María Sofía Giménez

Mechanism and functional role of XIAP and Mcl-1 down-regulation in flavopiridol/vorinostat antileukemic interactions.

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-κB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat–mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat–mediated transcriptional repression of XIAP, Mcl-1–enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials. [Mol Cancer Ther 2007;6(2):692–702]

Lipid metabolism in liver of rat exposed to cadmium.

We investigated the effect of exposition to cadmium (Cd, 15ppm for 8 weeks) through drinking water on liver lipid metabolism in adult male Wistar rats. As compared to metal non-exposed (control) rats, the serum triglycerides, cholesterol and LDL+VLDL cholesterol concentrations increased. This was associated to a decrease of lipoprotein lipase activity in post heparinic plasma. The VLDL secretion from liver was not modified. Cd treatment increased triglycerides and decreased esterified cholesterol contents in liver. The high triglyceride mass was related to the increased glycerol-3-phosphate acyltransferase mRNA expression. In addition, the liver fatty acids synthesis increased, as determined by an increment of fatty acid synthetase and isocitrate dehydrogenase activities, and [(14)C]-acetate incorporation into saponifiable lipid fraction. The relative percentage of palmitic acid (16:0) and total saturated fatty acids were increased compared with control. Hepatic glucose-6-phosphate dehydrogenase, malic dehydrogenase and cholesteryl ester hydrolase activities were unchanged. In liver, the Cd treatment decreased triglyceride and cholesterol in mitochondria, also increased triglyceride in cytosol, and cholesterol and phospholipid contents in nuclei, compared with control. In addition, an increase of nuclei phosphatidylcholine synthesis was observed. Cd exposure alters directly or indirectly the serum lipid content and liver lipid metabolism.

Myeloperoxidase-induced genomic DNA-centered radicals

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.

Induction of redox changes, inducible nitric oxide synthase and cyclooxygenase-2 by chronic cadmium exposure in mouse peritoneal macrophages☆

Redox changes and the secretion of inflammatory mediators were investigated in resident peritoneal macrophages of mice chronically exposed to cadmium (Cd, 15 ppm for 2 months) through drinking water. Our results showed that in vivo Cd exposure altered the redox balance in mouse peritoneal macrophages, leading to excessive production of reactive oxygen species (ROS) that overwhelmed the antioxidant defenses. It also led to increased lipid peroxidation and arachidonic acid (AA) release, higher nitric oxide and prostaglandin E(2) (PGE(2)) production, and induction of inducible nitric oxide synthase and cyclooxygenase-2 compared with control macrophages. Oxidative stress and inflammation could be important processes operating in the modulation of mouse macrophage physiology induced by chronic Cd exposure.

Vitamin A deficiency modifies lipid metabolism in rat liver

Liver fatty acid metabolism of male rats fed on a vitamin A-deficient diet for 3 months from 21 d of age was evaluated. Vitamin A restriction produced subclinical plasma and negligible liver retinol concentrations, compared with the control group receiving the same diet with 4000 IU vitamin A (8 mg retinol as retinyl palmitate)/kg diet. Vitamin A deficiency induced a hypolipidaemic effect by decreasing serum triacylglycerol, cholesterol and HDL-cholesterol levels. The decrease of liver total phospholipid was associated with low phosphatidylcholine synthesis observed by lower [14C]choline incorporation into phosphatidylcholine, compared with control. Also, liver fatty acid synthesis decreased, as was indicated by activity and mRNA expression of acetyl-CoA carboxylase (ACC), and incorporation of [14C]acetate into saponified lipids. A decrease of the PPARalpha mRNA expression was observed. Liver mitochondria of vitamin A-deficient rats showed a lower total phospholipid concentration coinciding with a decrease of the cardiolipin proportion, without changes in the other phospholipid fractions determined. The mitochondria fatty acid oxidation increased by 30 % of the control value and it was attributed to a high activity and mRNA expression of carnitine palmitoyltransferase-I (CPT-I). An increase in serum beta-hydroxybutyrate levels was observed in vitamin A-deficient rats. Vitamin A deficiency alters the mitochondria lipid composition and also enhances fatty acid oxidation by modifying the production of malonyl-CoA, the endogenous inhibitor of CPT-I, due to decreased activity of liver ACC. The incorporation of vitamin A into the diet of vitamin A-deficient rats reverted all the changes observed.

Lipid modification in mouse peritoneal macrophages after chronic cadmium exposure

The effect of cadmium (Cd) exposure through drinking water on lipid status in mouse peritoneal macrophages (pM) was studied. After 2 months, adult male Balb/c mice that had drunk water with 15 ppm of Cd, showed tissue damage mediated by oxidative stress, as assessed by serum measuring of tissue damage and lipoperoxidation indicators. Resident pM obtained from Cd-exposed mice showed diminution in total lipids, total cholesterol, free cholesterol/esterified cholesterol ratio (FC/EC) and phospholipids in relation to control pM. On a percentage basis, the phospholipid composition showed that phosphatidylcholine (PC) and phosphatidylglycerol decreased, phosphatidylethanolamine (PE) increased, while phosphatidylinositol, sphingomyeline and phosphatidylserine did not change. The incorporation in vitro of [14C]-methyl-choline and [14C]-phosphorylcholine, as well as the activity of regulatory enzyme CTP-phosphocholine cytidylyltransferase, decreased in PC after 60 min. The incorporation of [14C]-linoleic acid increased after 1 h and the incorporation of [14C]-ethanolamine increased after 90 min in PC. The incorporation in vitro of [3H]-cholesterol in total lipids decreased after 120 min of incubation. Besides, the stearic acid and arachidonic acid content increased, while the contents of palmitoleic acid and linoleic acid decreased. Chronic Cd exposure alters the lipid composition in resident pM of Balb/c mice.

