- Email: [email protected]
6 mice
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Abstracts / Toxicology Letters 196S (2010) S37–S351
24 h exposure we found a slight DNA damage at the lower concentrations and a DNA damage similar to that found at the shorter times of exposure at 100 g/mL MWCNTs. Moreover at the used MWCNTs concentrations a slight oxidative DNA damage induction was shown only after 4 h exposure. The results show cytotoxic effects in terms of LDH release and cell viability reduction after 24 h exposure to the highest MWCNTs concentration. The findings also show genotoxic effects induced on BEAS-2B cells by MWCNTs particularly after 2 and 4 h that decrease after 24 h suggesting the capability of this cellular type to repair DNA damage at low concentrations. The findings demonstrate the suitability of this experimental model to point out early cytotoxic/genotoxic effects induced on target organ by carbon nanotubes and furnish useful information for the evaluation of potential health risks of nanomaterials. doi:10.1016/j.toxlet.2010.03.1158
P303-038 In vitro toxicity assessment of silver nanoparticles in Chang liver cells and J-774 macrophages
exposing them to gold np with and without coating, and a gold salt as a control, for 10 days. Secondly, internalization of np in 3T3 cells was assessed by transmission electronic microscopy (TEM), in time kinetic. Results showed a higher sensitivity of stem cells when exposed to gold np coated with hyaluronic acid (IC50 3T3 = 2873 g/mL and IC50 D3 = 852 g/mL), while exposure to uncoated np showed higher sensitivity in differentiated cells (IC50 3T3 = 1757 g/mL and IC50 D3 = 2632 g/mL). Treatment with gold salts resulted in an IC50 almost equal for both cell lines (IC50 3T3 = 7.9 g/mL and IC50 D3 = 7.7 g/mL. TEM imaging revealed that at 15, 30 and 60 min there were no gold np inside the cells. For coated nanoparticles the entry started after 4 h, being completed by 24 h when all np were inside the lysosomes, some of which appeared damaged. In other way, uncoated nanoparticles were aggregated in the lysosomes after 4 h. This work suggests that the different presentations of np can have different responses in differentiated cells or stem cells. Moreover the onset of active internalization by the 3T3 fibroblast cell line can vary depending on nanoparticles’ coating, being completed after 4 or 24 h of exposition for uncoated or hyaluronic coated np respectively.
˜ 1 , M. Ramirez-Cabrera 1 , M.T. Zanatta-Calderon 1 , L. Garza-Ocanas R. Lujan-Rangel 1 , D.A. Ferrer 2 , M.J. Yacaman 3
doi:10.1016/j.toxlet.2010.03.1160
1
Fac de Medicina Universidad Autonoma de Nuevo Leon, Mexico, The University of Texas at Austin, USA, 3 The University of Texas at San Antonio, USA
P303-040 Exposure to nickel nanoparticles alters vascular reactivity in C57BL/6 mice
Silver nanoparticles have been widely used in medicinal and biological fields and little is yet known about their toxicity. In this study we evaluated the toxic effects of silver nanoparticles (2 nm) in Chang liver cells and J-774 macrophages. For toxicity evaluations cellular morphology, mitochondrial function (MTT assay), cell membrane viability (Neutral Red assay), reactive oxygen species production (DCFDA fluorescent probe), and nitric oxide production were assessed under control and exposed conditions. Cells were grown as monolayer in 96-well plates and exposed to 2–800 g/mL for 24 h and 48 h. Results: The microscopic studies demonstrated that nanoparticle-exposed cells became abnormal in size, displaying cellular shrinkage and irregular shape. Silver nanoparticles produced cell oxidative stress and a significant viability decrease (60%) was observed in Chang liver cells exposed to 27 g/ml. An increase of more than 5-fold of nitric oxide production was detected in macrophages exposed to 400 g/ml. Silver nanoparticles induce a pro-oxidant environment in the cell suggesting that their cytotoxicity is likely to be mediated through oxidative stress.
