Dosimetry and toxicology of inhaled ultrafine particles.

Both epidemiological and toxicological studies indicate that inhalation and subsequent deposition of airborne particles into the lungs have adverse health effects. Recently, the ultrafine particle (UfP) fraction (diameter < 100 nm) has received particular attention, as their small size may lead to more toxic properties. In this study we summarize the current knowledge on the dosimetry of inhaled particles (including UfPs) with a focus on recent data on translocation of UfPs into secondary target organs (such as brain and heart) suggesting that the lifetime dose of ambient UfPs in secondary target organs is about 10(11) particles. Furthermore, we highlight the main pathways of particle induced toxicity and the reasons for the potentially higher toxicity of UfPs. Finally, we discuss recent evidence indicating that (BET) surface area is the single most relevant dose metric for the toxicity of UfPs, which has important implications for regulatory measures on the toxicity of ambient and engineered particles.

Erythropoiesis-stimulating agent withdrawal and oxidative stress in hemodialysis.

AIMS: Variation of the action of erythropoiesis-stimulating agent (ESA) may modify oxidative stress in hemodialyzed (HD) patients. Our aim was to follow changes of oxidative stress during withdrawal and subsequent resumption of ESA therapy. PATIENTS AND METHODS: After a 14-day suspension of epoietin-beta treatment, 11 HD patients received epoietin-beta and 10 patients darbepoietin-alpha. The whole blood oxidized and reduced glutathione (GSSG, GSH) and erythrocyte malondialdehyde (E-MDA) concentrations and the erythrocyte superoxide dismutase (E-SOD) and catalase (E-CAT) activities were determined before the ESA-free interval (baseline) and at Weeks 2, 6, 10 and 14. RESULTS: In both groups, the ratios GSSG/ GSH were increased at Weeks 2 and 6 (p < 0.001). The E-MDA levels were elevated (p < 0.01) and the E-SOD activities were decreased (p < 0.001) at Week 6. By Week 14, these markers had returned to the baseline, whereas the GSH (p < 0.001) and E-CAT activity levels (p < 0.001) had increased. CONCLUSIONS: An increase in oxidative stress was revealed by the ratio GSSG/GSH directly after the short-term withdrawal of epoietin-b therapy in HD. This new finding may have implications in conditions involving transiently depressed ESA action. For both ESAs, the early phase of readministration was associated with similarly increased oxidative stress, with a subsequent return to the baseline level.

Long-term responses of canine lungs to acidic particles.

Sixteen beagle dogs were housed in four large chambers under minimum restraint. They were exposed for 16 months to clean air and individual baseline data of markers were obtained. For 13 months, eight dogs were further exposed to clean air and eight dogs for 6 h/d to 1-microm MMAD (mass median aerodynamic diameter) acidic sulfate particles carrying 25 micromol H(+) m(-3) into their lungs. To establish functional responses (lung function, cell and tissue integrity, redox balance, and non-specific respiratory defense capacity), each exposed animal served as its own control. To establish structural responses, the eight non-exposed animals served as controls. Acidic particles were produced by nebulization of aqueous sodium hydrogen sulfate at pH 1.5. Only subtle exposure-related changes of lung function and structure were detected. A significant increase in respiratory burst function of alveolar macrophages points to a marginal inflammatory response. This can be explained by the significant production of prostaglandin E(2), activating cyclooxygenase-dependent mechanisms in epithelia and thus inhibiting lung inflammation. The non-specific defense capacity was slightly affected, giving increased tracheal mucus velocity and reduced in vivo dissolution of moderately soluble test particles. Hypertrophy and hyperplasia of bronchial epithelia were not observed, but there was an increase in volume density of bronchial glands and a shift from neutral to acidic staining of epithelial secretory cells in distal airways. The acidic exposure had thus no pathophysiological consequences. It is therefore unlikely that long-term inhalation of acidic particles is associated with a health risk.

Comparison of tetrahydrofuran and ethyl acetate as extraction solvents for urinary organic acid analysis.

The analysis of urinary organic acids is crucial for the diagnosis of many inborn errors of metabolism. A vital part of the analytical process is the extraction procedure. The sensitivity and linearity of the analysis of 26 diagnostically important urinary metabolites with tetrahydrofuran (THF) and ethyl acetate (EtOAc) as extraction solvents were determined by gas chromatography-mass spectrometry. Good linearity (r (2) > 0.90) was observed for all of the compounds in the investigated concentration range (290-900 mumol/L) for both solvents. For less polar compounds, THF extraction yielded lower or similar sensitivities as compared with EtOAc (sensitivity ratio: 0.6-1.3). For more polar compounds, however, much higher sensitivities were observed when THF was used (sensitivity ratio: 1.8-17.2). Our results provide information concerning the use of THF for the sensitive quantitative analysis of polar urinary metabolites which are difficult to quantify using EtOAc.

