A toxicogenomic data space for system-level understanding and prediction of EDC-induced toxicity.

Endocrine disrupting compounds (EDCs) are a persistent threat to humans and wildlife due to their ability to interfere with endocrine signaling pathways. Inspired by previous work to improve chemical hazard identification through the use of toxicogenomics data, we developed a genomic-oriented data space for profiling the molecular activity of EDCs in an in silico manner, and for creating predictive models that identify and prioritize EDCs. Predictive models of EDCs, derived from gene expression data from rats (in vivo and in vitro primary hepatocytes) and humans (in vitro primary hepatocytes and HepG2), achieve testing accuracy greater than 90%. Negative test sets indicate that known safer chemicals are not predicted as EDCs. The rat in vivo-based classifiers achieve accuracy greater than 75% when tested for invitro to in vivoextrapolation. This study reveals key metabolic pathways and genes affected by EDCs together with a set of predictive models that utilize these pathways to prioritize EDCs in dose/time dependent manner and to predict EDCevokedmetabolic diseases.

PDGF-C and -D and their receptors PDGFR-alpha and PDGFR-beta in atherosclerotic human arteries.

BACKGROUND: Platelet derived growth factors (PDGFs) are mitogens for fibroblasts and smooth muscle cells. This growth factor family contains four members PDGF-A, PDGF-B, PDGF-C and PDGF-D. Biology of recently discovered PDGF-C and PDGF-D is not well-established. Here we studied the expression of PDGF-C and PDGF-D and their receptors PDGFR-alpha and PDGFR-beta in normal and atherosclerotic human arteries. MATERIALS AND METHODS: Human arterial samples from amputations and autopsies were classified according to the atherosclerotic stage and the expression of PDGF-C and PDGF-D proteins and their receptors was studied by immunohistochemistry. In situ hybridization and reverse transcriptase-PCR were used to study mRNA expression. RESULTS: Both growth factors were expressed in medial smooth muscle cells (SMCs) in normal arteries and atherosclerotic lesions. However, clear differences were found in the expression profiles in endothelium: PDGF-C was strongly expressed in endothelial cells in both normal arteries and lesions whereas PDGF-D was only weakly expressed in endothelium. PDGF-C expression was very prominent in lesion macrophages. PDGF-D was expressed throughout the artery wall in lesions. PDGFR-alpha expression was strong in endothelium and in lesion macrophage-rich areas, whereas PDGFR-beta was mostly expressed in SMCs. CONCLUSIONS: Our results suggest that PDGF-C may play an important role in endothelium in normal and atherosclerotic arteries and in macrophages in lesions. PDGF-D was expressed in all types of lesions with the same intensity and thus differs from the expression of PDGF-C.

Oxidative stress-inducible lentiviral vectors for gene therapy.

Oxidative stress is important in several pathologies, including cardiovascular diseases such as atherosclerosis and cardiac ischemia-reperfusion injury. An important mechanism for adaptation to oxidative stress is induction of genes through the antioxidant response element (ARE), which regulates the expression of antioxidant and cytoprotective genes via the transcription factor Nrf2 (nuclear factor E2-related factor 2). As Nrf2-regulated genes are induced during oxidant stress occurring, for example, in reperfusion after ischemia, we took a novel approach to exploit ARE for the development of oxidative stress-inducible gene therapy vectors. To this end, one, two or three ARE-containing regions from human NAD(P)H:quinone oxidoreductase-1, glutamate-cysteine ligase modifier subunit and mouse heme oxygenase-1 were cloned into a vector expressing luciferase under a minimal SV40 promoter. The construct, which was the most responsive to ARE-inducing agents, was chosen for further studies in which a lentiviral vector was produced for an efficient transfer to endothelial cells. Heme oxygenase-1 (HO-1), which has well-characterized anti-inflammatory properties, was used as the therapeutic transgene. In human endothelial cells, ARE-driven HO-1 overexpression inhibited nuclear factor-kappaB activation and subsequent vascular cell adhesion molecule-1 expression induced by tumor necrosis factor-alpha. We conclude that the ARE element is a promising alternative for the development of oxidative stress-inducible gene therapy vectors.

Biphasic effects of 15-deoxy-delta(12,14)-prostaglandin J(2) on glutathione induction and apoptosis in human endothelial cells.