A. Cuevas, E. Liberda, L.C. Chen
2
doi:10.1016/j.toxlet.2010.03.1159
P303-039 In vitro cytotoxicity and cellular uptake of gold nanoparticles J. De Lapuente, D. Ramos, C. Porredon, C. Di Guglielmo, M. Borràs Parc Cientific, Barcelona, Spain In recent years, the extensive use of nanoparticles (np) is arising some questions about its toxicity and possible risks for human health. The high cost of experimentation in vivo and the development of in vitro alternative techniques are leading to a better knowledge of them. The aim of this work, encompassed in a broader project, was to know, first, the cytotoxicity in 3T3 fibroblasts and D3 stem cells
New York University, United States There is growing interest in engineered nanoparticles (NPs) for various medical and technological applications, but the long term environmental and health effects of these NPs (particles with a diameter <100 nm) are still being discovered. Since there have been associations reported between inhaled ambient ultrafine particles and increased risk of cardiopulmonary disease, it has been suggested that inhaled NPs may also induce adverse effects on the cardiovascular system. Acute exposure to nickel NPs, specifically has been suggested to induce pulmonary and systemic inflammation and, long term studies demonstrate exacerbation of atherosclerosis in apoprotein E knockout (ApoE KO) mice. However the exact mechanisms are not well understood. Few of these studies have specifically measured vascular function, specifically, arterial vasoconstriction and endothelial dysfunction. Nickel hydroxide nanoparticles (NH-NPs) were used as a test material and thoroughly characterized for various physicochemical properties related to toxicity. A series of preliminary inhalation studies were conducted using wild-type C57BL/6 mice to identify acute toxic potential of NH-NPs. Utilizing a whole body inhalation exposure system, C57BL/6 mice were exposed for 1 or 3 days to either NH-NPs (diameter of primary particle: 5 nm, count median diameter of agglomerates: approximately 40 nm) at approximately 150 g Ni/cubic meter (approximately 10% of the current occupational standard), or to filtered air. Carotid arteries were isolated from each animal and 2 mm segments were mounted on a tension myograph system to measure contractile response. Compared to the filtered air exposed mice, no significant differences in the contractile response to phenylephrine (PE) was observed at either time point. However, at both time points, data indicates decreased relaxation after administration of acytelcholine (ACh) compared to the controls. The results suggest that short-term exposure to NH-NPs is associated with acute endothelial response and vasoconstric-
Abstracts / Toxicology Letters 196S (2010) S37–S351
tion, consistent with similar findings from findings from studies examining aorta. doi:10.1016/j.toxlet.2010.03.1161
P303-041 Safety assessment of nanomedicines J.A. Vericat Noscira & European Technology Platform on Nanomedicines, Spain The fight against disease is developing new approaches which may solve some of the today’s limitations in the R&D process for new treatments. An example is the development of nanomedicines. Three areas of research are covered by the term NANOMEDICINES according to the European Technology Platform on Nanomedicines: (1) Regenerative Medicine, based in the use of cells, tissues, and scaffolds to facilitate regeneration. (2) Drug Delivery, to improve the treatment targeting and reduce side effects. It also covers the application of nanotechnology to develop improved pharmaceutical forms. (3) Diagnosis, applying nanotechnology to facilitate analysis, either by reducing size of some instruments or by miniaturizing ex vivo systems to facilitate the task. Nanomedicines, at term, will be complex devices incorporating several of these features. They will rest long time in the body as devices and will deliver adequate drug doses in response to specific determinations. Thus, it is important to define which regulation must be applied: DRUGS and/or DEVICES. Today’s approach for safety assessment is, in general, based on single oral administration with elimination after one day. Accordingly, a step-by-step approach for safety assessment after repeated administration is followed to allow clinical studies. It is difficult to apply standard approaches to long-term resting nanomedicines. Finally, nanomedicines will be complex micro or nanodevices. Today we follow up the fate and metabolism of a chemical by established procedures. It is difficult to imagine how to apply these approaches to nanomedicines. Regulatory Authorities are well aware of the impact of these nanomedicines. Since there is not existing experience in their safety evaluation, Authorities are extremely kind to organize meetings to exchange results with reserachers and deciding together the best way for safety assessment. The recommendation is to interact with Authorities as soon as possible to avoid unnecessary delays in the project. doi:10.1016/j.toxlet.2010.03.1162
