Hibernation induces glutathione redox imbalance in ground squirrel intestine.

Glutathione (GSH) is the major thiol-disulfide redox buffer in cells and is a critical component of antioxidant defense. Here we examined GSH redox balance in the intestinal mucosa during the annual cycle of 13-lined ground squirrels (Spermophilus tridecemlineatus). The ratio of reduced GSH to its oxidized form (glutathione disulfide, GSSG), which is an index of oxidative stress, was five-fold lower in hibernating compared with summer-active squirrels, an effect due primarily to elevated GSSG concentration in hibernators. During hibernation the total pool of GSH equivalents was lowest in squirrels undergoing arousal and highest in squirrels during interbout arousals. Hibernation decreased intestinal GSSG reductase activity by approximately 50%, but had no effect on activities of glutathione peroxidase or glucose-6-phosphate dehydrogenase. Within the hibernation season, expression of the stress protein HSP70 in intestinal mucosa was highest in squirrels entering torpor and early in a torpor bout, and lowest in squirrels arousing from torpor and during interbout euthermia. The results suggest that hibernation in ground squirrels is associated with a shift in intestinal GSH redox balance to a more oxidized state. Higher levels of HSP70 during the early phases of torpor may reflect induction of the stress response due to aberrations in protein folding or may be a mechanism to increase enterocyte tolerance to subsequent stress imposed by extended torpor or the arousal process.

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells.

Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 microm cysteine and/or 200-400 microm cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 mm BSO treatment significantly increased cell proliferation as measured by 3H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G1 phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G1-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.

NF kappa b signaling in posthypoxic endothelial cells: relevance to E-selectin expression and neutrophil adhesion.

Our previous studies have implicated the nuclear transcription factor kappa B (NF kappa B) 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 NF kappa B activation by examining I kappa B alpha 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 I kappa B alpha 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 I kappa B alpha degradation within 30 min and the concomitant appearance of p65 in the nucleus, consistent with rapid cytosolic activation of NF kappa B and subsequent nuclear translocation of the activated p65 subunit. Inhibition of PKC blocked I kappa B alpha degradation and p65 translocation in A/R-challenged, but not redox-altered, endothelial cells. However, both A/R- and redox-induced NF kappa B 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 I kappa B 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 NF kappa B. This A/R-induced NF kappa B signaling response appears to be mediated, at least in part, by intracellular redox imbalance.

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.

Determinants of hydroperoxide detoxification in diabetic rat intestine: effect of insulin and fasting on the glutathione redox cycle.

The capacity for hydroperoxide detoxification in diabetic (DM) intestine was studied in streptozocin-induced DM rats by quantification of the intestinal glutathione (GSH) redox cycle, a key cellular pathway for peroxide elimination. A role for luminal glucose in regulation of redox cycle activity was examined in insulin-treated or 24-hour-fasted DM animals. Intestinal activities of the redox enzymes, GSH peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase (G6PD), were significantly decreased by 17 hours' insulin treatment, whereas only G6PD was decreased by fasting. Mucosal GSH levels were also markedly decreased under these conditions. These results are consistent with an overall suppression of intestinal GSH redox cycle function by short-term administration of insulin. Insulin treatment for 7 consecutive days increased hepatic G6PD activity by fourfold but was without effect on intestinal G6PD, suggesting tissue specificity in insulin regulation of G6PD. The rate of metabolism of tert-butyl hydroperoxide (tBH) in isolated enterocytes was low in the absence of substrates (0.51 +/- 0.07 nmol/10(6) cells/min) but was increased fivefold by exogenous glucose (2.70 +/- 0.11 nmol/10(6) cells/min), indicating that glucose availability is an important contributor to intestinal detoxification of toxic hydroperoxides. Collectively, the current results show that GSH redox cycle enzymes in DM intestine are under coordinate insulin control, and that this control appears to be downregulated by short-term insulin treatment.

Ethanol as a possible cofactor in the development of murine AIDS.

Chronic ethanol (EtOH) abuse in humans leads to a variety of immunomodulatory events that can alter resistance to a number of infectious agents. Whether alcohol abuse affects the susceptibility to human immunodeficiency virus infection or the subsequent development of acquired immune deficiency syndrome (AIDS) is a matter of extreme importance; however, available information in humans or animal models is limited. The goal of this study was to evaluate the effect of chronic EtOH feeding in mice on the development of immunodeficiency in the murine model of AIDS (MAIDS). C57BI/6 mice were placed on the Lieber-DeCarli liquid EtOH diet (25% or 31% total caloric intake) or a nutrient-matched isocaloric liquid control diet. Seven days later, mice were infected with the LP-BM5 murine leukemia virus mixture, and groups of infected and noninfected mice were assayed at defined time points postinfection for antigen-specific and nonspecific immune responses. In the absence of retroviral infection, chronic EtOH feeding (5-8 weeks) led to reductions in spleen weights, compared with isocaloric controls. In spite of reduced spleen size, mitogenic responses of spleen cells to concanavalin A (ConA) and lipopolysaccharide (LPS) were elevated in EtOH-fed mice, as compared with mice fed the control diet. Chronic EtOH feeding also enhanced the allogeneic mixed lymphocyte response and increased antigen-specific priming of both B-cells and CD4+ T-cells to the antigen, sheep red blood cells. In MAIDS-infected mice, chronic EtOH feeding delayed but did not prevent the onset of virus-induced immunodeficiency and MAIDS-induced autoantibody synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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.