Cadmium-induced oxidative stress and histological damage in the myocardium. Effects of a soy-based diet

Cd exposure has been associated to an augmented risk for cardiovascular disease. We investigated the effects of 15 and 100 ppm of Cd on redox status as well as histological changes in the rat heart and the putative protective effect of a soy-based diet. Male Wistar rats were separated into 6 groups and treated during 60 days as follows: groups (1), (2) and (3) were fed a casein-based diet; groups (4), (5) and (6), a soy-based diet; (1) and (4) were given tap water; (2) and (5) tap water containing 15 ppm of Cd²⁺; and (3) and (6) tap water containing 100 ppm of Cd²⁺. Serum lipid peroxides increased and PON-1 activity decreased in group (3). Lipoperoxidation also increased in the heart of all intoxicated groups; however protein oxidation only augmented in (3) and reduced glutathione levels diminished in (2) and (3). Catalase activity increased in groups (3) and (6) while superoxide dismutase activity increased only in (6). Glutathione peroxidase activity decreased in groups (3) and (6). Nrf2 expression was higher in groups (3) and (6), and MTI expression augmented in (3). Histological examination of the heart tissue showed the development of hypertrophic and fusion of cardiomyocytes along with foci of myocardial fiber necrosis. The transmission electron microscopy analysis showed profound ultra-structural damages. No protection against tissue degeneration was observed in animals fed the soy-based diet. Our findings indicate that even though the intake of a soy-based diet is capable of ameliorating Cd induced oxidative stress, it failed in preventing cardiac damage.

Alterations in the Lipid Metabolism of Rat Aorta: Effects of Vitamin A Deficiency

Antioxidants are known to reduce cardiovascular disease by reducing the concentration of free radicals in the vessel wall and by preventing the oxidative modification of low-density lipoproteins. The prooxidative effect of a vitamin-A-deficient diet on the aorta has previously been demonstrated by us. In this study, the lipid metabolism in the aorta of rats fed on a vitamin-A-deficient diet was evaluated. Vitamin A deficiency induced a hypolipidemic effect (lower serum triglyceride and cholesterol levels) and a decreased serum paraoxonase 1/arylesterase activity. The concentrations of triglycerides, total cholesterol, free and esterified cholesterol, and phospholipids were increased in the aorta of vitamin-A-deficient rats. The phospholipid compositions showed an increase in phosphatidylcholine (PC), phosphatidylinositol plus phosphatidylserine and phosphatidylethanolamine, a decrease in sphingomyelin, and no change in phosphatidylglycerol. In the aorta, the increase in triglycerides was associated with an increased fatty acid synthesis and mRNA expression of diacylglycerol acyltransferase 1. The increased PC content was attributed to an increased synthesis, as measured by [methyl-14C]choline incorporation into PC and high CTP:phosphocholine cytidylyltransferase-α mRNA expression. The cholesterol synthesis, evaluated by [1-14C]acetate incorporated into cholesterol and mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, did not change. The lipoprotein lipase and lectin-like oxidized low-density lipoprotein receptor 1 mRNA expression levels increased in the aorta of vitamin-A-deficient animals. The incorporation of vitamin A into the diet of vitamin-A-deficient rats reverted all the changes observed. These results indicate that a vitamin-A-deficient diet,in addition to having a prooxidative effect, alters the aorta lipid metabolism.

Biphasic effect of cadmium in non-cytotoxic conditions on the secretion of nitric oxide from peritoneal macrophages.

The effects of cadmium (Cd) in non-cytotoxic conditions on the nitric oxide (NO) production in peritoneal macrophages (pM) were studied. Peritoneal macrophages from Balb/c mice were incubated over 18 h with 5, 10, 20, or 25 microM Cd2+ (as CdCl2 21:2 H2O) in the culture medium. Concentrations of 20 microM Cd2+ and over had cytotoxic effects, measured by MTT assay. Cell viability with 10 microM Cd2+ in the medium was above 90% after 18 h of incubation, and above 80% after 72 h. At this same Cd2+ concentration, NO production increased from 6 to 18 h. At 24 h production decreased but was still above control levels. At 48 h production NO was near control levels, and continued to decrease until the end of the experiment (72 h). NO levels produced with Cd2+ concentrations of 5, 10 and 20 microM in the medium were above the control at 18 h. NO production and lipoperoxidation increased simultaneously after 18 h with 10 microM of Cd in the medium. Amounts of inducible nitric oxide synthase (iNOS) protein and iNOS activity also increased. At a concentration of 10 microM Cd has a biphasic effect on NO production over time.

Alterations in the lipid content of pituitary gland and serum prolactin and growth hormone in cadmium treated rats.

The present study was undertaken to assess whether chronic exposition to cadmium (Cd, 0.133 mM per liter for 2 months) through drinking water may affect the lipid contents in the pituitary anterior lobe (PAL) of adult male Wistar rats. As compared to metal non-exposed controls, PALs exposed to cadmium showed an increase in total phospholipid contents, which was associated to an increase of the incorporation of [1−14C]-methyl choline into phosphatidylcholine and of [U−14C]-glucose into total phospholipids. The incorporation of [1−14C]-methyl choline into sphingomyelin was not changed. Incorporation of [1−14C]-acetate into total fatty acids also increased but incorporation of [1−14C]-acetate into cholesterol did not change. The activity of phospholipase D decreased both in PALs from Cd exposed rats and in PAL dispersed cells treated with Cd in the culture medium from Cd non-exposed rats. In PALS from Cd exposed rats, a decrease of serum prolactin and growth hormone concentrations was determined. The results shown that cadmium modifies the lipid contents of pituitary gland and directly or indirectly the levels of prolactin and growth hormone in serum.