The lipid products derived from the cyclooxygenase pathway can have diverse and often contrasting effects on vascular cell function. Cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, have been reported to cause endothelial cell apoptosis, yet in other cell types, cyPGs induce cytoprotective mediators, such as heat shock proteins, heme oxygenase-1, and glutathione (GSH). Herein, we show in human endothelial cells that low micromolar concentrations of 15d-PGJ(2) enhance GSH-dependent cytoprotection through the upregulation of glutamate-cysteine ligase, the rate-limiting enzyme of GSH synthesis, as well as GSH reductase. The effect of 15d-PGJ(2) on GSH synthesis is attributable to the cyPG structure but is independent of PPAR, inasmuch as the other cyPGs, but not PPARgamma or PPARalpha agonists, are able to increase GSH. The increase in cellular GSH is accompanied by abrogation of the proapoptotic effects of 4-hydroxynonenal, a product of lipid peroxidation present in atherosclerotic lesions. However, higher concentrations of 15d-PGJ(2) (10 micromol/L) cause endothelial cell apoptosis, which is further enhanced by depletion of cellular GSH by buthionine sulfoximine. We propose that the GSH-dependent cytoprotective pathways induced by 15d-PGJ(2) contribute to its antiatherogenic effects and that these pathways are distinct from those leading to apoptosis.

Mechanisms of the pro- and anti-oxidant actions of nitric oxide in atherosclerosis.

The association of nitric oxide (NO) with cardiovascular disease has long been recognized and the extensive research on this topic has revealed both pro- and anti-atherosclerotic effects. While these contradictory findings were initially perplexing recent studies offer molecular mechanisms for the integration of these data in the context of our current understanding of the biochemistry of NO. The essential findings are that the biochemical properties of NO allow its exploitation as both a cell signaling molecule, through its interaction with redox centers in heme proteins, and an extremely rapid reaction with other biologically relevant free radicals. The direct reaction of NO with free radicals can have either pro- or antioxidant effects. In the cell, antioxidant properties of NO can be greatly amplified by the activation of signal transduction pathways that lead to the increased synthesis of endogenous antioxidants or down regulate responses to pro-inflammatory stimuli. These findings will be discussed in the context of atherosclerosis.

Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases.

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

Down-regulation of renal glutathione synthesis by systemic nitric oxide synthesis inhibition in spontaneously hypertensive rats.

Nitric oxide stimulates in vitro the synthesis of glutathione, an abundant thiol with a number of functions such as detoxification of xenobiotics and reactive oxygen species. In order to study this relationship in an animal model of hypertension, we treated spontaneously hypertensive rats (SHR) either with a nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) or with a nitric oxide donor isosorbide-5-mononitrate (IS-5-MN). Inhibition of nitric oxide synthesis led to malignant hypertension and to a marked decrease in glutathione synthesis through down-regulation of the rate-limiting enzyme gamma-glutamylcysteine synthetase (GCS). The reduction in GCS activity was further augmented in SHR on a high sodium diet. Renal GCS activity in untreated SHR was 234 +/- 14 and 240 +/- 18 nmol/min/mg protein (mean +/- SD) on a low and high sodium diet, respectively. When L-NAME was included in the diet, the activities dropped to 173 +/- 28 and 123 +/- 28 for the low and high sodium diets, respectively. IS-5-MN attenuated the rise in blood pressure induced by sodium chloride, but did not affect the GCS activity. The mechanism of GCS stimulation by nitric oxide is not known, but our results combined with the literature suggest that a relatively high concentration of nitric oxide is needed.

Thiol metabolism in preterm infants during the first week of life.

BACKGROUND: Oxidative stress is implicated in the pathogenesis of several complications of prematurity. The glutathione cycle is one of the most important intracellular antioxidant systems. The synthesis of glutathione may not be adequate in preterm neonates because of the low levels of cysteine available. The aim of this study was to evaluate cysteine and glutathione metabolism during the first week of life in preterm infants. METHODS: Plasma and erythrocyte thiol concentrations were measured in 78 preterm infants with a birthweight of 500-1500 g, and erythrocyte glutamate-cysteine ligase (GCL), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferases (GST) and glucose 6-phosphatedehydrogenase (G6PDH) in 26 infants with a birthweight of 1000-1500 g. RESULTS: The mean (SD) plasma glutathione concentration increased from day 0 to day 1 (14.9 (7.1) vs. 27.7 (11.9) micromol/L, p < 0.001), and then decreased. The plasma cysteine concentration changed in the opposite direction (172 (59) vs. 129 (42) micromol/L, p < 0.01). In infants with respiratory distress syndrome (RDS) the mean plasma glutathione concentration, but not cysteine, was lower on day 0 compared with infants without RDS (11.7 (5.2) vs. 21.4 (5.6) micromol/L, p < 0.01). Erythrocyte glutathione concentration decreased during the first week of life, whereas erythrocyte cysteine concentration increased significantly from day 3 to day 7 (p < 0.01). Erythrocyte cysteine and glutathione concentrations had a positive correlation. The GCL and GR activities did not change, but GST and G6PDH activities decreased during the first week (p < 0.01). GPx activity decreased until day 3 (p < 0.01) and was higher on day 0 and day 1 in infants with RDS. CONCLUSIONS: Very low birthweight infants have an initial increase in plasma glutathione and initial decrease in plasma cysteine level during the first week of life, and also a positive correlation between erythrocyte cysteine and glutathione levels.