P303-042 Comparison between two different exposure methods, via culture medium and exposure at the air–liquid interface, of ZnO nanoparticles to A-549 cells S. Wagner 1 , N. Möhle 2 , O. Krischenowski 2 , S. Röker 2 , D. Bahnemann 1 , T. Scheper 1 , M. Aufderheide 2 , K. Cornelia 1 1
Institut für Technische Chemie, Leibniz Universität Hannover, Germany, 2 Cultex Laboratories GmbH, Germany The technological progress allows the assembly of new, tiny material structures having the size of only a few nanometers. In this
S285
range the materials often exhibit different physical and chemical properties as compared with their bulk counterparts and can thus be employed for specific applications. One of this materials used in nanotechnology is zinc oxide, which is a semiconductor with a direct band gap of 3.27 eV. It is used as an ingredient in sun creams and it is interesting for photocatalytical applications. It is also used as a white pigment for wall paint. In this work ZnO nanoparticles have been tested concerning their toxicity on A-549 cells, a human lung adenocarcinoma epithelial cell line. Two different exposure methods have been applied: the direct exposure of the cells at the air–liquid interface which was carried out with the Cultex® -Radial Flow System, and the exposure to a suspension of ZnO particles in culture medium. To characterize ZnO particles sizes, SEM picture were taken. To make sure, that the different nanoparticles do not agglomerate in the cell culture medium, DLS measurements of particle suspensions in water and in cell culture medium were performed. The viability of the cells after exposure was examined by the MTT-assay, a photometric method to determine the cell metabolism. Membrane integrity measurements were also performed after cell treatment by the measurements of LDH release. Both methods resulted in comparable effects. It was shown that ZnO nanoparticles have a toxic effect on A-549 cells in a concentration range between 10 ppm and 50 ppm in the culture medium. doi:10.1016/j.toxlet.2010.03.1163
P303-043 The effects of silver nanoparticles on male rats sperm quantity and quality 1 , A. Gajowik 1 , J. Radzikowska 1 , J. ´ M. Dobrzynska 2 , G. ´ Gromadzka-Ostrowska 2 , M. Oczkowski 2 , A. Krawczynska 3 4 5 5 Brunborg , M. Dusinska , A. Lankoff , M. Kruszewski 1
National Institute of Public Health - National Institute of Hygiene, United States, 2 Warsaw University of Life Sciences, Poland, 3 Norwegian Institute of Public Health, Norway, 4 Norwegian Institute of Air Research, Norway, 5 Institute of Nuclear Chemistry and Technology, Poland Nowadays silver nanoparticles (AgNPs) are widely used in consumer and biomedical products. The study was designed to assess the effects of acute exposure to different doses and particle sizes of AgNPs on the gonads and gametes of adult male rats. Adult Wistar rats were injected intravenously with 5 mg/kg or 10 mg/kg AgNPs of size 20 nm or 5 mg/kg of size 200 nm. Animals were sacrificed at 24 h, 1 week and 4 weeks after exposure. Testes and epididymes were removed and weighed; sperm count, morphology and DNA damage in male mice germ cells were estimated. There were no significant differences between testes and epididymes weights of exposed and unexposed rats. Sperm counts were slightly reduced 24 h after exposure to 5 mg/kg of 20 nm Ag. One and four weeks following the exposure, sperm counts were similar in all the AgNPs treated and the control groups. There were no significant differences between percentages of abnormal spermatozoa in exposed and unexposed groups. At 24 h after injection, comet tail moments in male germ testicular cells were significantly enhanced in all groups exposed to AgNPs. One week later, lower levels of DNA damage compared to results of 24 h were noted. Four weeks after the exposure, DNA damage in all experimental groups was lower than in control. At all time points, the highest values of comet tail moment were observed after exposure to 5 mg/kg of 20 nm Ag.
