In vitro toxicity of cationic micelles and liposomes in cultured human hepatocyte (HepG2) and lung epithelial (A549) cell lines.

The aim of this study was to compare the effects of cationic micelle and liposome drug delivery systems on liver and lung cells in a toxicological in vitro screening model, with observations on cytotoxicity and genotoxicity. A screening battery was established for assessment of a broad range of parameters related to adverse effects. Clear concentration response effects were observed related to impairment of mitochondrial function, membrane integrity and oxidative stress markers, but no effect was observed on genotoxicity. The adverse effects were highest for the liposomes. The High Content Screening seems optimal for initial screening of adverse effects, and combined with standard cytotoxicity measurements initial screening can be performed for predictive toxicological screening.

Accumulation of lipids and oxidatively damaged DNA in hepatocytes exposed to particles.

Exposure to particles has been suggested to generate hepatosteatosis by oxidative stress mechanisms. We investigated lipid accumulation in cultured human hepatocytes (HepG2) and rat liver after exposure to four different carbon-based particles. HepG2 cells were exposed to particles for 3h and subsequently incubated for another 18h to manifest lipid accumulation. In an animal model of metabolic syndrome we investigated the association between intake of carbon black (CB, 14nm) particles and hepatic lipid accumulation, inflammation and gene expression of Srebp-1, Fasn and Scd-1 involved in lipid synthesis. There was a concentration-dependent increase in intracellular lipid content after exposure to CB in HepG2 cells, which was only observed after co-exposure to oleic/palmitic acid. Similar results were observed in HepG2 cells after exposure to diesel exhaust particles, fullerenes C60 or pristine single-walled carbon nanotubes. All four types of particles also generated oxidatively damaged DNA, assessed as formamidopyrimidine DNA glycosylase (FPG) sensitive sites, in HepG2 cells after 3h exposure. The animal model of metabolic syndrome showed increased lipid load in the liver after one oral exposure to 6.4mg/kg of CB in lean Zucker rats. This was not associated with increased iNOS staining in the liver, indicating that the oral CB exposure was associated with hepatic steatosis rather than steatohepatitis. The lipid accumulation did not seem to be related to increased lipogenesis because there were unaltered gene expression levels in both the HepG2 cells and rat livers. Collectively, exposure to particles is associated with oxidative stress and steatosis in hepatocytes.

Pulmonary exposure to particles from diesel exhaust, urban dust or single-walled carbon nanotubes and oxidatively damaged DNA and vascular function in apoE(-/-) mice.

This study compared the oxidative stress level and vasomotor dysfunction after exposure to urban dust, diesel exhaust particles (DEP) or single-walled carbon nanotubes (SWCNT). DEP and SWCNT increased the production of reactive oxygen species (ROS) in cultured endothelial cells and acellullarly, whereas the exposure to urban dust did not generate ROS. The apoE(-/-) mice, which were exposed twice to 0.5 mg/kg of the particles by intratracheal (i.t.) instillation, had unaltered acetylcholine-elicited vasorelaxation in aorta segments. There was unaltered pulmonary expression level of Vcam-1, Icam-1, Hmox-1 and Ogg1. The levels of oxidatively damaged DNA were unchanged in lung tissue. The exposure to SWCNT significantly increased the expression of Ccl-2 in the lung tissue of the mice. The exposure to DEP and SWCNT was associated with elevated ROS production in cultured cells, whereas i.t. instillation of the same particles had no effect on biomarkers of pulmonary oxidative stress and dilatory dysfunction in the aorta.

Carbon black nanoparticles and vascular dysfunction in cultured endothelial cells and artery segments.

Exposure to small size particulates is regarded as a risk factor for cardiovascular disease. We investigated effects of exposure to nanosized carbon black (CB) in human umbilical vein endothelial cells (HUVECs) and segments of arteries from rodents. The CB exposure was associated with increased surface expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) in HUVECs at 100µg/ml. CB exposure was also associated with increased reactive oxygen species production and damage to the cell membranes in the form of increased lactate dehydrogenase leakage, whereas it did not alter the mitochondrial enzyme activity (WST-1) or the nitric oxide level in HUVECs. Incubation of aorta segments with 10µg/ml of CB increased the endothelial-dependent vasorelaxation, induced by acetylcholine, and shifted the endothelium-independent vasorelaxation, induced by sodium nitroprusside, towards a decreased sensitivity. In mesenteric arteries, the exposure to 10µg/ml was associated with a reduced pressure-diameter relationship. Incubation with 100µg/ml CB significantly decreased both acetylcholine and sodium nitroprusside responses as well as decreased the receptor-dependent vasoconstriction caused by phenylephrine. In conclusion, nanosized CB exposure activates endothelial cells and generates oxidative stress, which is associated with vasomotor dysfunction.

