Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb2+) in immortalized mouse hypothalamic GT1-7 neuronal cells.

We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1-7 cells, it clearly enhances Pb2+-induced cytotoxicity. It is likely that Pb2+ must enter cells to exert most of its toxic effects. Pb2+ is known to substitute for Ca2+ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb2+ into GT1-7 neuronal cells with a special focus on the role of extracellular calcium (Ca2+), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb2+ (1 microM or 10 microM), i.e. without any external stimulus, clearly increased in nominally Ca2+-free buffer and was partially abolished by 13 mM Ca2+ when compared to uptake in the presence of a physiological concentration of extracellular Ca2+ (1.3 mM). Depolarization by 25 mM K+, or antagonists of VSCCs, verapamil (10 microM) or flunarizine (10 microM) had no clear effect on basal Pb2+ uptake. Glutamate (1 mM) increased Pb2+ uptake, but only when cells were treated with 1 microM Pb2+ in the presence of 1.3 mM Ca2+. Our data suggest that Pb2+ competes for the same cellular uptake pathways with Ca2+, although not via VSCCs. In addition, enhancement of Pb2+-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb2+.

Effects of 2-ethylhexanoic acid on the production of reactive oxygen species in human polymorphonuclear leukocytes in vitro.

2-Ethylhexanoic acid (2-EHA), is an industrial chemical and a toxic biotransformation product of the plasticizer di(2-ethylhexyl)phthalate. Its immunological effects are unknown. 2-EHA resembles structurally C18 fatty acids, which are known activators of respiratory burst in human polymorphonuclear leukocytes (PMNL). Therefore, we exposed PMNL to 2-EHA in vitro and measured the production of reactive oxygen species (ROS) and explored the associated cellular mechanisms. 2-EHA (10-2000 microM) inhibited dose-dependently formyl-methionyl-leucyl-phenylalanine (FMLP)-induced respiratory burst in PMNL. Moreover, 2-EHA decreased oxidative burst evoked by the protein kinase C (PKC) activators, phorbol myristate acetate (PMA) and dioctanoyl-s,n-glycerol (DIC(8)). 2-EHA affected neither the levels of free intracellular calcium nor inhibited PKC. The results indicate that 2-EHA inhibits activation of PMNL to produce ROS, i.e. has an immunosuppressive effect in vitro. The site of action in the PKC is after activation of this enzyme.

Production of reactive oxygen species by man-made vitreous fibres in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) or erythrocytes, isolated from human blood, were exposed to graded doses of asbestos (chrysotile), quartz, or man-made vitreous fibres (MMVF), i.e. refractory ceramic fibres (RCF), glasswool, or rockwool fibres. None of the MMVF affected either the viability of PMNL, as measured by trypan blue exclusion test, or induced haemolysis, whereas the positive controls, quartz and chrysotile, dose-dependently induced haemolysis in PMNL. MMVF did not increase the release of lactate dehydrogenase (LDH) from the PMNL, whereas the positive controls, chrysotile and quartz, induced a marked and dose-dependent release of LDH. When PMNL were exposed to MMVF, some of the fibre types slightly increased the levels of free intracellular calcium ([Ca2+]i) within the cells in a manner similar to that induced by chrysotile or quartz. All MMVF induced a dose-dependent production of reactive oxygen species (ROS) in PMNL, with RCF-induced production of ROS being the most marked. Production of ROS by MMVF seemed to depend on the availability of extracellular calcium because it could be attenuated with a Ca2+ channel blocker, verapamil, or a Ca2+ chelating agent, EGTA. Production of ROS may be a common pathway through which PMNL respond to MMVF-induced cell activation, but alterations of levels of free intracellular Ca2+ do not seem to be an absolute prerequisite for this effect. Fibre length seemed not to be an important factor in affecting the ability of MMVF to induce ROS production in PMNL. However, the balance between different elements in the fibre seemed importantly to affect the biological activity of a fibre.

Registration of toxicologists in Europe.

