-day-old rat foetuses. This therefore suggests that gliadin peptides may directly damage the small intestine mucosa only during an early phase of its morphogenesis (Auricchio et al., 1982; de Ritis et al., 1979). Another biological activity of gliadin peptides that is probably related to their toxicity for CD patients is their ability to agglutinate undifferentiated human K 562 cells, a line of chronic myeloid leukaemia (Auricchio et al., 1985). tAuthor for correspondence. Abbreviations: AP = aDica1; BL = basolateral; BSA = bovine serum albumin; CD = coeliac disease; DMEM = Dulbecco’s modified Eagle’s medium; PBS = phosphate buffered saline. The culture of organs from untreated CD patients has also been proposed as an in vitro model of CD (Falchuck et al., 1974; Jos et al., 1975). Gliadin peptides are able to inhibit the improvement of intestinal epithelium after in vitro culture. Both non- immunomediated cytotoxic activity and immunologi- cal mechanisms could be responsible for this phenomenon. These three in vitro models have been used not only to study the mechanisms underlying the coeliac lesion, but also to identify toxic proteins and pep- tides. In in vivo and/or in vitro studies on the coeliac intestine, the toxic activity of a large series of proteins, mixtures of peptides and pure A-gliadin peptides appears to correlate very well with the cell agglutinating activity and the damaging effect on the in vitro developing foetal rat intestine. This suggests that unknown mechanism(s) related to the direct cytotoxic activity of gliadin peptides on undifferenti- ated cells may be involved in the pathogenesis of the intestinal lesion in CD. In this study we investigated the potentiality of another in vitro system as a possible model to study the mechanism of toxicity in CD-the long-term culture of Caco-2 cells, a line derived from human colon adenocarcinoma, which is capable of express- ing some differentiated characteristics of small intestine enterocytes. The cytotoxic effects of various prolamin-derived peptides have been detected on Caco-2 cells by measuring the alterations of several parameters at different stages of cell differentiation. 251