Abstract

Background: Superoxide (O₂⁻) generation through the activity of reduced nicotinamide dinucleotide (NADH) or reduced nicotinamide dinucleotide phosphate (NADPH) oxidases has been demonstrated in a variety of cell types, but not in human colonic epithelial cells.

Aims: To measure O₂⁻ production and effects of modulators of NAD(P)H oxidase activity and inhibitors of potential O₂⁻ generating enzymes in cultures of human colonic epithelial cells. Expression of the catalytic subunits of NAD(P)H oxidase, Nox1 and gp91^phox (phox, phagocytic oxidase), and the membrane bound subunit p22^phox was assessed.

Methods: The transformed colonic epithelial cell lines (DLD-1, HT-29, and Caco-2) were studied at subconfluence, confluence, and after differentiation. Primary colonic epithelial cells were isolated from mucosal biopsies from the normal human colon. Extracellular O₂⁻ production was measured by the cytochrome c reduction assay or luminol enhanced luminescence. Nox1, gp91^phox, and p22^phox mRNA expression was assessed in colonic epithelial cells and blood neutrophils by reverse transcriptase-polymerase chain reaction.

Results: Production rates of O₂⁻ were higher in subconfluent transformed cells (mean (SEM) 35.8 (4.2) nmol/mg of protein/h) and primary cells (40.4 (5.9)) than in confluent transformed cells (6.0 (0.9); p<0.01). The oxidoreductase inhibitor diphenylene iodonium significantly inhibited O₂⁻ production whereas NADPH and NADH increased production rates. In contrast, O₂⁻ was unaffected by phorbol myristate ester, N^G-nitro-l-arginine methyl ester, indomethacin, or allopurinol. Nox1 mRNA was expressed in all colonic epithelial cells whereas gp91^phox was detected only in HT-29 cells and neutrophils. p22^phox was expressed in all cell types.

Conclusions: Cultures of transformed and primary epithelial cells from human colon may produce extracellular O₂⁻ through an NAD(P)H oxidase expressing Nox1 and p22^phox.