Recently several European Toxicological Societies have established national toxicology registers. Such societies include the British, Dutch, Finnish, French, Hungarian, and Swiss societies of toxicology. The basis for registration in these societies is peer review evaluation of the applicants. A key-criterion for acceptance is theoretical training covering the main areas of toxicology. Furthermore, job experience is required. After reviewing the merits of the applicant, the register then accepts the applicant to the register usually for five years. As a consequence of this development, EUROTOX, European Societies of Toxicology, has established a European Register of Toxicologists which will be officially inaugurated during 1997. The register already contains more than two hundred British, Dutch, Finnish, and German toxicologists. The European Register of Toxicologists already has had an impact on the recognition of toxicology in Europe, and will strive to promote toxicology education and professional status in other parts of the world.

Glutamate-stimulated ROS production in neuronal cultures: interactions with lead and the cholinergic system.

Oxidative stress may be an important factor in several pathological brain conditions. A contributing factor in many such conditions is excessive glutamate release, and subsequent glutamatergic neuronal stimulation, that causes increased production of reactive oxygen species (ROS), oxidative stress, excitotoxicity and neuronal damage. Glutamate release is also associated with illnesses such as Alzheimer's disease, stroke, and brain injury. Glutamate may interact with an environmental toxin, lead, and this interaction may result in neuronal damage. Glutamate-induced ROS production is greatly amplified by lead in cultured neuronal cells. Alterations in protein kinase C (PKC) activity seem to be important both for glutamate-induced ROS production, and for the amplification of glutamate-induced ROS production by lead. It is possible that the neurotoxic effects of lead are amplified through glutamate-induced neuronal excitation. Cholinergic stimulation can also trigger ROS production in neuronal cells. PKC seems to play a key-role also in cholinergic-induced ROS production superoxide anion being the primary reactive oxygen species. There seems to be a close relationship between the responses of cholinergic muscarinic and glutamatergic receptors because glutamate receptor antagonists inhibit cholinergic-induced activation of human neuroblastoma cells. Glutamatergic neuronal stimulation may be a common final pathway in several brain conditions in which oxidative stress and ensuing excitotoxicity plays a role.

Modification of glutamate-induced oxidative stress by lead: the role of extracellular calcium.

The role of extracellular calcium in glutamate-induced oxidative stress, and the role of glutamatergic neuronal stimulation and oxidative stress in lead neurotoxicity were explored in mouse hypothalamic GT1-7 cells. Glutamate increased the production of reactive oxygen species (ROS) whether or not extracellular calcium was present. Glutamate-induced ROS production was amplified by lead acetate (PbAc), but only in the absence of extracellular calcium. However, PbAc on its own did not increase the production of ROS. A PKC inhibitor (Ro 31-8220) and superoxide dismutase (SOD) abolished the amplification of glutamate-induced production of ROS by PbAc, but did not inhibit ROS production induced by glutamate alone. Both glutamate and PbAc decreased the levels of intracellular glutathione (GSH), and amplified each other's effect on GSH depletion. Glutamate did not decrease cell viability, whereas the cytotoxicity of PbAc was amplified by glutamate. Extracellular calcium, a PKC inhibitor, or SOD did not modify the effects of glutamate, PbAc or their combination on the levels of GSH or cell viability. These data indicate that in GT1-7 cells extracellular calcium is not essential for glutamate-induced ROS production, which is amplified by PbAc, but only without extracellular calcium. The joint cytotoxicity of glutamate and PbAc is mainly induced by PbAc, preferentially through mechanisms other than ROS production.

Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead.