Abstracts / Toxicology Letters 196S (2010) S37–S351
24 h exposure we found a slight DNA damage at the lower concentrations and a DNA damage similar to that found at the shorter times of exposure at 100 g/mL MWCNTs. Moreover at the used MWCNTs concentrations a slight oxidative DNA damage induction was shown only after 4 h exposure. The results show cytotoxic effects in terms of LDH release and cell viability reduction after 24 h exposure to the highest MWCNTs concentration. The findings also show genotoxic effects induced on BEAS-2B cells by MWCNTs particularly after 2 and 4 h that decrease after 24 h suggesting the capability of this cellular type to repair DNA damage at low concentrations. The findings demonstrate the suitability of this experimental model to point out early cytotoxic/genotoxic effects induced on target organ by carbon nanotubes and furnish useful information for the evaluation of potential health risks of nanomaterials. doi:10.1016/j.toxlet.2010.03.1158
P303-038 In vitro toxicity assessment of silver nanoparticles in Chang liver cells and J-774 macrophages
exposing them to gold np with and without coating, and a gold salt as a control, for 10 days. Secondly, internalization of np in 3T3 cells was assessed by transmission electronic microscopy (TEM), in time kinetic. Results showed a higher sensitivity of stem cells when exposed to gold np coated with hyaluronic acid (IC50 3T3 = 2873 g/mL and IC50 D3 = 852 g/mL), while exposure to uncoated np showed higher sensitivity in differentiated cells (IC50 3T3 = 1757 g/mL and IC50 D3 = 2632 g/mL). Treatment with gold salts resulted in an IC50 almost equal for both cell lines (IC50 3T3 = 7.9 g/mL and IC50 D3 = 7.7 g/mL. TEM imaging revealed that at 15, 30 and 60 min there were no gold np inside the cells. For coated nanoparticles the entry started after 4 h, being completed by 24 h when all np were inside the lysosomes, some of which appeared damaged. In other way, uncoated nanoparticles were aggregated in the lysosomes after 4 h. This work suggests that the different presentations of np can have different responses in differentiated cells or stem cells. Moreover the onset of active internalization by the 3T3 fibroblast cell line can vary depending on nanoparticles’ coating, being completed after 4 or 24 h of exposition for uncoated or hyaluronic coated np respectively.
˜ 1 , M. Ramirez-Cabrera 1 , M.T. Zanatta-Calderon 1 , L. Garza-Ocanas R. Lujan-Rangel 1 , D.A. Ferrer 2 , M.J. Yacaman 3
doi:10.1016/j.toxlet.2010.03.1160
1
Fac de Medicina Universidad Autonoma de Nuevo Leon, Mexico, The University of Texas at Austin, USA, 3 The University of Texas at San Antonio, USA
P303-040 Exposure to nickel nanoparticles alters vascular reactivity in C57BL/6 mice
Silver nanoparticles have been widely used in medicinal and biological fields and little is yet known about their toxicity. In this study we evaluated the toxic effects of silver nanoparticles (2 nm) in Chang liver cells and J-774 macrophages. For toxicity evaluations cellular morphology, mitochondrial function (MTT assay), cell membrane viability (Neutral Red assay), reactive oxygen species production (DCFDA fluorescent probe), and nitric oxide production were assessed under control and exposed conditions. Cells were grown as monolayer in 96-well plates and exposed to 2–800 g/mL for 24 h and 48 h. Results: The microscopic studies demonstrated that nanoparticle-exposed cells became abnormal in size, displaying cellular shrinkage and irregular shape. Silver nanoparticles produced cell oxidative stress and a significant viability decrease (60%) was observed in Chang liver cells exposed to 27 g/ml. An increase of more than 5-fold of nitric oxide production was detected in macrophages exposed to 400 g/ml. Silver nanoparticles induce a pro-oxidant environment in the cell suggesting that their cytotoxicity is likely to be mediated through oxidative stress.
