Theodore A. Sarafian

Oxidative stress modulates osteoblastic differentiation of vascular and bone cells

Oxidative stress may regulate cellular function in multiple pathological conditions, including atherosclerosis. One feature of the atherosclerotic plaque is calcium mineral deposition, which appears to result from the differentiation of vascular osteoblastic cells, calcifying vascular cells (CVC). To determine the role of oxidative stress in regulating the activity of CVC, we treated these cells with hydrogen peroxide (H(2)O(2)) or xanthine/xanthine oxidase (XXO) and assessed their effects on intracellular oxidative stress, differentiation, and mineralization. These agents increased intracellular oxidative stress as determined by 2,7 dichlorofluorescein fluorescence, and enhanced osteoblastic differentiation of vascular cells, based on alkaline phosphatase activity and mineralization. In contrast, H(2)O(2) and XXO resulted in inhibition of differentiation markers in bone osteoblastic cells, MC3T3-E1, and marrow stromal cells, M2-10B4, while increasing oxidative stress. In addition, minimally oxidized low-density lipoprotein (MM-LDL), previously shown to enhance vascular cell and inhibit bone cell differentiation, also increased intracellular oxidative stress in the three cell types. These effects of XXO and MM-LDL were counteracted by the antioxidants Trolox and pyrrolidinedithiocarbamate. These results suggest that oxidative stress modulates differentiation of vascular and bone cells oppositely, which may explain the parallel buildup and loss of calcification, seen in vascular calcification and osteoporosis, respectively.

INVITED COMMENTARY IS APOPTOSIS MEDIATED BY REACTIVE OXYGEN SPECIES

Apoptosis is a common mode of programmed cell death occurring during development as well as in many pathological conditions, in which the cell plays an active role in its own demise. Although the morphological and biochemical hallmarks of apoptosis are conserved across phyla and cell type, the mechanism(s) of apoptosis is unknown. However, data recently published demonstrate that expression of the anti-apoptotic gene bcl-2 decreases the net cellular generation of reactive oxygen species, and that reactive oxygen species serve as mediators of apoptosis in at least some cases.

Oxidative mechanisms underlying methyl mercury neurotoxicity

Cerebellar granule cells from 5–12‐day‐old rats can be incubated in suspension at 37°C for up to 3 hr with minimal decline in viability. Methyl mercury was found to produce time‐ and concentrationdependent cell killing with >85% cell death after 3 hr exposure to a concentration of 20 μM. Previously characterized inhibition of protein and RNA synthesis as well as known methyl mercury‐induced defects in cellular ATP production have been shown to be incapable of causing this degree of cell death. Here we report that methyl mercury induced a concentration‐dependent increase in membrane lipoperoxidation and a rapid decline in reduced glutathione in this cerebellar neuronal preparation. Hydrogen peroxide at 5 mM was found to closely reproduce each of the cytotoxic effects manifested by methyl mercury suggesting that oxidizing conditions produced by methyl mercury may account for the observed cell death. Methyl mercury‐induced lipoperoxidation was not the cause of cell death since malonaldehyde production could be blocked by α‐tocopherol or EDTA without appreciably protecting against cell death. Significant protection from methyl mercury‐induced cell death was observed, with EGTA, deferoxamine and KCN. We propose that oxidative events contribute to the toxic mechanism of action of methyl mercury in isolated cerebellar granule neurons.

bcl-2 expression decreases methyl mercury-induced free-radical generation and cell killing in a neural cell line

Methyl mercury neurotoxicity is associated with a broad range of neuropathologic and biochemical disturbances which include induction of oxidative injury. Treatment of the hypothalamic neural cell line GT1-7 with 10 microM methyl mercury (MeHg) for 3 h resulted in increased formation of reactive oxygen species (ROS), and decreased levels of reduced glutathione (GSH), associated with 20% cell death. Cells transfected with an expression construct for the anti-apoptotic proto-oncogene, bcl-2, displayed attenuated ROS induction and negligible cell death. Twenty-four-h exposure to 5 microM MeHg killed 56% of control cells, but only 19% of bcl-2-transfected cells. By using diethyl maleate to deplete cells of GSH, we demonstrate that the differential sensitivity to MeHg was not due solely to intrinsically different GSH levels. The data suggest that MeHg-mediated cell killing correlates more closely with ROS generation than with GSH levels and that bcl-2 protects MeHg-treated cells by suppressing ROS generation.