Increased glutamate release is associated with serious neurological disorders such as epilepsy, stroke, Alzheimer's disease and other brain injuries. Excessive glutamate release and subsequent glutamatergic neuronal stimulation increase the production of reactive oxygen species (ROS), which in turn induce oxidative stress, excitotoxicity and neuronal damage. A number of studies have shown that co-exposure of neuronal cells to glutamate, and an environmental toxin, lead, can greatly amplify glutamate excitotoxicity and cell death through apoptosis or necrosis. Even though the mechanisms of excitotoxicity or those of glutamate-lead interactions have not been exhaustively delineated, there is ample evidence to suggest that increased production of ROS may play an important role in both events. Subsequently, increased DNA binding of redox-regulated transcription factors, NF-kappaB and AP-1, seems to be associated with these events. Induction of an immediate early gene, c-fos, is seen in neuronal cells exposed to glutamate or lead. Immediate early genes are important in regulating the expression of other neuronal genes; Elevated expressions of the genes encoding Hsp70 or cyclo-oxygenase-2 seem to be involved in the apoptosis or necrosis induced by glutamate, and may be associated with induction of several of the genes in cells exposed to lead, or to the glutamate-lead combination. Further studies are required to clarify the mechanisms of glutamate-lead neurotoxicity.

Effects of 3-phenylamino-1,2-propanediol and its mono- and dioleylesters on the production of reactive oxygen metabolites in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) were exposed to 3-phenylamino-1,2-propanediol (PAP) or to its mono- or dioleylesters. The dioleylester of PAP (140 microM) slightly increased the production of reactive oxygen metabolites (ROM) from 8 +/- 2 mV to 18 +/- 3 mV, whereas PAP and its mono-oleylester were without any effect on ROM production in PMNL. None of the compounds were able to modulate formyl-Methionyl-Leucyl-Phenylalanine-induced production of ROM. Moreover, PAP did not have any effect on the production of ROM induced by phorbol myristate acetate (PMA). On the other hand, both mono- and dioleylesters of PAP dose-dependently inhibited PMA-induced production of ROM. The dioleylester of PAP, at a concentration of 14 microM, inhibited PMA-induced production of ROM by 79%, whereas a 2.3 mM concentration of mono-oleylester of PAP was required for 65% inhibition of PMA-induced production of ROM. Moreover, both esters of PAP shifted the peak production of ROM by PMA to a later time-point. Our results suggest that mono- and dioleylesters of PAP modulate the responses of PMNL upon stimulation with PMA, whereas PAP does not have any effect on PMA-induced ROM production in PMNL.

Palmitic acid anilide-induced respiratory burst in human polymorphonuclear leukocytes is inhibited by a protein kinase C inhibitor, Ro 31-8220.

Human polymorphonuclear leukocytes (PMNL) were exposed to palmitic acid anilide, an impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981, and to the corresponding fatty acid, palmitic acid. The effects of these compounds were studied on the production of reactive oxygen metabolites (ROM) and changes in the levels of free intracellular calcium. Palmitic acid anilide induced the production of reactive oxygen metabolites in PMNL. Interestingly, the palmitic acid anilide-induced respiratory burst was completely blocked by a protein kinase C inhibitor, Ro 31-8220. Moreover, palmitic acid anilide additively amplified the production of ROM caused by a chemotactic peptide, formyl-Methionyl-Leucyl-Phenylalanine (FMLP). In contrast, palmitic acid anilide did not have any effect on the production of ROM induced by a tumor promoter, phorbol myristate acetate (PMA). Palmitic acid, in turn, did not markedly induce the production of ROM nor did it amplify the agonist-induced respiratory burst. Neither of the compounds, alone or in combination with FMLP, affected the levels of intracellular calcium in PMNL. These results indicate that the aniline moiety in palmitic acid modifies its effects on the activation of human PMNL, and the subsequent oxidative burst. The present results also suggest that palmitic acid anilide may activate PMNL through a protein kinase C-dependent mechanism.

Effect of linoleic acid, linoleic acid anilide, and arachidonic acid on the expression of adhesion molecules on human neutrophils.

