Krishnaswamy Kannan

Oxidative stress and apoptosis.

Apoptosis or programmed cell death, is essential for the normal functioning and survival of most multi-cellular organisms. The morphological and biochemical characteristics of apoptosis, however, are highly conserved during the evolution. It is currently believed that apoptosis can be divided into at least three functionally distinct phases, i.e. induction, effector and execution phase. Recent studies have demonstrated that reactive oxygen species (ROS) and the resulting oxidative stress play a pivotal role in apoptosis. Antioxidants and thiol reductants, such as N-acetylcysteine, and overexpression of manganese superoxide (MnSOD) can block or delay apoptosis. Bcl-2, an endogenously produced protein, has been shown to prevent cells from dying of apoptosis apparently by an antioxidative mechanism. Taken together ROS, and the resulting cellular redox change, can be part of signal transduction pathway during apoptosis. It is now established that mitochondria play a prominent role in apoptosis. During mitochondrial dysfunction, several essential players of apoptosis, including pro-caspases, cytochrome C, apoptosis-inducing factor (AIF), and apoptotic protease-activating factor-1 (APAF-1) are released into the cytosol. The multimeric complex formation of cytochrome C, APAF-1 and caspase 9 activates downstream caspases leading to apoptotic cell death. All the three functional phases of apoptosis are under the influence of regulatory controls. Thus, increasing evidences provide support that oxidative stress and apoptosis are closely linked physiological phenomena and are implicated in pathophysiology of some of the chronic diseases including AIDS, autoimmunity, cancer, diabetes mellitus, Alzheimers and Parkinsons and ischemia of heart and brain.

Ketosis (acetoacetate) can generate oxygen radicals and cause increased lipid peroxidation and growth inhibition in human endothelial cells

Elevated level of cellular lipid peroxidation can increase the incidence of vascular disease. The mechanism by which ketosis causes accelerated cellular damage and vascular disease in diabetes is not known. This study was undertaken to test the hypothesis that elevated levels of ketone bodies increase lipid peroxidation in endothelial cells. Human umbilical venous endothelial cells (HUVEC) were cultured for 24 h at 37 degrees C with ketone bodies (acetoacetate, beta-hydroxybutyrate). Acetoacetate, but not beta-hydroxybutyrate, caused an increase in lipid peroxidation and growth inhibition in cultured HUVEC. To determine whether ketone bodies generate oxygen radicals, studies using cell-free buffered solution were performed. They showed a significant superoxide dismutase (SOD) inhibitable reduction of cytochrome C by acetoacetate, but not by beta-hydroxybutyrate, suggesting the generation of superoxide anion radicals by acetoacetate. Additional studies show that Fe2+ potentiates oxygen radical generation by acetoacetate. Thus, elevated levels of ketone body acetoacetate can generate oxygen radicals and cause lipid peroxidation in endothelial cells, providing a possible mechanism for the increased incidence of vascular disease in diabetes.

Effect of elevated glucose concentrations on cellular lipid peroxidation and growth of cultured human kidney proximal tubule cells.

This study has examined whether elevated glucose can induce lipid peroxidation and contribute to the inhibition of cell growth in human kidney proximal tubule(HPT) cells. HPT cells were cultured in media containing glucose concentrations of 8 mM (control), 25 mM, and 50 mM. Lipid peroxidation was assessed by the thiobarbituric acid reactivity and cell growth was assessed by 3H-thymidine uptake. Results show decreased (59%, p < 0.01) growth of HPT cells cultured in 50 mM glucose. Cells cultured in 50 mM mannitol did not show any growth inhibition, suggesting that the decreased cell growth associated with glucose is not due to osmolarity changes. There was an increase (108%, p < 0.02) in lipid peroxidation in cells cultured with high levels of glucose (50 mM) compared with controls and cells cultured with 50 mM mannitol. To examine if membrane lipid peroxidation or malondialdehyde (MDA, an end product of lipid peroxidation) has any role in the inhibition of cell growth, we examined the effect of tertiary butylhydroperoxide (TBH, known to cause lipid peroxidation and generate MDA) on the growth of HPT cells. TBH decreased cell growth (49, 17 and 3% of controls at 0.1, 0.25, and 0.5 [mole TBH/ml medium). Similarly, a marked reduction in the growth was observed with exogenous MDA (72, 69 and 34% of controls at 0.1, 0.25, and 0.5 μmole MDA/ml medium). This suggests that elevated glucose can induce membrane lipid peroxidation and accumulation of MDA, which in turn can inhibit cellular growth and contribute to the altered structure and function of HPT cells in diabetes.