Respiratory and Immunologic Consequences of Marijuana Smoking

Habitual smoking of marijuana has a number of effects on the respiratory and immune systems that may be clinically relevant. These include alterations in lung function ranging from no to mild airflow obstruction without evidence of diffusion impairment, an increased prevalence of acute and chronic bronchitis, striking endoscopic findings of airway injury (erythema, edema, and increased secretions) that correlate with histopathological alterations in bronchial biopsies, and dysregulated growth of the bronchial epithelium associated with altered expression of nuclear and cytoplasmic proteins involved in the pathogenesis of bronchogenic carcinoma. Other consequences of regular marijuana use include ultrastructual abnormalities in human alveolar macrophages along with impairment of their cytokine production, antimicrobial activity, and tumoricidal function. Cannabinoid receptor expression is altered in leukocytes collected from the blood of chronic smokers, and experimental models support a role for Δ9‐tetrahydrocannabinol in suppressing T cell function and cell‐mediated immunity. The potential for marijuana smoking to predispose to the development of respiratory malignancy is suggested by several lines of evidence, including the presence of potent carcinogens in marijuana smoke and their resulting deposition in the lung, the occurrence of premalignant changes in bronchial biopsies obtained from smokers of marijuana in the absence of tobacco, impairment of antitumor immune defenses by Δ9‐tetrahydrocannabinol, and several clinical case series in which marijuana smokers were disproportionately overrepresented among young individuals who developed upper or lower respiratory tract cancer. Additional well‐designed epidemiological and immune monitoring studies are required to determine the potential causal relationship between marijuana use and the development of respiratory infection and/or cancer.

DIFFERENTIAL EXPRESSION OF PEROXIREDOXIN SUBTYPES IN HUMAN BRAIN CELL TYPES

The peroxiredoxin (Prx) protein is expressed widely in animal tissues and serves an antioxidant function associated with removal of cellular peroxides. We have cloned two Prx genes and observed differential expression of Prx-I and Prx-II (formerly NKEF-A and NKEF-B) in purified rat brain cell cultures (Sarafian et al. [1998] Mol. Chem. Neuropathol. 34:39-51). We have examined regional and cell-type-specific expression of Prx-I and Prx-II in paraffin sections of human brain using immunohistochemical methods. These studies revealed a clear segregation of expression of these two gene products in different brain cell types. In the cerebral cortex, cerebellum, basal ganglia, substantia nigra, and spinal cord, Prx-I was expressed primarily in astrocytes, while Prx-II was expressed exclusively in neurons. Prx-I was also prominently expressed in ependymal cells and subependymal matrix of substantia nigra and basal ganglia. Prx-II was not expressed at uniform density in all neurons. In general, small neurons such as cerebellar granule neurons displayed little or no staining, while large neurons, such as hippocampal pyramidal and Purkinje neurons were heavily stained. The absence of expression of Prx-I in neurons and the selective expression of Prx-II in large neurons suggest that these antioxidant enzymes serve distinct functional roles that may reflect the different functions and biochemical activities of these cell types. Restricted expression of these genes may also contribute to the selective vulnerability of these cells to a wide variety of neuropathologic conditions.

Disruption of Astrocyte STAT3 Signaling Decreases Mitochondrial Function and Increases Oxidative Stress In Vitro

Background Astrocytes exert a wide variety of functions in health and disease and respond to a wide range of signaling pathways, including members of the Janus-kinase signal transducers and activators of transcription (Jak-STAT) family. We have recently shown that STAT3 is an important regulator of astrocyte reactivity after spinal cord injury in vivo [1]. Methodology/Principal Findings Here, we used both a conditional gene deletion strategy that targets the deletion of STAT3 selectively to astrocytes (STAT3-CKO), and a pharmacological inhibitor of JAK-2, AG490, in cultured astrocytes in vitro, to investigate potential functions and molecules influenced by STAT3 signaling in relation to mitochondrial function and oxidative stress. Our findings show that the absence of STAT3 signaling in astrocytes leads to (i) increased production of superoxide anion and other reactive oxygen species and decreased level of glutathione, (ii) decreased mitochondrial membrane potential and decreased ATP production, and (iii) decreased rate of cell proliferation. Many of the differences observed in STAT3-CKO astrocytes were distinctly altered by exposure to rotenone, suggesting a role for complex I of the mitochondrial electron transport chain. Gene expression microarray studies identified numerous changes in STAT3-CKO cells that may have contributed to the identified deficits in cell function. Conclusions/Significance Taken together, these STAT3-dependent alterations in cell function and gene expression have relevance to both reactive gliosis and to the support and protection of surrounding cells in neural tissue.

