Russell P. Saneto

Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line

Catalase, superoxide dismutase, and dimethylsulfoxide were tested for their ability to prevent the cytotoxic effect of 6-hydroxydopamine (6-OHDA) on the human neuroblastoma line SY5Y. Viability was measured at two time points after 6-OHDA treatment: at 3 hr by means of amino acid incorporation and at 24 hr by trypan blue dye exclusion. Survival of cells treated concomitantly with catalase (50 microgram/ml) and 6-OHDA was at least 90 per cent that of untreated controls. Cells receiving 6-OHDA alone showed less than 30 per cent survival relative to untreated controls. Superoxide dismutase (50 microgram/ml) temporarily protected cells from a high concentration of 60-OHDA. Dimethylsulfoxide treatment increased survival from the control level 24 hr after treatment with 6-OHDA. Two other cell lines (A1B1 human glial cells and CHO fibroblasts) had intermediate and high resistance to the drug, respectively, compared to the low resistance of SY5Y cells. CHO and SY5Y cells had similar responses to 6-OHDA and to H2O2 when tested at twice the molarity of 6-OHDA. Specific activities of three enzymes known to detoxify H2O2 or H2O2-generated organic hydroperoxides (catalase, glutathione S-transferase, and glutathione peroxidase) were compared in the three cell lines. Catalase activity was 2.5 times as high as in A1B1 and CHO cells as in SY5Y cells when expressed as units/mg protein and 7 times as high in units/culture dish. Other enzyme activities showed no correlation to 6-OHDA resistance.

Purification and properties of bovine lens glutathione S-transferase

Abstract Glutathione S-transferase (EC 2.5.1.18) has been implicated in the detoxification of drugs and atmospheric pollutants bearing an electrophylic group. The presence of glutathione S-transferase (GSH S-transferase) has been demonstrated for the first time in bovine ocular lens and cornea. The one major species of GSH S-transferase from bovine lens has been purified to homogeneity. The enzyme has a molecular weight of 49 000 and is a dimer of equal size subunits. Kinetic properties and substrate specificity of the enzyme have been studied. The lens GSH S-transferase is probably present as a single species which is highly deamidated but catalytically active. GSH S-transferase of bovine lens does not have any GSH-peroxidase (GSH-Px II) activity which has been shown to be present in rat liver GSH S-transferase.

Mercapturic acid pathway enzymes in bovine ocular lens, cornea, retina and retinal pigmented epithelium

Analogous to the liver, ocular tissues contain large concentrations of glutathione and are exposed to potentially damaging chemical compounds. Since glutathione has been shown to have a detoxification function, via mercapturic acid production in the liver, we investigated whether glutathione has a similar function in ocular tissues. We have demonstrated the presence of all of the enzymes involved in the mercapturic acid pathway i.e. glutathione S-transferase, gamma-glutamyl transpeptidase, cysteinylglycinase, and N-acetyl transferase, in the ocular tissues of bovine lens, cornea, retina, and retinal pigmented epithelium. Therefore glutathione may have another function in ocular tissues, that of the detoxification of xenobiotics.

Lithium Uptake at Physiological Ion Concentrations in a Human Clonal Neuroblastoma Cell Line