Distribution pattern of inhaled ultrafine gold particles in the rat lung.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Dose-controlled exposure of A549 epithelial cells at the air-liquid interface to airborne ultrafine carbonaceous particles.

The geometry of commercially available perfusion chambers designed for harbouring three membrane-based cell cultures was modified for reliable and dose-controlled air-liquid interface (ALI) exposures. Confluent A549 epithelial cells grown on membranes were integrated in the chamber system and supplied with medium from the chamber bottom. Cell viability was not impaired by the conditions of ALI exposure without particles. Expression of the inflammatory cytokines interleukin 6 and interleukin 8 by A549 cells during ALI exposure to filtered air for 6h and subsequent stimulation with tumor necrosis factor was not altered compared to submersed controls, indicating that the cells maintained their functional integrity. Ultrafine carbonaceous model particles with a count median mobility diameter of about 95+/-5 nm were produced by spark discharge at a stable concentration of about 2 x 10(6) cm(-3) and continuously monitored for accurate determination of the exposure dose. Delivery to the ALI exposure system yielded a homogeneous particle deposition over the membranes with a deposition efficiency of 2%. Mid dose exposure of A549 cells to this aerosol for 6h yielded a total particle deposition of (2.6+/-0.4) x 10(8) cm(-2) corresponding to (87+/-23) ng cm(-2). The 2.7-fold (p < or = 0.05) increased transcription of heme oxygenase-1 indicated a sensitive antioxidant and stress response, while cell viability did not reveal a toxic mechanism.

Hydrogen peroxide as a mediator of dopac-induced effects on melanoma cells.

Dopac increases tyrosinase activity and exerts cytotoxic effects in cultures of human melanoma cells. The possible role of hydrogen peroxide in these actions was examined. Catalase (100 micrograms/ml) completely reversed the cytotoxic action of 0.3 mM dopac and reduced its tyrosinase-stimulating effect by approximately one half. The results show that extracellular hydrogen peroxide is a mediator of both the tyrosinase-stimulating and cytotoxic actions of dopac. Analysis of the degradation products of melanin from dopac-treated melanoma cells after hydriodic acid (HI) hydrolysis revealed the presence of aminohydroxy-phenylacetic acid (AHPAc). This substance is obtained by HI hydrolysis of melanin formed by oxidation of cysteinyl-dopac. Thus, the presence of AHPAc indicates that dopac is transported into the melanocytes where it serves as a substrate for tyrosinase.

Hydrogen peroxide as an inducer of elevated tyrosinase level in melanoma cells.

The effects of systems generating active oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) on tyrosinase have been studied in cultured human melanoma cells. Tyrosinase activity was determined by measuring the quantity of 5-S-L-cysteinyl-L-dopa (5-S-CD) formed in the presence of D,L-dopa and L-cysteine. In some experiments, the enzyme protein was determined by radio immunoassay [RIA]. Exposure of cells to xanthine/xanthine oxidase or glucose/glucose oxidase resulted in a dose-related elevation of tyrosinase. Catalase, but not superoxide dismutase, prevented this increase indicating that hydrogen peroxide may be the agent responsible for the action, whereas superoxide anion is not involved. Hydroxyl radicals formed by the Haber-Weiss or Fenton type reactions were not found to produce elevation of tyrosinase. Catalase determinations showed no enzyme in the medium but a high concentration in the cells. Inhibition of intracellular catalase by 3-amino-1,2,4-triazole caused an increase in the tyrosinase level. The effects of dopac, xanthine/xanthine oxidase, and glucose/glucose oxidase all producing hydrogen peroxide, and increasing tyrosinase, were enhanced by the inhibition of catalase. It is concluded that hydrogen peroxide, formed by the systems, accounts for the elevation of tyrosinase level. When tyrosinase activities determined by 5-S-CD formation were compared to enzyme amounts found by RIA, the ratios of these values were always constant. This fact indicates that the increase in the tyrosinase activities was not due to an activation of the enzyme, but mirrored the quantities of enzyme protein present in the samples. On the basis of our findings, it is assumed that hydrogen peroxide is a regulator of tyrosinase in normal melanocytes and melanoma cells.