The effects of linoleic acid, linoleic acid anilide, and arachidonic acid on the expression of CD11b/ CD18, CD11c/CD18 integrins and L-selectin on human neutrophils were studied by flow cytometry in a whole blood assay. None of these compounds had any effect on the basal expression of CD11b, CD11c, or L-selectin in the concentration range of 20-100 microM. However, linoleic acid at a concentration of 1000 microM slightly up-regulated CD11b and CD11c by a factor of 2.1 and 1.7, respectively. Linoleic acid, linoleic acid anilide, and arachidonic acid did not affect the formyl-methionyl-leucyl-phenylalanine induced up-regulation of CD11b or CD11c. However, linoleic acid and linoleic acid anilide slightly inhibited the phorbol myristate acetate (PMA)-induced expression of CD11b, which was decreased by 27 and 21% at concentrations of 100 and 1000 microM, respectively. Likewise, arachidonic acid at 40 microM inhibited the PMA-induced expression of CD11b by 19%. Our results suggest that linoleic acid, linoleic acid anilide, and arachidonic acid do not dramatically affect the expression of leukocyte adhesion molecules in a whole blood assay.

Erucic acid and erucic acid anilide-induced oxidative burst in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) were exposed to erucic acid or erucic acid anilide to explore their effects on the production of reactive oxygen species (ROS) and the levels of free intracellular calcium. The compounds did not change the levels of intracellular calcium, but both dose-dependently induced respiratory burst in PMNL. Maximal production of ROS by erucic acid exceeded that induced by its anilide 13-fold. A protein kinase C inhibitor, Ro 31-8220, completely inhibited erucic acid and erucic acid anilide-induced production of ROS. Neither erucic acid nor erucic acid anilide modified FMLP-induced production of ROS. However, erucic acid (500 microM) amplified 5 nM PMA-induced ROS production 1.8-fold, but did not have this effect at a lower PMA concentration. On the contrary, erucic acid anilide inhibited PMA-induced oxidative burst, and shifted the peak ROS production induced by PMA to a later time-point. The present results show that aniline moiety modifies the effects of erucic acid on the activation of PMNL, and suggest that both erucic acid and erucic acid anilide may activate PMNL through a protein kinase C-dependent mechanism.

Formyl-methionyl-leucyl-phenylalanine and a calcium ionophore A23187 reverse the inhibition of phorbol myristate acetate-induced oxidative burst by linoleic and oleic acid anilides.

Linoleic and oleic acid anilides profoundly inhibited the production of reactive oxygen metabolites (ROM) in human polymorphonuclear leukocytes (PMNL) induced by a tumor promoter, phorbol myristate acetate (PMA). The addition of a Ca2+ ionophore, A23187, or a chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP), readily reversed linoleic and oleic acid anilide-induced inhibiton of PMA-evoked respiratory burst in PMNL without affecting PMA-induced respiratory burst. fMLP or A23187 caused a marked increase in the production of ROM in PMNL that did not produce ROM after their co-exposure to PMA and cis-fatty acid anilides. This suggests a role for Ca2+ in this restoration of respiratory burst activity in PMNL. Oleic and linoleic acid anilides enhanced also respiratory burst in PMNL subsequent to their stimulation with fMLP. Interestingly, corresponding fatty acids, linoleic and oleic acid, also inhibited PMA-induced production of ROM in PMNL, but this inhibition was not reversed by A23187 or fMLP. These findings suggest that the aniline moiety of cis-fatty acids significantly modifies the effects of linoleic and oleic acids in the production of ROM in PMNL. Moreover, free intracellular Ca2+ may play a critical role in the activation of PMNL to produce ROM, and in the modulation of the effects of cis-fatty acid anilides.

Cellular effects of particles--impact of dissolution on toxicity of man-made mineral fibers.