Lithium is perhaps the most widely used drug in the therapy of acute mania and in the prevention of depression recurrence (Gershon and Shopsin, 1973). The probable site of its therapeutic action is the central nervous system; however, the mechanism of action of lithium, as well as its transport into neuronal cells, is poorly understood. Lithium transport has in the past been studied using human erythrocytes, where at least three transport components of lithium influx have been demonstrated (Pandey et al., 1978). The ratio of the net erythrocyte to plasma lithium content has been suggested as an index to differentiate lithium-responsive from nonresponsive patients (Mendels and Frazer, 1973). Although there are conflicting results, it is likely that there is an inheritable lithium transport component which affects the ratio of erythrocyte to plasma lithium content (Mendels and Frazer, 1973; Pandey et al., 1977; Mendlewicz and Verbanck, 1977). To investigate lithium transport, a more recent approach has been to use cultured mouse neuroblastoma cells, where significant differences in the components of lithium transport were found when compared to human erythrocytes (Richelson, 1977). A more meaningful approach might be to study neurons of human origin in cell culture. We have, therefore, investigated lithium influx in a human neuroblastoma clonal line which is nearly diploid and is known to express many in vivo adrenergic neuronal functions (Perez-Polo et al., 1979). Whereas human erythrocytes have been shown to have a ouabain-sensitive component, a phloretin-sensitive component, and a component of lithium transport not sensitive to either drug, C1300 mouse neuroblastoma cells lack the ouabain-sensitive component (Richelson, 1977: Pandey et al., 1978). In contrast to this, we wish to report that human neuroblastoma cells cultured at physiological ion concentrations exhibit a ouabain-sensitive component of lithium uptake. However, there were significant differences in this ouabain-sensitive component as compared to erythrocytes. We would propose this system as a more relevant model system for the study of lithium transport components in target tissues relevant to lithium therapy. The SY-5Y clone of human neuroblastoma cell line SK-N-SH was grown in nutrient media F-12 (GIBCO) with 15% fetal calf serum (GIBCO) in the presence of humidified 1.5% CO, at 37°C. The nutrient media were changed for new media every other day. For each experiment, the cells were plated at a density of 200,000 cells per 60-mrn Petri dish (Falcon) and grown to confluency. Cells were washed three times in Hanks’ baianced salt solution (HBSS), pH 7.2, and incubated in 2.5 ml of the appropriate incubation media for 10 min. The incubation medium was HBSS containing 10 mM-LiC1 with or without 0.1 mwouabain andlor phloretin, pH 7.2; phloretin was dissolved in a small amount of 0.1 M-NaOH and brought to a final concentration of 0.1 mM with the incubation medium. The HBSS contains physiological concentrations of Na+, K+, and C1-. The medium was removed by careful suction, and the cells washed three additional times. Cells were then scraped off the Petri dishes in the presence of 2 ml of 5% trichloroacetic acid, sonicated, and centrifuged at 600 x g for 5 min. The supernatant was assayed for lithium content by atomic absorption spectrophotometry (Perkin-Elmer model 303) and protein was determined by a modification of the method of Lowry et al. (1951). Lithium transport components were selectively investigated by using the drugs ouabain and phloretin (Pandey et al., 1978). Ouabain is known to specifically inhibit the sodium-potassium (Na, K) ATPase, and K+ reversibly inhibits ouabain binding (Choi and Akera, 1977; Matsui et al., 1977). Phloretin probably associates with high affinity to plasma membrane proteins and with low affinity to membrane lipids, and is insensitive to K+ (Jennings et al., 1976; Pandey et al., 1978). Therefore, ouabain and phloretin have been suggested to have different sites of action on the cell membrane. Since lithium uptake by the SY-5Y cells was linear for 20 min, a 10-min incubation period was used in all experiments. In the presence of HBSS the uptake of lithium by the SY-5Y cells was significantly inhibited by 0.1 KIMouabain (n = 6 , p < 0.0005), whereas 0.1 mM-phloretin

Purification and characterization of glutathione S-transferases from the bovine cornea

We have purified two cationic (pI 7·2 and >10·0) forms of glutathione S-transferase from the bovine cornea through a combination of gel filtration, affinity chromatography, and isoelectric focusing. Both these glutathione S-transferases conjugate glutathione (GSH) to 1-chloro-2,4-dinitrobenzene, have an absolute requirement of GSH as the thiol donor, and do not express selenium-independent glutathione peroxidase activity. The conjugation of GSH to 1-chloro-2,4-dinitrobenzene catalyzed by the less cationic transferase is inhibited by bilirubin. Both glutathione S-transferases of the cornea have a broader spectrum of substrates that are conjugated to GSH than the glutathione S-transferases of bovine lens and retina.

Interferon inhibition of hexose monophosphate shunt activity as the mechanism of blocking differentiation of a preadipose cell line.

Mouse L cell interferon (IFN αβ) inhibited the differentiation of mouse 3T3-Li fibroblasts into adipocytes. IFN αβ also inhibited hexose monophosphate shunt (HMP) activity in these cells. HMP activity is required for the reducing power necessary for the conversion of 3T3-Li fibroblasts to adipocytes. Both IFN blockage of differentiation and HMP activity were reversed by the reducing agent 2-mercaptoethanol, probably through interaction with membrane receptors and not through direct inactivation of IFN. Several non-antiviral effects of IFN αβ on cellular function, including differentiation and immunoregulation, may be mediated at the biochemical level through blockage of HMP activity.

Dissociation of protein kinase activity and the induction of the antiviral state in a cell line responsive to the antiviral effects of interferon

Abstract Interferons or oxidized glutathione were found to induce double-stranded RNA-dependent protein kinase activity in mouse L cells that phosphorylates the α subunit of eukaryotic peptide initiation factor 2. A mixture of leukocyte/fibroblast interferons as well as immune interferon induced the protein kinase and also suppressed virus replication in the L cells. Oxidized glutathione was equally effective in inducing protein kinase activity, but it did not induce an antiviral state in these cells. The data suggest that a simple cause and effect relationship does not exist between protein kinase induction and the establishment of the antiviral state in a cell that is responsive to the antiviral effects of interferon.