Glutathione status in retinopathy of prematurity.

This study examines the glutathione status of red blood cells in patients with retinopathy of prematurity (ROP) both in vivo and after an in vitro oxidative challenge. Fifty ROP patients of different ages (between 6 weeks and 6 years), born prematurely (gestational age: 28.7 +/- 1.3 weeks; birth weight: 1210 +/- 313 g; mean +/- SD) suffering either from active ROP (<3 months old; n = 12) or from a visual handicap due to preceding ROP (3 months-6 years; n = 38) as well as control patients of similar age and maturity (n = 56) were included. Infants with active disease have the lowest levels of reduced glutathione (GSH), the highest levels of oxidized form (GSSG), the highest GSSG/GSH ratios and the greatest fall in GSH after an in vitro oxidative challenge. After an in vitro oxidative stress, defective glutathione recycling was found in patients with preceding ROP and was suggested as a factor predisposing to oxidative hemolysis. The glutathione redox ratio was warranted as a biochemical screen for active ROP in premature infants.

Pulmonary and systemic distribution of inhaled ultrafine silver particles in rats.

The cardiovascular system is currently considered a target for particulate matter, especially for ultrafine particles. In addition to autonomic or cytokine mediated effects, the direct interaction of inhaled materials with the target tissue must be examined to understand the underlying mechanisms. In the first approach, pulmonary and systemic distribution of inhaled ultrafine elemental silver (EAg) particles was investigated on the basis of morphology and inductively coupled plasma mass spectrometry (ICP-MS) analysis. Rats were exposed for 6 hr at a concentration of 133 microg EAg m(3) (3 x 10(6) cm(3), 15 nm modal diameter) and were sacrificed on days 0, 1, 4, and 7. ICP-MS analysis showed that 1.7 microg Ag was found in the lungs immediately after the end of exposure. Amounts of Ag in the lungs decreased rapidly with time, and by day 7 only 4% of the initial burden remained. In the blood, significant amounts of Ag were detected on day 0 and thereafter decreased rapidly. In the liver, kidney, spleen, brain, and heart, low concentrations of Ag were observed. Nasal cavities, especially the posterior portion, and lung-associated lymph nodes showed relatively high concentrations of Ag. For comparison, rats received by intratracheal instillation either 150 microL aqueous solution of 7 microg silver nitrate (AgNO(3) (4.4 microg Ag) or 150 microL aqueous suspension of 50 microg agglomerated ultrafine EAg particles. A portion of the agglomerates remained undissolved in the alveolar macrophages and in the septum for at least 7 days. In contrast, rapid clearance of instilled water-soluble AgNO(3) from the lung was observed. These findings show that although instilled agglomerates of ultrafine EAg particles were retained in the lung, Ag was rapidly cleared from the lung after inhalation of ultrafine EAg particles, as well as after instillation of AgNO(3), and entered systemic pathways.

Agglomerates of ultrafine particles of elemental carbon and TiO2 induce generation of lipid mediators in alveolar macrophages.

Agglomerates of ultrafine particles (AUFPs) may cause adverse health effects because of their large surface area. To evaluate physiologic responses of immune cells, we studied whether agglomerates of 77-nm elemental carbon [(EC); specific surface area 750 m2/g] and 21 nm titanium dioxide (TiO(2) particles (specific surface area 50 m(2)/g) affect the release of lipid mediators by alveolar macrophages (AMs). After 60-min incubation with 1 microg/mL AUFP-EC (corresponding to 7.5 cm(2) particle surface area), canine AMs (1 x 10(6) cells/mL) released arachidonic acid (AA) and the cyclooxygenase (COX) products prostaglandin E(2) (PGE(2), thromboxane B(2), and 12-hydroxyheptadecatrienoic acid but not 5-lipoxygenase (5-LO) products. AUFP-TiO(2) with a 10-fold higher mass (10 microg/mL) than AUFP-EC, but a similar particle surface area (5 cm(2) also induced AMs to release AA and COX products. Agglomerates of 250 nm TiO(2) particles (specific surface area 6.5 m(2)/g) at 100 microg/mL mass concentration (particle surface area 6.5 cm(2) showed the same response. Interestingly, 75 cm(2)/mL surface area of AUFP-EC and 16 cm(2)/mL surface area of AUFP-TiO(2) additionally induced the release of the 5-LO products leukotriene B(4) and 5-hydroxyeicosatetraenoic acid. Respiratory burst activity of stimulated canine neutrophils was partially suppressed by supernatants of AMs treated with various mass concentrations of the three types of particles. Inhibition of neutrophil activity was abolished by supernatants of AMs treated with COX inhibitors prior to AUFP-incubation. This indicates that anti-inflammatory properties of PGE(2) dominate the overall response of lipid mediators released by AUFP-affected AMs. In conclusion, our data indicate that surface area rather than mass concentration determines the effect of AUFPs, and that activation of phospholipase A(subscript)2(/subscript) and COX pathway occurs at a lower particle surface area than that of 5-LO-pathway. We hypothesize a protective role of PGE(2) in downregulating potential inflammatory reactions induced by ultrafine particles.