A. Cuevas, E. Liberda, L.C. Chen
2
doi:10.1016/j.toxlet.2010.03.1159
P303-039 In vitro cytotoxicity and cellular uptake of gold nanoparticles J. De Lapuente, D. Ramos, C. Porredon, C. Di Guglielmo, M. Borràs Parc Cientific, Barcelona, Spain In recent years, the extensive use of nanoparticles (np) is arising some questions about its toxicity and possible risks for human health. The high cost of experimentation in vivo and the development of in vitro alternative techniques are leading to a better knowledge of them. The aim of this work, encompassed in a broader project, was to know, first, the cytotoxicity in 3T3 fibroblasts and D3 stem cells
New York University, United States There is growing interest in engineered nanoparticles (NPs) for various medical and technological applications, but the long term environmental and health effects of these NPs (particles with a diameter <100 nm) are still being discovered. Since there have been associations reported between inhaled ambient ultrafine particles and increased risk of cardiopulmonary disease, it has been suggested that inhaled NPs may also induce adverse effects on the cardiovascular system. Acute exposure to nickel NPs, specifically has been suggested to induce pulmonary and systemic inflammation and, long term studies demonstrate exacerbation of atherosclerosis in apoprotein E knockout (ApoE KO) mice. However the exact mechanisms are not well understood. Few of these studies have specifically measured vascular function, specifically, arterial vasoconstriction and endothelial dysfunction. Nickel hydroxide nanoparticles (NH-NPs) were used as a test material and thoroughly characterized for various physicochemical properties related to toxicity. A series of preliminary inhalation studies were conducted using wild-type C57BL/6 mice to identify acute toxic potential of NH-NPs. Utilizing a whole body inhalation exposure system, C57BL/6 mice were exposed for 1 or 3 days to either NH-NPs (diameter of primary particle: 5 nm, count median diameter of agglomerates: approximately 40 nm) at approximately 150 g Ni/cubic meter (approximately 10% of the current occupational standard), or to filtered air. Carotid arteries were isolated from each animal and 2 mm segments were mounted on a tension myograph system to measure contractile response. Compared to the filtered air exposed mice, no significant differences in the contractile response to phenylephrine (PE) was observed at either time point. However, at both time points, data indicates decreased relaxation after administration of acytelcholine (ACh) compared to the controls. The results suggest that short-term exposure to NH-NPs is associated with acute endothelial response and vasoconstric-
Abstracts / Toxicology Letters 196S (2010) S37–S351
tion, consistent with similar findings from findings from studies examining aorta. doi:10.1016/j.toxlet.2010.03.1161
P303-041 Safety assessment of nanomedicines J.A. Vericat Noscira & European Technology Platform on Nanomedicines, Spain The fight against disease is developing new approaches which may solve some of the today’s limitations in the R&D process for new treatments. An example is the development of nanomedicines. Three areas of research are covered by the term NANOMEDICINES according to the European Technology Platform on Nanomedicines: (1) Regenerative Medicine, based in the use of cells, tissues, and scaffolds to facilitate regeneration. (2) Drug Delivery, to improve the treatment targeting and reduce side effects. It also covers the application of nanotechnology to develop improved pharmaceutical forms. (3) Diagnosis, applying nanotechnology to facilitate analysis, either by reducing size of some instruments or by miniaturizing ex vivo systems to facilitate the task. Nanomedicines, at term, will be complex devices incorporating several of these features. They will rest long time in the body as devices and will deliver adequate drug doses in response to specific determinations. Thus, it is important to define which regulation must be applied: DRUGS and/or DEVICES. Today’s approach for safety assessment is, in general, based on single oral administration with elimination after one day. Accordingly, a step-by-step approach for safety assessment after repeated administration is followed