Alteration in lymphocyte phenotype associated with administration of adjuvant levamisole and 5-fluorouracil

Levamisole (LMS) and 5-fluorouracil (5FU) administered adjuvantly are effective in reducing the relapse rate following surgical resection of Dukes stage C colon carcinoma. It has been postulated that LMS acts to stimulate the immune system and that this is one mechanism through which this drug exerts its antitumor effects. In this study, peripheral blood mononuclear cells (PBMC) were analyzed in nine patients with surgically resected colon carcinoma prior to initiation of adjuvant LMS/5FU and at several subsequent times while patients were on therapy. Changes in lymphocyte phenotype and soluble interleukin-2 receptor (sIL-2R) between pre-study samples and samples obtained during adjuvant LMS/5FU were evaluated. Significant increases were seen in the proportion of PBMC expressing natural killer (NK) antigen CD56 (14.7±2.4% versus 18.1±2.6%;P<0.05) and surface IL-2R (CD25; 0% versus 0.42±0.15%;P<0.05), in sIL-2R (314±86 U/ml versus 736±173 U/ml;P<0.05), and in the CD4∶CD8 ratio (2.34±0.93 versus 3.47±1.23;P<0.01). A significant decrease in the proportion of CD8+ PBMC (24.7±3.8% versus 18.8±2.6%;P<0.01) and total CD8+ PBMC (537±118 versus 324±37;P<0.01) was seen. The increase in CD56+ cells correlated with sIL2R levels (r=0.46;P<0.05). No changes were noted for CD3, CD4, CD5, CD14, CD16, CD19, CDw49a, or TCRδ. The greatest increase in CD56+ cells and the smallest reduction in CD8+ cells were seen in the subgroup of patients who remained disease-free following adjuvant chemotherapy. This study suggests that adjuvant LMS/5FU has significant stimulatory effects on the immune system, which correlate with patient outcome and may account at least in part for its clinical efficacy.

Ketosis and the generation of oxygen radicals in diabetes mellitus.

The long-term complications of diabetes remain a major public health issue. Over the last decade, many of the biochemical pathways by which hyperglycemia may cause cellular damage have been studied. These include increased polyol pathway and associated changes in intracellular redox state, increased diacylglycerol synthesis with consequent activation of specific protein kinase C isoforms, increased nonenzymatic glycation of both intra-and extracellular proteins, and increased oxidative stress (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Tissue injury then results from acute changes in protein function and chronic changes in protein expression. However, the molecular pathophysiology of altered membrane function and gene expression leading to tissue injury is still unclear. Type 1 diabetics frequently experience ketosis (hyperketonemia) because, in a state of insulin deficiency, body fuel is derived mainly from fat (12). The blood concentration of ketone bodies may reach 10 mM in diabetics with severe ketosis, compared with concentrations of less than 0.5 mM in normal individuals (12). It is known that ketosis can accelerate microangiopathy and underlying vascular disease and precipitate neuropathy in patients with long-duration diabetes (12). However, the underlying mechanisms by which ketosis promotes vascular disease in type 1 diabetic patients are unclear This chapter is focussed on the mechanisms that underlie the accelerated vascular disease and mortality in diabetic patients. To better differentiate among the complex interactions of various cell types, hormones and dynamic changes in the blood, we have used a cell culture model to accomplish the stated objectives. Specifically, this review discusses whether ketosis increases cellular oxidative stress/damage, and thereby promotes cell surface changes and adhesion between the monocytes and endothelial cells, a crucial step in the pathogenesis of vascular disease in diabetes.