Carol A. Rhoads

Lipid hydroperoxide-induced apoptosis in human colonic CaCo-2 cells is associated with an early loss of cellular redox balance

Apoptosis plays a critical role in maintaining homeostasis of the intestinal epithelium. Dietary oxidants like peroxidized lipids could perturb cellular redox status and disrupt mucosal turnover. The objective of this study was to delineate the role of lipid hydroperoxide (LOOH) ‐induced redox shifts in intestinal apoptosis using the human colonic CaCo‐2 cell. We found that subtoxic concentrations of LOOH increased CaCo‐2 cell apoptosis. This LOOH‐induced apoptosis was associated with a significant decrease in the ratio of reduced glutathione‐to‐oxidized glutathione (GSH/GSSG), which preceded DNA fragmentation by 12 to 14 h, suggesting a temporal relationship between the two events. Oxidation of GSH with the thiol oxidant diamide caused significant decreases in cellular GSH and GSH/GSSG at 15 min that correlated with the activation of caspase 3 (60 min) and cleavage of PARP (120 min), confirming a temporal link between induction of cellular redox imbalance and initiation of apoptotic cell death. These kinetic studies further reveal that oxidant‐mediated early redox change (within 1 h) was a primary inciting event of the apoptotic cascade. Once initiated, the recovery of redox balance did not prevent the progression of CaCo‐2 cell apoptosis to its biological end point at 24 h. Collectively, the study shows that subtoxic levels of LOOH disrupt intestinal redox homeostasis, which contributes to apoptosis. These results provide insights into the mechanism of hydroperoxide‐induced mucosal turnover that have important implications for understanding oxidant‐mediated genesis of gut pathology.–Wang, T.‐G., Gotoh, Y., Jennings, M. H., Rhoads, C. A., Aw, T. Y. Lipid hydroperoxide‐induced apoptosis in human colonic CaCo‐2 cells is associated with an early loss of cellular redox balance. FASEB J. 14, 1567–1576 (2000)

Chronic Exposure to Subtoxic Levels of Peroxidized Lipids Suppresses Mucosal Cell Turnover in Rat Small Intestine and Reversal by Glutathione

Oxidative challenge can compromise intestinal growth and death responses. This study examines the effect of chronic consumption of subtoxic levels of peroxidized lipids on intestinal redox balance and turnover and the effect of glutathione (GSH) supplementation. Male Sprague-Dawley rats were fed standard rat chow or 4% peroxidized menhaden oil chow (2–8 weeks). Intestinal GSH and glutathione disulfide (GSSG), GSH synthetic and redox enzymes as well as proliferative (ornithine decarboxylase, ODC) and apoptotic activities were evaluated. Chronic peroxide intake did not affect overall animal growth, but decreased intestinal GSH/GSSG ratio that directly correlated with decreased GSH and increased GSSG, and suppressed peak circadian ODC activities and postprandial mucosal apoptosis. Supplementation with GSH restored the mucosal GSH/GSSG ratio and abrogated the peroxide-induced suppression of intestinal cell turnover. Collectively, the results show that chronic lipid peroxide consumption induces intestinal GSH redox imbalance that interferes with regulation of enterocyte death and proliferation in vivo. These disruptive effects of lipid peroxides were reversed by GSH supplementation in accordance with the normalization of tissue GSH/GSSG redox balance.

Lipid peroxide-induced redox imbalance differentially mediates CaCo-2 cell proliferation and growth arrest.

Abstract. Dietary oxidants like lipid hydroperoxides (LOOH) can perturb cellular glutathione/glutathione disulphide (GSH/GSSG) status and disrupt mucosal turnover. This study examines the effect of LOOH on GSH/GSSG balance and phase transitions in the human colon cancer CaCo‐2 cell. LOOH at 1 or 5 µm were noncytotoxic, but disrupted cellular GSH/GSSG and stimulated proliferative activity at 6 h that paralleled increases in ornithine decarboxylase activity, thymidine incorporation, expression of cyclin D1/cyclin‐dependent kinase 4, phosphorylation of retinoblastoma protein, and cell progression from G0/G1 to S. At 24 h, LOOH‐induced sustained GSH/GSSG imbalance mediated growth arrest at G0/G1 that correlated with suppression of proliferative activity and enhanced oxidative DNA damage. LOOH‐induced cell transitions were effectively blocked by N‐acetylcysteine. Collectively, the study shows that subtoxic LOOH levels induce CaCo‐2 GSH/GSSG imbalance that elicits time‐dependent cell proliferation followed by growth arrest. These results provide insights into the mechanism of hydroperoxide‐induced disruption of mucosal turnover with implications for understanding oxidant‐mediated genesis of gut pathology.

