Facing the key workplace challenge: assessing and preventing exposure to nanoparticles at source.

Nanomaterials present new challenges to understanding, predicting, and managing potential health risks in occupational environments. In this study, we characterize the key physical processes related to formation and growth of nanoparticles. The main focus is on various occupational environments, as these are known to be major environments with nanoparticles in indoor air. The protection of people potentially to be exposed to nanoparticles is one of the key issues in terms of risk assessment and prevention. Two of the main protection techniques that are discussed and characterized are ventilation and filtration, which are widely used in practical applications.

Effects of fumonisin B(1) on the expression of cytokines and chemokines in human dendritic cells.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other crops across the world. Exposure to FB(1) is known to have toxic and carcinogenic effects in animals, and to express toxicity in cells. In this study, we investigated mechanisms whereby FB(1) may induce immunotoxic effects in human dendritic cells (DC) differentiated from human peripheral blood mononuclear cells. mRNA and protein levels of a number of cytokines and chemokines were analyzed in DC, after exposure to 100 microM FB(1), 10 ng/ml LPS, or a combination of 100 microM FB(1) and 10 ng/ml LPS for 6h or 24h. Exposure to FB(1) resulted in an increase in the expression of IFNgamma and CXCL9. Moreover, FB(1) inhibited the LPS-induced expression of IL-6, IL-1beta, CCL3 and CCL5. The other cytokines studied (TNFalpha, IL-12, IL-18 and IL-23) were not affected by FB(1) in DC. The results of this study indicate that FB(1) has an impact on the expression of cytokines and chemokines in human DC, and in addition to its other toxic effects, FB(1) may also be potentially immunotoxic to humans.

A review of the toxic effects and mechanisms of action of fumonisin B1.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other agricultural products. Fusarium species can also be found in moisture-damaged buildings, and, therefore, exposure of humans to Fusarium mycotoxins including FB(1) may take place. FB(1) bears a clear structural similarity to the cellular sphingolipids, and this similarity has been shown to disturb the metabolism of sphingolipids by inhibiting the enzyme ceramide synthase leading to accumulation of sphinganine in cells and tissues. FB(1) is neurotoxic, hepatotoxic, and nephrotoxic in animals, and it has been classified as a possible carcinogen to humans. The cellular mechanisms behind FB(1)-induced toxicity include the induction of oxidative stress, apoptosis, and cytotoxicity, as well as alterations in cytokine expression. The effects of FB(1) on different parameters vary markedly depending on what types of cells are studied or what species they originate from. These aspects are important to consider when evaluating the toxic potential of FB(1).

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+.

Two-site enzyme immunoassay for measuring intact human osteocalcin in serum.

We developed a sensitive two-site sandwich ELISA for quantitative analysis of human osteocalcin in serum or plasma. Our method is based on two different highly specific antibodies recognizing epitopes at different ends of the protein so that only intact osteocalcin is detected. The method is fast (total analysis time less than 6 h/96 wells), precise (intraassay variation less than 2.3% at four different levels; n = 10, and interassay variation less than 2.5%, n = 5, respectively), and accurate, with a mean recovery of 105%. The detection limit in serum is approximately 0.1 micrograms/liter. The mean concentration of osteocalcin in normal serum with this assay is 3.3 micrograms/liter (SD 3.7 micrograms/liter; range 0.1-13.1 micrograms/liter; n = 41), and the reference range is 0.28-10.1 micrograms/liter (10 and 90% confidence limits). The method shows a reasonable positive linear correlation with other osteocalcin assays (Incstar, r = 0.55, p < 0.05, n = 13; Henning Oscatest, r = 0.52, p < 0.005, n = 34). A good correlation (r = 0.70, p < 0.001) between individual osteocalcin and bone-specific alkaline phosphatase serum concentrations was observed in normal subjects. We found a low or undetectable concentration of intact osteocalcin in serum of all four of our patients with acute primary hyperparathyroidism, and in all five patients with hypocalcemic secondary hyperparathyroidism, which suggests that PTH effectively inhibited the synthesis of osteocalcin in osteoblasts. The serum concentration of intact osteocalcin was elevated in two of three patients with chronic primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Excitatory amino acid-induced slow biphasic responses of free intracellular calcium in human neuroblastoma cells.

Effects of an excitatory amino acid, glutamate, and of ionotropic and metabotropic glutamate receptor agonists on the levels of free intracellular calcium, and their specific receptor binding in human SH-SY5Y neuroblastoma cells were studied. The calcium response was always biphasic, except for AMPA, suggesting both stimulatory and inhibitory effects on free intracellular calcium upon glutamate receptor stimulation, both with ionotropic and metabotropic glutamate receptor agonists. Specific binding of glutamate and other glutamate receptor agonists, together with the biphasic calcium response, suggests that human SH-SY5Y neuroblastoma cells express both ionotropic and metabotropic glutamate receptors. These findings shed new light on the use of human SH-SY5Y neuroblastoma cells as a human neuronal tumor cell model.