Glutathione in human melanoma cells. Effects of cysteine, cysteine esters and glutathione isopropyl ester.

Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of L-cysteine methyl and octyl esters, of N,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The L-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl-L-cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.

Nonenzymatic antioxidants of blood in multiple sclerosis.

Free radical action has been suggested as a causal factor in multiple sclerosis. We investigated the plasma level of lipid peroxides expressed in terms of malone dialdehyde and changes in blood nonenzymatic antioxidants (glutathione, alpha-tocopherol, retinol, plasma sulfhydryl groups, and uric acid) in multiple sclerosis patients with exacerbation or in remission, including a group treated with beta-interferon. The malone dialdehyde level was increased by 38% (n.s.) during exacerbations. The blood concentration of oxidized glutathione was likewise elevated (P<0.05), while the ratio of plasma alpha-tocopherol to cholesterol plus triglyceride was decreased (P<0.001). These changes suggest increased free radical production and consumption of the scavenger molecules during the active phase of the disease. Blood reduced glutathione level was increased (P<0.01) during exacerbation and remission as well. The rise in this thiol is likely to be a compensatory mechanism defending the cells from further oxidant injuries. Beta-interferon increased plasma alpha-tocopherol levels (P<0.001) but not the lipid corrected alpha-tocopherol value. Other parameters were not influenced by the drug.

Ferroxidases and xanthine oxidase in plasma of healthy newborn infants.

In the neonatal period, there is a high iron load, while both the level and molar oxidase activity of ceruloplasmin are low. On the other hand, the neonatal xanthine oxidase (XO) activity is higher than later in life and XO has a significant iron-oxidizing capacity. We therefore studied the physiological contribution of XO to the ferroxidase activity of the plasma in 20 full-term newborn infants. Ferroxidase activity was measured spectrophotometrically, with Fe++ as substrate. The uric acid formed by XO was assayed by means of HPLC, with electrochemical detection. The total ferroxidase activity in the plasma was about one-fourth of the adult level and rapidly increased doubling within 3 days after birth. About 90% of the plasma ferroxidase activity was due to ceruloplasmin, the remainder being accounted for by ferroxidase II. The XO activity underwent a 30% (statistically non-significant) elevation at 24 h, though ferroxidase activity attributable to XO was not detected at any time. Accordingly, XO does not seem to add substantially to the total iron-oxidizing capacity of the plasma in the neonatal period. The high molar ferroxidase activity is probably of importance at the endothelial cell surface.

Melanin-related biochemistry of IGR 1 human melanoma cells.

Cultured melanoma cells have been of great value in the study of pigment metabolism. IGR 1 human melanoma cells, established by Dr Christian Aubert, produce melanin in large quantities. These cells have been used for isolation of human tyrosinase which enzyme has not previously been obtained in a pure form. IGR 1 cells contain large amounts of 5-S-cysteinyldopa which is the quantitatively most important catecholic amino acid. This review deals with the metabolism of dopa, cysteinyldopa, glutathionyldopa, cysteine and glutathione, compounds of central importance in pigment metabolism. The information available on tyrosinase, catecholic compounds and on thiols in IGR 1 melanoma cells makes these cells most suitable for further investigation of the metabolism of human melanoma cells.

Morphologic effects of a sulfur(IV) aerosol on the nasal cavity of beagle dogs.

Morphologic changes were observed in nasal cavities of beagle dogs after long-term exposure to a respirable sulfur(IV) aerosol at a concentration equivalent to a sulfur dioxide (SO2) concentration of 0.6 mg/m3. The changes were characterized by a thickened epithelial layer resulting from epithelial proliferation, by a loss of secretory material, and by moderate mononuclear cell infiltration.