THE ROLE OF GSH EFFLUX IN STAUROSPORINE-INDUCED APOPTOSIS IN COLONIC EPITHELIAL CELLS

Staurosporine (STP) was shown to induce cell apoptosis through formation of reactive oxygen species, but a role for cellular redox has not been defined. In this study, we report that STP (2 microM) caused apoptosis (24+/-3% at 24 h) of human colon adenocarcinoma epithelial cell line HT29 that was preceded by significant glutathione (GSH) and glutathione disulfide (GSSG) efflux (6 h), but independent of changes in cellular glutathione/glutathione disulfide (GSH/GSSG) redox status. The blockade of GSH efflux by gamma-glutamyl glutamate (gamma-GG) or ophthalmic acid was associated with apoptosis attenuation; however, gamma-GG administration after peak GSH efflux (8 h) did not confer cytoprotection. Moreover, lowering cellular GSH through inhibition of its synthesis prevented extracellular GSH accumulation and cell apoptosis, thus validating a link between cellular GSH export and the trigger of cell apoptosis. Inhibition of gamma-glutamyl transferase (GGT1, EC 2.3.2.2)-catalyzed extracellular GSH degradation with acivicin significantly blocked GSH efflux, suggesting that GSH breakdown is a driving force for GSH export. Interestingly, acivicin treatment enhanced extracellular GSSG accumulation, consistent with GSH oxidation. STP-induced HT29 cell apoptosis was associated with caspase-3 activation independent of caspase-8 or caspase-9 activity; accordingly, inhibitors of the latter caspases were without effect on STP-induced apoptosis. STP similarly induced GSH efflux and apoptosis in a non-malignant human NCM460 colonic cell line in association with caspase-3 activation. Collectively, our results demonstrate that STP induction of apoptosis in malignant and non-malignant colonic cells is temporally linked to the export of cellular GSH and the activation of caspase-3 without caspase-8 or -9 involvement.

Glucose regulation of hydroperoxide metabolism in rat intestinal cells. Stimulation of reduced nicotinamide adenine dinucleotide phosphate supply.

The regulation of intestinal metabolism of t-butylhydroperoxide by glucose was examined in isolated enterocytes from proximal rat intestine. The basal rate of hydroperoxide elimination in control cells was 0.57 +/- 0.05 nmol/min per 10(6) cells, and was increased threefold by 10 mM exogenous glucose (1.74 +/- 0.14 nmol/min per 10(6) cells). Concurrently, cellular NADPH levels increased threefold (1.62 +/- 0.40 nmol/10(6) cells vs 0.57 +/- 0.14 nmol/10(6) cells in controls). The glucose effect was blocked by 6-aminonicotinamide and by 1,3-bis-(2-chloroethyl) 1-nitrosourea, consistent with glucose stimulation of NADPH production by the pentose phosphate shunt, and of NADPH utilization for glutathione disulfide reduction. The NADPH supply rate was quantified by controlled infusions of diamide, a thiol oxidant. At diamide infusion of 0.05 nmol/min per 10(6) cells, GSH and protein thiols in control cells were decreased significantly, consistent with a limited capacity for glutathione disulfide reduction. With glucose, cell GSH and protein thiols were preserved at a 10-fold higher diamide infusion which was reversed by 6-aminonicotinamide, supporting the view that glucose promotes glutathione disulfide reduction by increased NADPH supply. Collectively, the results demonstrate that intestinal metabolism of hydroperoxides subscribes to regulation by glucose availability. This responsiveness to glucose suggests that nutrient availability would be an important contributing factor in the detoxication of toxic hydroperoxides by the small intestine.

NFκB Signaling in Posthypoxic Endothelial Cells: Relevance to E-Selectin Expression and Neutrophil Adhesion

Our previous studies have implicated the nuclear transcription factor ĸB (NFĸB) in the regulation of adhesion molecule expression in endothelial cells exposed to anoxia-reoxygenation (A/R) or a redox imbalance. The objectives of this study were (1) to define the kinetics of NFĸB activation by examining IĸBα degradation and the nuclear translocation of p65 in response to A/R or redox imbalance (induced by treatment of cells with diamide and buthionine sulfoximine) and (2) to determine whether the signal for IĸBα degradation, nuclear translocation of p65, and E-selectin-mediated neutrophil adhesion is related to the activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTPase) and/or protein kinase C (PKC). The results demonstrate that both A/R and redox imbalance led to IĸBα degradation within 30 min and the concomitant appearance of p65 in the nucleus, consistent with rapid cytosolic activation of NFĸB and subsequent nuclear translocation of the activated p65 subunit. Inhibition of PKC blocked IĸBα degradation and p65 translocation in A/R-challenged, but not redox-altered, endothelial cells. However, both A/R- and redox-induced NFĸB activation was blocked by inhibition of PTK. Similarly, A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion were blocked by inhibition of PKC or PTK, while only PTK inhibited the redox-induced adhesion response. Pretreatment of cells with N-acetyl cysteine effectively blocked A/R- or redox-induced IĸB degradation and significantly attenuated the respective neutrophil adhesion responses. Collectively, these findings indicate that A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion are mediated by both PKC and PTK, which signal rapid activation of NFĸB. This A/R-induced NFĸB signaling response appears to be mediated, at least in part, by intracellular redox imbalance.