Expression of gamma-glutamylcysteine synthetase during development.

Prematurity has been associated with low glutathione (GSH) concentrations in bronchoalveolar lavage fluid as well as in leukocytes from tracheal aspirates and peripheral blood. To elucidate whether this is caused by deficient GSH synthesis, the expression and activity of gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, GCS, EC 6.3.2.2), the rate-limiting enzyme for GSH synthesis, were measured from fetal, neonatal, and adult human liver, lung, and kidney samples. The highest activity was measured in the liver, in which mRNA expression of the catalytic GCS heavy and the regulatory light subunits, as well as activity, were, on average, similar in the various stages of development. Although GCS light subunit mRNA concentrations in the lung were higher in neonates than in fetuses and adults, enzyme activities were similar. In the adult kidney, mean enzyme activity was somewhat higher than in fetal or neonatal kidney, but this may be accounted for by the variation in the small number of samples. In conclusion, GCS is expressed and active already in the second trimester and thus low GSH concentrations found in preterm neonates appear not to be explained by deficient GSH synthesis. Other factors, such as limited availability of the GSH precursor cysteine or increased GSH consumption, may account for the lower concentrations of GSH found in preterm infants.

Human cystathionine gamma-lyase: developmental and in vitro expression of two isoforms.

Cystathionine gamma-lyase (CGL) is the last enzyme of the trans-sulphuration pathway, which converts methionine into cysteine. To study the possible differences in enzymic activity of the two human cystathionine gamma-lyase isoforms characterized earlier, these were separately expressed in human kidney embryonic 293T cells. Furthermore, developmental changes in the expression of the two mRNA forms as well as the enzymic activity in human liver were studied, as it has been postulated that a change in the relative expression of CGL isoforms causes the postnatal increase in CGL activity. Transfection with the longer isoform increased the CGL activity 1.5-fold, while the activity of the cells transfected with the shorter form did not differ from the basal activity. In human liver samples, CGL activity was only detected in adult tissue (68+/-9 nmol of cysteine/h per mg of protein), whereas activity in fetal, premature and full-term neonatal liver tissue was undetectable. In contrast, strong mRNA expression of both mRNA isoforms was detected from the 19th gestational week onwards and the longer form of CGL appeared to be predominant. The expression of the two mRNA forms varied in parallel. In conclusion, we have shown that only cells overexpressing the longer form of CGL have increased activity, and CGL appears to be regulated at the post-transcriptional level during development.

Cellular redox state and its relationship to the inhibition of clonal cell growth and the induction of apoptosis during all-trans retinoic acid exposure in acute myeloblastic leukemia cells.

BACKGROUND AND OBJECTIVE: All-trans retinoic acid (ATRA) induces growth arrest and apoptosis in acute myeloblastic leukemia (AML) cells. Since cellular redox state regulates these events, we were interested in studying whether it has any role in the responsiveness of AML cells to ATRA. DESIGN AND METHODS: Two human AML cell lines, the ATRA-sensitive OU-AML-3, and the ATRA-resistant OU-AML-7, were used as models. Clonogenic cell culture assay, annexin V method, and measurement of mitochondrial membrane potential were used for the determination of cell growth and apoptosis. Peroxide formation was analyzed by flow cytometry, glutathione and g-glutamylcysteine synthetase (g-GCS) activity was determined spectrophotometrically, and the expression of manganese superoxide dismutase (MnSOD) by Western blotting. RESULTS: ATRA inhibited clonogenic cell growth and induced apoptosis particularly in OU-AML-3 cells. The OU-AML-7 cells had a higher basal level of glutathione and g-GCS activity than the OU-AML-3 cells. ATRA enhanced the generation of peroxides after 24h exposure, which was more prominent in the sensitive than the resistant cell line and was not preventable by N-acetyl-L-cysteine. ATRA also increased the activity of g-GCS, which was associated with increased intracellular glutathione in the resistant cell line, while the glutathione level was maintained in the sensitive cell line. During ATRA exposure, MnSOD was induced in the sensitive cell line, but not until after 72 h. Buthionine sulfoximine significantly increased the inhibitory effect of ATRA on colony formation in both cell lines, but only marginally enhanced the effect of ATRA on the induction of apoptosis. INTERPRETATION AND CONCLUSIONS: The balance between oxidative and antioxidative actions of ATRA, as well as the basal redox state of the cells seem to have a definite influence on the responsiveness of AML cells to ATRA.

Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.