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.

Production of inositol phosphates and reactive oxygen metabolites in quartz-dust-stimulated human polymorphonuclear leukocytes.

The present paper explores phosphoinositide turnover in quartz-stimulated human polymorphonuclear leukocytes. Separation of inositol phosphates was carried out with a new ion-pair, reverse-phase high performance liquid chromatographic method applying a gentle tetrabutyl ammonium phosphate buffer gradient. The method separates inositol monophosphates, inositol 1,4-bisphosphate, inositol trisphosphates and inositol 1,3,4,5-tetrakisphosphate. Reactive oxygen metabolites, indices for leukocyte activation, were measured with a luminometric assay. Quartz increased the production of reactive oxygen metabolites, preceded by facilitated inositol phosphate turnover. This finding provides evidence that inositol phosphate second messengers may be involved in quartz-induced leukocyte activation and subsequent production of reactive oxygen metabolites.

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.

Metabolic and nutritional effects of long-term use of guar gum in the treatment of noninsulin-dependent diabetes of poor metabolic control.

Thirty-nine patients with noninsulin-dependent diabetes on oral drug treatment were randomly allocated to either guar gum or placebo treatments for 3 mo. After 3 mo the placebo group was switched to guar gum treatment and both groups were followed for 10 mo (open trial). No significant difference occurred in the fasting blood glucose or glycosylated hemoglobin A1 levels between the two groups at 3 mo. Serum total cholesterol level decreased in the guar gum group from 6.55 +/- 1.45 to 5.69 +/- 1.2 mmol/L (p less than 0.001) but no changes were observed in the placebo group (6.55 +/- 1.2 vs 6.26 +/- 1.4 mmol/L, NS) during 3 mo. At the end of the open trial (n = 33), serum cholesterol was still approximately 7% lower than before guar gum treatment. No consistent changes occurred in serum HDL-cholesterol or triglycerides. Serum vitamin A level was slightly lowered and plasma zinc level elevated during the open trial. Serum vitamin E level was decreased only in the group switched to guar gum at 3 mo.

Dissolution of man-made vitreous fibers in rat alveolar macrophage culture and Gamble's saline solution: influence of different media and chemical composition of the fibers.

The effect of different chemical compositions of man-made vitreous fibers (MMVF) on their dissolution by alveolar macrophages (AM) in culture and in Gamble's solution was studied. The fibers were exposed to cultured rat AMs, culture medium alone; or Gamble's saline solution for 2, 4, or 8 days. The dissolution of the fibers was studied by measuring the amount of silicon (Si), iron (Fe), and aluminum (Al) in each medium. The AMs in culture dissolved Fe and Al from the fibers but the dissolution of Si was more marked in the cell culture medium without cells and in the Gamble's solution. The dissolution of Si, Fe, and Al was different for different fibers, and increased as a function of time. The Fe and Al content of the fibers correlated negatively with the dissolution of Si by AMs from the MMVF, i.e., when the content of Fe and Al of the fibers increased the dissolution of Si decreased. These results suggest that the chemical composition of MMVFs has a marked effect on their dissolution. AMs seem to affect the dissolution of Fe and Al from the fibers. This suggests that in vitro models with cells in the media rather than only culture media or saline solutions would be preferable in dissolution studies of MMVFs.

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.

An improved method for routine determination of vitamin D and its hydroxylated metabolites in serum from children and adults.

A method for routine determination of vitamin D and its major metabolites 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1,25-dihydroxy-vitamin D (1,25(OH)2D) in serum samples from normal children and adults has been developed. Methodological improvements enable a rapid and accurate analysis of 25(OH)D and also the microscale screening of other metabolites present in large concentrations in serum. Vitamin D and its metabolites are extracted from serum samples using hexane/propan-2-ol, which allows a convenient separation of the water soluble and lipid soluble fractions from each other and also from proteins. Preparative silicic acid chromatography was used to separate vitamin D from its metabolites and also from the major portion of co-eluting lipid contaminants. An automated LC solvent delivery and sample introduction system was used to achieve the rapid separation of metabolites. Vitamin D was further purified using adsorption high-performance liquid chromatography and assayed using reverse phase high-performance liquid chromatography connected with UV detection. The 25(OH)D fraction from the preparative chromatography was measured by a competitive protein-binding assay along with 24,25(OH)2D, which was purified by reverse phase high-performance liquid chromatography along with 1,25(OH)2D. A diluted human serum from a pregnant woman (3rd trimester of pregnancy) was used as source of the binding protein for 25(OH)D and 24,25(OH)2D. 1,25(OH)2D was determined by a competitive protein-binding assay using a diluted cytoplasmic 1,25(OH)2D receptor protein isolated from the intestinal mucosa of rachitic chicks. Vitamin D and its metabolite levels were assayed in serum samples from normal children and adults.