Methyl Mercury Increases Intracellular Ca2+ and Inositol Phosphate Levels in Cultured Cerebellar Granule Neurons

Abstract: In an effort to explain the previously observed methyl mecury (MeHg)‐induced stimulation of protein phosphorylation in cerebellar granule neuron cultures, the effect of MeHg on protein kinase activities in cell‐free assays and on second messenger systems in cultured neurons has been examined. Using cell‐free assays for several protein kinases, no stimulation of enzyme activity was found at any concentration of MeHg tested. After 24 h exposure, 1–5 μM MeHg was found to have no significant effect on neuronal cyclic AMP levels. In contrast, intracellular levels of Ca2+ and rates of 45Ca2+ uptake were elevated 2.2‐fold and 3.6‐fold, respectively, by 5 μM MeHg. These effects were not observed with mercuric chloride, triethyllead, or lead acetate. Measurement of inositol phosphate production in granule cell cultures revealed a sensitive, pretoxic effect of MeHg with twofold stimulation following 30‐min exposure to 5 μM MeHg and 1.6‐fold after 24‐h exposure to 3 μM MeHg. Detection of inositol phosphate production after 30 min of MeHg was largely neuron‐specific. These results suggest that second messenger‐mediated activation of select protein kinase enzymes may be the mechanism underlying MeHg‐induced stimulation of protein phosphorylation in cerebellar neuronal culture. In addition, these findings indicate a specific interference with neuronal signal transduction and suggest a basis for the selective neurotoxic action of this agent.

Modulation of Pulmonary Leukotriene B4 Production by Cyclooxygenase-2 Inhibitors and Lipopolysaccharide

Purpose: Emerging data continue to link carcinogenesis to inflammatory events involving the eicosanoid metabolic pathways. We therefore evaluated the effects of cyclooxygenase (COX)-2 inhibition on leukotriene (LT) B4 synthesis in the lungs of active smokers, as part of a pilot lung cancer chemoprevention study with celecoxib (Celebrex), an oral COX-2 inhibitor. Experimental Design: Bronchoalveolar lavage was performed before celecoxib treatment and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from ex-smoker control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. Results: Treatment with oral celecoxib only modestly increased LTB4 levels in bronchoalveolar lavage, without increasing the mRNA transcription of 5-lipoxygenase (5-LOX) or 5-LOX-activating protein in AMs, whereas the acute calcium ionophore-stimulated LTB4 production from smokers’ AMs was markedly increased by 10.6-fold. In addition, smokers’ AMs were twice as responsive in producing LTB4 when exposed to lipopolysaccharide compared with ex-smokers’ AMs. Concomitant COX-2 inhibition with SC58236, however, did not significantly impact these changes, whereas the 5-LOX inhibitor Zileuton blocked the generation of LTB4 in a dose-responsive manner. Finally, cycloheximide increased the production of LTB4 under all conditions, suggesting a shunting phenomenon and/or the presence of pathway inhibitors. Conclusions: Our findings suggest that whereas oral celecoxib is capable of modulating LTB4 production in the lung microenvironment, under physiologic conditions, this effect is probably not functionally significant.

Retinoic Acid-Induced Differentiation of Human Neuroblastoma: A Cell Variant System Showing Two Distinct Responses

Retinoic acid (RA) has been shown to induce the differentiation of human neuroblastoma cells in vitro. In this study, we describe two variants of the SK-N-SH human neuroblastoma cell line that have dramatically different responses to RA. RA induces neuronal-like differentiation characterized by extensive neurite outgrowth, thick neurite bundles, and large cellular aggregates of SK-N-SH-N (SH-N) cells. In contrast, RA treatment of SK-N-SH-F (SH-F) cultures transforms the small neuroblast cells into large flattened, fibroblastic or epithelial-like cells. Karyotype analysis verified that the SH-N and SH-F cultures were derived from a common precursor cell. Confirmation of their markedly different responses to RA was obtained by metabolic labelling of glycoproteins and SDS-PAGE analysis. While both sublines showed very similar Coomassie-labelled protein bands and glycoprotein profiles in control cultures, dramatic differences between the lines were revealed following RA treatment. In contrast to their similar protein profiles, untreated SH-N and SH-F cells had quite different patterns of ganglioside biosynthesis in that GM3 was detected in SH-F cells but not in SH-N, while GM1 was only detected in SH-N. Cellular RA binding protein (CRABP) was detected in both SH-F and SH-N cells and their RA-transformed derivatives. These results demonstrate heterogeneity in the response to RA of neuroblastoma cells derived from a common origin that cannot be accounted for by differences in CRABP content. The SH-N and SH-F neuroblastoma sublines should provide a useful system for further studies of the molecular processes through which RA exerts its differentiation-inducing activity on this type of tumor.