The use of man-made vitreous fibers (MMVF) has grown rapidly because exposure to natural fibers, mainly asbestos, has proved harmful to humans. Biological activity of MMVF made of glass, rock, slag, or other minerals does not depend only on their respirability, but also on their chemical durability and persistency. In the use of MMVF, the goal is to decrease harmful effects of fibers by increasing their dissolution and removal from the lungs. The dissolution of Fe and Al from MMVF is more marked by rat alveolar macrophages (AMs) in culture than by mere medium, whereas medium is more effective than AMs in dissoluting silicon (Si) from MMVF, Fe and Al content of the fibers correlate negatively with the fiber Si dissolution by the AMs. Scanning electron micrographs show that MMVF are readily phagocytized by rat AMs in culture. The phagocytosis begins within 30 min after the onset of the exposure and continues for a 96-h observation period. Short fibers, less than 20 microns in length, are readily phagocytized by the AMs whereas longer fibers are attacked with a large number of AMs. MMVF induce also non-lethal changes in the rat AM surface morphology. Before exposure the cells have continuous membranes. The exposed AMs produce extensions which fasten them to the fibers or to other cells to form clumps or clusters of cells and fibers, each cell engulfing a part of a fiber. Over 70% of the exposed cells are viable after 96 h of exposure suggesting that MMVF are not acutely toxic rat AMs. MMVF also slightly damage cell membrane and increase the production of reactive oxygen species.

Amplification of glutamate-induced oxidative stress.

Glutamate is a ubiquitous neurotransmitter which causes excess neuronal excitotoxicity and neurodegenerative insults such as stroke, trauma and seizures. A salient feature of the activation of glutamate receptors is the induction of oxidative burst. Moreover, glutamate stimulates Ca2+ influx and translocates protein kinase C (PKC). PKC mediates cellular processes mediated via phosphorylations which may be essential for oxidative burst in many cells. Subsequent oxidative stress may be a causal factor of neurodegenerative diseases. Increased glutamate release and oxidative burst may thus both be essential in the cascade of events leading to neuronal damage. Glutamate may also mediate neurotoxic effects of environmental toxic agents such as lead which amplify glutamate excitotoxicity. In these interactions, excessive activation of glutamate receptors and oxidative burst may converge into a common pathway leading to cell death through a cascade involving PKC or other protein important in oxidative burst in neurons.

Lead amplifies glutamate-induced oxidative stress.

Lead markedly amplified L-glutamate-induced oxidative stress, that is, increased L-glutamate-induced production of reactive oxygen species, decreased cellular glutathione, and induced cytotoxicity in human neuroblastoma cells. It was notable that oxidative burst induced by L-glutamate alone was observed only when neuronal glutathione was depleted. A role of protein kinase C (PKC) in glutamate-induced production of reactive oxygen species is likely because it was blocked by a PKC inhibitor. We suggest here that the mechanism whereby lead causes its neurotoxicity may be through the amplification of glutamate-induced oxidative stress, possibly through PKC activation.

Mineral fiber-induced leukocyte activation: the role of intra- and extracellular calcium.

The role of intra- and extracellular calcium in the activation of human polymorphonuclear leukocytes (PMNL) to produce reactive oxygen metabolites (ROM) were studied by using soluble, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), or particulate stimuli, quartz or chrysotile. A calcium channel inhibitor, verapamil, attenuated only quartz-induced elevation of free intracellular calcium ([Ca2+]i) and ROM production. Likewise, ethyleneglycol-bis (aminoethyl ether) tetraacetic acid (EGTA) attenuated quartz-, chrysotile- and fMLP-induced elevation of [Ca2+]i and ROM production. It also inhibited PMA-induced ROM production. A calcium ionophore, A23187 amplified ROM production by all of these stimuli. These results suggest that both intra- and extra-cellular calcium are required for the full activation of respiratory burst by soluble and particulate stimuli in human PMNL.

The effects of N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA) on the changes in free intracellular calcium and the production of reactive oxygen metabolites in human leukocytes.