Interactions of cis-fatty acids and their anilides with formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate and dioctanoyl-s,n-glycerol in human leukocytes.

Aniline-denaturated rape-seed food oils that contained anilides of linoleic and oleic acids caused a poisoning epidemic, known as Toxic Oil Syndrome, in Spain in 1981. Toxic Oil Syndrome affected mainly the lungs and the immune system of exposed individuals. Linoleic and oleic acids, and linoleic and oleic anilides increased the production of reactive oxygen metabolites in human polymorphonuclear leukocytes. Both cis-fatty acids inhibited a chemotactic peptide-, fMLP-induced production of reactive oxygen metabolites without affecting fMLP-induced elevation of intracellular calcium levels. Linoleic acid anilide slightly amplified fMLP-induced respiratory burst, whereas oleic acid anilide was without an effect. However, both fatty acid anilides decreased fMLP-induced elevation of levels of free intracellular calcium. Moreover, both cis-fatty acids and their anilides inhibited phorbol myristate acetate (PMA)- and dioctanoyl-s,n-glycerol (DiC8)-induced production of reactive oxygen metabolites. Thus, both cis-fatty acids and their anilides inhibited agonist-stimulated production of reactive oxygen metabolites; this is most likely due to interactions with cell signalling events. These results suggest that both linoleic and oleic acids and their anilides may inhibit immunological responses of leukocytes.

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.

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.

Changes in free intracellular calcium and production of reactive oxygen metabolites in human leukocytes by soluble and particulate stimuli.

Changes in free intracellular calcium concentration ([Ca2+]i) and in the production of reactive oxygen metabolites (ROM) were studied in human polymorphonuclear leukocytes (PMNL) exposed to graded concentrations of formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), quartz, or chrysotile. fMLP, quartz, and chrysotile induced a concentration-dependent elevation in [Ca2+]i. PMA did not affect [Ca2+]i. fMLP caused a rapid and transient increase in [Ca2+]i, whereas that caused by quartz or chrysotile was slow and sustained. fMLP, PMA and quartz all caused a concentration-dependent ROM production in PMNL. Chrysotile also caused a concentration-dependent increase in ROM production, but the slope of the curve was more gentle, with the maximum response being only 25% of that caused by the other stimuli. fMLP caused a rapid and transient peak in ROM production at 5 min, PMA a peak at 15 min, whereas quartz and chrysotile induced a biphasic ROM production with peaks at 5 and 20 min, and at 18 and 28 min, respectively. These results suggest that [Ca2+]i may have an important role in the production of ROM by PMNL after an exposure to fMLP, quartz, or chrysotile, and that the involvement of PKC in ROM production is possible.

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.

Malaoxon-induced neurotoxicity in old rats: alterations in cerebral inositol lipid signalling, brain tissue calcium levels and early neuronal injury.

Effects of malaoxon (MO) on brain regional inositol, inositol monophosphate and calcium levels, as well as on early neuronal injury, were studied in old (18 months) male rats. In old rats, a dose of 8.7 mg/kg of MO caused convulsions similar to those reported earlier in parallel experiments with young male (10 weeks) rats using a dose of 39.2 mg/kg. In the convulsing old male rats, MO caused a transient decrease of cerebral inositol 1 h post MO in the piriform cortex and thalamus, whereas more persistent decreases of inositol occurred in the frontal cortex and the cerebellum. In the non-convulsing rats, a decrease of inositol was only seen in the cerebellum. Cerebral inositol-1-phosphate (Ins1P) increased in all brain regions of convulsing rats, whereas Ins1P did not change in the non-convulsing rats. Brain Ca2+ increased post MO in convulsing and non-convulsing rats in the frontal cortex, caudate and thalamus; in the piriform cortex and hippocampus increases of Ca2+ were only seen in the convulsing rats. Inositol-4-phosphate (Ins4P) remained stable in all MO-exposed rats. MO-induced early neuronal injury occurred only in the convulsing rats and was most severe in the cortex, hippocampus and the subcortical structures. Qualitatively the effects of MO in the old and young rats were, however, similar and, therefore, probably due to cholinergic brain stimulation and subsequent increase in inositol lipid signalling. These results suggest that old rats are likely to be more sensitive than the young rats to the neurotoxic effects of MO.