[Reoxigenation after neonatal asphyxia with 21% or 100% oxygen in piglets]. / Ujszülöttkori asphyxiát követö reoxigenizáció sertéseken 21% vagy 100% oxigénnel.

Birth asphyxia represents a serious problem worldwide, resulting in 1 million deaths and an equal number of neurologic sequelae annually. It is therefore important to develop new and better ways to treat asphyxia. In the present study we tested the effect of reoxygenation with room air or 100% oxygen following experimental pneumothorax induced asphyxia on blood oxidative stress indicators, early neurologic outcome and cerebral histopathology of newborn piglets. 26 animals were studied in three experimental groups: sham-operated (SHAM, n = 6), reoxygenation with room air after pneumothorax (RORA, n = 10) and reoxygenation with 100% oxygen after pneumothorax (RO100, n = 10). In RORA and RO100 asphyxia was induced under anesthesia with bilateral intrapleural room air insufflation. Gasping, bradyarrhythmia, arterial hypotension, hypoxemia, hypercarbia and severe combined acidosis occurred 62 +/- 6 (RORA) and 65 +/- 7 min (RO100) after the start of the experiments, when the pneumothorax was relieved and ten min of reoxygenation period was started with mechanical ventilation with room air (RORA) or 100% oxygen (RO100). Then the spontaneously breathing animals were followed on room air during the next three hours. Blood oxidative stress indicators--as oxidized and reduced glutathione, plasma hemoglobin and malondialdehyde concentrations--were also measured at different stages of the experiments and early neurologic examinations (neurological score: 20 = normal, 5 = brain dead) were performed at the end of the study. Then the brains were fixed and stained. In SHAM blood gases and acid/base status differed significantly from values measured in RORA and RO100. In RO100 PaO2 was significantly higher at 5 (13.8 +/- 1.8 kPa) and 10 min (13.2 +/- 2.0 kPa) than in RORA (8.7 +/- 0.9, 9.2 +/- 1.0 kPa), respectively. All the measures of oxidative stress indicators remained unchanged in the study groups (SHAM, RORA, RO100). Neurologic examination scores from SHAM were 18 +/- 0, from RORA 13.5 +/- 1.0 and from RO100 9.5 +/- 1.3 (significant differences between SHAM and RORA and RO100, significant difference between RORA and RO100). Cerebral histopathology showed marked damage with similar severity in both asphyxiated groups. We conclude that blood oxidative stress indicators and cerebral histopathology did not differ significantly after 10 min reoxygenation either with room air or with 100% oxygen following pneumothorax induced asphyxia, but reoxygenation with 100% oxygen might impair the early neurologic outcome of newborn pigs.

Platelet adhesion and fibrinogen deposition in murine microvessels upon inhalation of nanosized carbon particles.

SUMMARY BACKGROUND: The translocation of nanoparticles in the lung toward effector organs via the circulation is considered an important direct pathway for systemic effects of nanoparticles after inhalation. Recently, we have reported that a moderate dose of systemically administered nanosized carbon black particles exerted thrombogenic effects in hepatic microvessels of healthy mice. OBJECTIVES: This study addresses the questions of whether similar thrombogenic effects are also evoked upon inhalation of nanosized carbon particles (NCP) and whether NCP-induced hepatic platelet accumulation is associated with pulmonary or systemic inflammation. METHODS: Two and 8 h after a 24-h exposure to either filtered air or to NCP, intravital fluorescence microscopy of the hepatic microcirculation was performed in C57Bl/6 mice. Parameters of pulmonary or systemic inflammatory response were determined in bronchoalveolar lavage and blood/plasma samples. RESULTS: Inhalative exposure to NCP caused platelet accumulation in the hepatic microvasculature, whereas leukocyte recruitment and sinusoidal perfusion did not differ from controls. Fibrinogen deposition was detected by immunohistochemistry in both hepatic and cardiac microvessels from NCP-exposed mice. In contrast, inhalation of NCP affected neither the plasma levels of proinflammatory cytokines nor blood cell counts. Moreover, the bronchoalveolar lavage data indicate that no significant inflammatory response occurred in the lung. CONCLUSIONS: Thus, exposure to NCP exerts thrombogenic effects in the microcirculation of healthy mice independent of the route of administration (i.e. inhalation or systemic intra-arterial administration). The NCP-induced thrombogenic effects are not liver specific, are associated with neither a local nor a systemic inflammatory response, and seem to be independent of pulmonary inflammation.