Pulmonary exposure to carbon black nanoparticles and vascular effects.

BACKGROUND: Exposure to small size particulates is regarded as a risk factor for cardiovascular diseases. METHODS: We exposed young and aged apolipoprotein E knockout mice (apoE-/-) to carbon black (Printex 90, 14 nm) by intratracheal instillation, with different dosing and timing, and measured vasomotor function, progression of atherosclerotic plaques, and VCAM-1, ICAM-1, and 3-nitrotyrosine in blood vessels. The mRNA expression of VCAM-1, ICAM-1, HO-1, and MCP-1 was examined in lung tissue. RESULTS: Young apoE-/- mice exposed to two consecutive 0.5 mg/kg doses of carbon black exhibited lower acetylcholine-induced vasorelaxation in aorta segments mounted in myographs, whereas single doses of 0.05-2.7 mg/kg produced no such effects. The phenylephrine-dependent vasocontraction response was shifted toward a lower responsiveness in the mice exposed once to a low dose for 24 hours. No effects were seen on the progression of atherosclerotic plaques in the aged apoE-/- mice or on the expression of VCAM-1 and ICAM-1 and the presence of 3-nitrotyrosine in the vascular tissue of either young or aged apoE-/- mice. The expression of MCP-1 mRNA was increased in the lungs of young apoE-/- mice exposed to 0.9-2.7 mg/kg carbon black for 24 hours and of aged apoE-/- mice exposed to two consecutive 0.5 mg/kg doses of carbon black seven and five weeks prior to sacrifice. CONCLUSION: Exposure to nano-sized carbon black particles is associated with modest vasomotor impairment, which is associated neither with nitrosative stress nor with any obvious increases in the expression of cell adhesion proteins on endothelial cells or in plaque progression. Evidence of pulmonary inflammation was observed, but only in animals exposed to higher doses.

Role of oxidative damage in toxicity of particulates.

Particulates are small particles of solid or liquid suspended in liquid or air. In vitro studies show that particles generate reactive oxygen species, deplete endogenous antioxidants, alter mitochondrial function and produce oxidative damage to lipids and DNA. Surface area, reactivity and chemical composition play important roles in the oxidative potential of particulates. Studies in animal models indicate that particles from combustion processes (generated by combustion of wood or diesel oil), silicate, titanium dioxide and nanoparticles (C60 fullerenes and carbon nanotubes) produce elevated levels of lipid peroxidation products and oxidatively damaged DNA. Biomonitoring studies in humans have shown associations between exposure to air pollution and wood smoke particulates and oxidative damage to DNA, deoxynucleotides and lipids measured in leukocytes, plasma, urine and/or exhaled breath. The results indicate that oxidative stress and elevated levels of oxidatively altered biomolecules are important intermediate endpoints that may be useful markers in hazard characterization of particulates.

Modest vasomotor dysfunction induced by low doses of C60 fullerenes in apolipoprotein E knockout mice with different degree of atherosclerosis.

BACKGROUND: Exposure to small size particulate matter in urban air is regarded as a risk factor for cardiovascular effects, whereas there is little information about the impact on the cardiovascular system by exposure to pure carbonaceous materials in the nano-size range. C60 fullerenes are nano-sized particles that are expected to have a widespread use, including cosmetics and medicines. METHODS: We investigated the association between intraperitoneal injection of pristine C60 fullerenes and vasomotor dysfunction in the aorta of 11-13 and 40-42 weeks old apolipoprotein E knockout mice (apoE-/-) with different degree of atherosclerosis. RESULTS: The aged apoE-/-mice had lower endothelium-dependent vasorelaxation elicited by acetylcholine in aorta segments mounted in myographs and the phenylephrine-dependent vasoconstriction response was increased. One hour after an intraperitoneal injection of 0.05 or 0.5 mg/kg of C60 fullerenes, the young apoE-/- mice had slightly reduced maximal endothelium-dependent vasorelaxation. A similar tendency was observed in the old apoE-/- mice. Hampered endothelium-independent vasorelaxation was also observed as slightly increased EC50 of sodium nitroprusside-induced vasorelaxation response in young apoE-/- mice. CONCLUSION: Treatment with C60 fullerenes affected mainly the response to vasorelaxation in young apoE-/- mice, whereas the vasomotor dysfunction in old apoE-/- mice with more advanced atherosclerosis was less affected by acute C60 fullerene treatment. These findings represent an important step in the hazard characterization of C60 fullerenes by showing that intraperitoneal administration is associated with a moderate decrease in the vascular function of mice with atherosclerosis.