Formaldehyde metabolism and formaldehyde‐induced stimulation of lactate production and glutathione export in cultured neurons

Formaldehyde is endogenously produced in the human body and brain levels of this compound are elevated in neurodegenerative conditions. Although the toxic potential of an excess of formaldehyde has been studied, little is known on the molecular mechanisms underlying its neurotoxicity as well as on the ability of neurons to metabolize formaldehyde. To address these topics, we have used cerebellar granule neuron cultures as model system. These cultures express mRNAs of various enzymes that are involved in formaldehyde metabolism and were remarkably resistant toward acute formaldehyde toxicity. Cerebellar granule neurons metabolized formaldehyde with a rate of around 200 nmol/(h × mg) which was accompanied by significant increases in the cellular and extracellular concentrations of formate. In addition, formaldehyde application significantly increased glucose consumption, almost doubled the rate of lactate release from viable neurons and strongly accelerated the export of the antioxidant glutathione. The latter process was completely prevented by inhibition of the known glutathione exporter multidrug resistance protein 1. These data indicate that cerebellar granule neurons are capable of metabolizing formaldehyde and that the neuronal glycolysis and glutathione export are severely affected by the presence of formaldehyde.     

Formaldehyde stimulates Mrp1-mediated glutathione deprivation of cultured astrocytes

Ionizing radiations can induce oxidative stress on target tissues, acting mainly through reactive oxygen species (ROS). The aim of this work was to investigate if 17-β-estradiol (βE) was able to prevent hippocampal-related behavioral and biochemical changes induced by neonatal ionizing radiation exposure and to elucidate a potential neuroprotective mechanism. Male Wistar rats were irradiated with 5 Gy of X-rays between 24 and 48 h after birth. A subset of rats was subcutaneously administered with successive injections of βE or 17-α-estradiol (αE), prior and after irradiation. Rats were subjected to different behavioral tasks to evaluate habituation and associative memory as well as anxiety levels. Hippocampal ROS levels and protein kinase C (PKC) activity were also assessed. Results show that although βE was unable to prevent radiation-induced hippocampal PKC activity changes, most behavioral abnormalities were reversed. Moreover, hippocampal ROS levels in βE-treated irradiated rats approached control values. In addition, αE administered to irradiated animals was effective in preventing radiation-induced alterations. In conclusion, βE was able to counteract behavioral and biochemical changes induced in irradiated animals, probably acting through an antioxidant mechanism.     

Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules

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Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures

Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2 h with arsenate in concentrations of up to 10 mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a time- and concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3 mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity.     

Formaldehyde induces rapid glutathione export from viable oligodendroglial OLN-93 cells

Formaldehyde is a neurotoxic environmental pollutant that can also be produced in the body by certain enzymatic reactions. To test for the potential consequences of an exposure of oligodendrocytes to formaldehyde, we used OLN-93 cells as a model system. Treatment with formaldehyde altered the cellular glutathione (GSH) content of these cells by inducing a rapid time- and concentration-dependent export of GSH. Half-maximal effects were observed for a formaldehyde concentration of about 0.2 mM. While the basal GSH efflux from OLN-93 cells was negligible even when the cellular GSH content was doubled by pre-incubation of the cells with cadmium chloride, the formaldehyde-stimulated export increased almost proportionally to the cellular GSH content. In addition, the stimulated GSH export required the presence of formaldehyde and was almost completely abolished after removal of the aldehyde. Analysis of kinetic parameters of the formaldehyde-induced GSH export revealed similar K_m and V_max values of around 100 nmol/mg and 40 nmol/(h mg), respectively, for both OLN-93 cells and cultured astrocytes. The transporter responsible for the formaldehyde-induced GSH export from OLN-93 cells is most likely the multidrug resistance protein 1 (Mrp1), since this transporter is expressed in these cells and since the inhibitor MK571 completely prevented the formaldehyde-induced GSH export. The rapid export of GSH from formaldehyde-treated viable oligodendroglial cells is likely to compromise the cellular antioxidative and detoxification potential which may contribute to the known neurotoxicity of formaldehyde.     

Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells

Adriamycin-resistant (AdrR) human breast cancer cells have been selected which exhibit cross-resistance to a wide range of anti-cancer drugs. This multidrug-resistant phenotype is associated with increases in the activities of glutathione peroxidase and glutathione transferase. The 45-fold increase in glutathione transferase activity is associated with the appearance of a new anionic isozyme in AdrR cells which is immunologically related to the anionic glutathione transferase present in human placenta. The increase in transferase and the level of drug resistance is relatively stable during passage of AdrR cells in the absence of adriamycin for over 10 months. A similar anionic glutathione transferase isozyme is also found in rat hyperplastic liver nodules, a preneoplastic state resulting from exposure to carcinogens. A rat cDNA which codes for the anionic glutathione transferase in rat hyperplastic nodules hybridizes to a 1.1-kilobase pair mRNA which is overexpressed in the AdrR MCF-7 cells. The anionic transferase has been purified from the AdrR cells and found to have characteristics which distinguish it from other anionic human glutathione transferases, including high levels of intrinsic peroxidase activity. The overexpression of a similar anionic glutathione transferase in human breast cancer cells selected for multidrug resistance and in rat hyperplastic liver nodules, which develop resistance to various hepatotoxins, suggests a possible role for this drug-conjugating enzyme in the mechanism of resistance in both of these states.