Human leukocytes were exposed to N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA), a postulated impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981. Changes induced by 5-VTPA alone and together with a chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), a tumor promoter, phorbol myristate acetate (PMA), or a synthetic diacylglycerol, dioctanoyl-s,n-glycerol (DiC8) in free intracellular calcium levels ([Ca2+]i) and in the induction of oxidative burst were measured. 5-VTPA elevated dose-dependently [Ca2+]i and induced the production of reactive oxygen metabolites in leukocytes. 5-VTPA also amplified FMLP-induced increase in [Ca2+]i, but was without an effect on FMLP-induced oxidative burst. On the contrary, 5-VTPA amplified dose-dependently PMA- and DiC8-induced respiratory burst. The present results indicate that 5-VTPA may interfere with transmembrane signalling in human leukocytes. 5-VTPA may elevate [Ca2+]i by acting directly on the membrane, or by acting through Ca(2+)-mobilizing receptors. Moreover, 5-VTPA also clearly amplified responses produced through protein kinase C stimulation. Thus, 5-VTPA may act on human leukocytes by affecting Ca(2+)-metabolism and the activity of protein kinase C.

The role of G-proteins in the activation of human leukocytes by particulate stimuli to produce reactive oxygen metabolites.

Effects of pertussis toxin (PTX), cholera toxin (CTX) and an anhydrolyzable GTP analogue, GTP gamma S, on the levels of free intracellular calcium ([Ca2+]i) and the production of reactive oxygen metabolites (ROM) in human polymorphonuclear leukocytes (PMNL) were studied during cell activation. Cells were stimulated by particulate stimuli, quartz or chrysotile, and soluble stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). Pretreatment of PMNL with PTX decreased fMLP-induced elevations of [Ca2+]i but not those induced by quartz or chrysotile. CTX, in turn, decreased both quartz- and fMLP-induced elevations of [Ca2+]i. Likewise, PTX inhibited only fMLP-induced production of ROM, whereas CTX inhibited also those induced by quartz, chrysotile or fMLP. PTX or CTX did not, however, have an impact on PMA-induced production of ROM. GTP gamma S alone did not elevate [Ca2+]i or amplify fMLP-, quartz- or chrysotile-induced [Ca2+]i elevation. However, GTP gamma S alone increased the production of ROM and amplified ROM production induced by fMLP and quartz. The present results suggest that a CTX-sensitive G-protein may be involved in quartz-induced PMNL activation whereas an fMLP-induced neutrophil activation may be regulated by G-proteins sensitive to both PTX and CTX. The involvement of G-protein in chrysotile-induced leukocyte activation is not likely. There may be, however, a relationship between G-protein-mediated cell signalling and quartz-induced production of reactive oxygen metabolites in these cells.

Production of reactive oxygen metabolites by opsonized fungi and bacteria isolated from indoor air, and their interactions with soluble stimuli, fMLP or PMA.

Changes in the levels of free intracellular calcium ([Ca2+]i) and the production of reactive oxygen metabolites (ROM) induced by opsonized indoor air fungi and bacteria in human polymorphonuclear leukocytes (PMNL) were measured. Moreover, modification of a chemotactic peptide (fMLP)-and a tumor promoter (PMA)-induced production of ROM by opsonized fungi and bacteria were studied. The cells were exposed to graded doses of opsonized Candida sp., Aspergillus sp., Cladosporium sp., Stachybotrys sp., Penicillium sp., Paecilomyces sp., or A4 or A91 Streptomyces sp. alone, or together with fMLP or PMA. All the organisms were isolated from air samples of mold-problem buildings. None of the fungi or bacteria induced changes in [Ca2+]i or the production of ROM without opsonization with human serum. Of all opsonized fungi and bacteria, only Candida sp. elevated [Ca2+]i. All fungi and bacteria, except Paecilomyces sp. and Stachybotrys sp., markedly increased the production of ROM in PMNL. Furthermore, A91 Streptomyces sp. and Aspergillus sp. amplified fMLP-induced production of ROM. Only Candida sp. increased PMA-induced phenomen that normally occurs in the lung, was required for biological activity of the fungi and bacteria. Amplification by opsonization of fungi- or bacteria-induced leukocyte activation revealed remarkable changes between these biologically active particles. The present results suggest that many indoor air fungi and bacteria may activate leukocytes to produce oxidative stress, perhaps associated with harmful effects in exposed individuals.