to allow clinical studies. It is difficult to apply standard approaches to long-term resting nanomedicines. Finally, nanomedicines will be complex micro or nanodevices. Today we follow up the fate and metabolism of a chemical by established procedures. It is difficult to imagine how to apply these approaches to nanomedicines. Regulatory Authorities are well aware of the impact of these nanomedicines. Since there is not existing experience in their safety evaluation, Authorities are extremely kind to organize meetings to exchange results with reserachers and deciding together the best way for safety assessment. The recommendation is to interact with Authorities as soon as possible to avoid unnecessary delays in the project. doi:10.1016/j.toxlet.2010.03.1162
P303-042 Comparison between two different exposure methods, via culture medium and exposure at the air–liquid interface, of ZnO nanoparticles to A-549 cells S. Wagner 1 , N. Möhle 2 , O. Krischenowski 2 , S. Röker 2 , D. Bahnemann 1 , T. Scheper 1 , M. Aufderheide 2 , K. Cornelia 1 1
Institut für Technische Chemie, Leibniz Universität Hannover, Germany, 2 Cultex Laboratories GmbH, Germany The technological progress allows the assembly of new, tiny material structures having the size of only a few nanometers. In this
S285
range the materials often exhibit different physical and chemical properties as compared with their bulk counterparts and can thus be employed for specific applications. One of this materials used in nanotechnology is zinc oxide, which is a semiconductor with a direct band gap of 3.27 eV. It is used as an ingredient in sun creams and it is interesting for photocatalytical applications. It is also used as a white pigment for wall paint. In this work ZnO nanoparticles have been tested concerning their toxicity on A-549 cells, a human lung adenocarcinoma epithelial cell line. Two different exposure methods have been applied: the direct exposure of the cells at the air–liquid interface which was carried out with the Cultex® -Radial Flow System, and the exposure to a suspension of ZnO particles in culture medium. To characterize ZnO particles sizes, SEM picture were taken. To make sure, that the different nanoparticles do not agglomerate in the cell culture medium, DLS measurements of particle suspensions in water and in cell culture medium were performed. The viability of the cells after exposure was examined by the MTT-assay, a photometric method to determine the cell metabolism. Membrane integrity measurements were also performed after cell treatment by the measurements of LDH release. Both methods resulted in comparable effects. It was shown that ZnO nanoparticles have a toxic effect on A-549 cells in a concentration range between 10 ppm and 50 ppm in the culture medium. doi:10.1016/j.toxlet.2010.03.1163
P303-043 The effects of silver nanoparticles on male rats sperm quantity and quality 1 , A. Gajowik 1 , J. Radzikowska 1 , J. ´ M. Dobrzynska 2 , G. ´ Gromadzka-Ostrowska 2 , M. Oczkowski 2 , A. Krawczynska 3 4 5 5 Brunborg , M. Dusinska , A. Lankoff , M. Kruszewski 1
National Institute of Public Health - National Institute of Hygiene, United States, 2 Warsaw University of Life Sciences, Poland, 3 Norwegian Institute of Public Health, Norway, 4 Norwegian Institute of Air Research, Norway, 5 Institute of Nuclear Chemistry and Technology, Poland Nowadays silver nanoparticles (AgNPs) are widely used in consumer and biomedical products. The study was designed to assess the effects of acute exposure to different doses and particle sizes of AgNPs on the gonads and gametes of adult male rats. Adult Wistar rats were injected intravenously with 5 mg/kg or 10 mg/kg AgNPs of size 20 nm or 5 mg/kg of size 200 nm. Animals were sacrificed at 24 h, 1 week and 4 weeks after exposure. Testes and epididymes were removed and weighed; sperm count, morphology and DNA damage in male mice germ cells were estimated. There were no significant differences between testes and epididymes weights of exposed and unexposed rats. Sperm counts were slightly reduced 24 h after exposure to 5 mg/kg of 20 nm Ag. One and four weeks following the exposure, sperm counts were similar in all the AgNPs treated and the control groups. There were no significant differences between percentages of abnormal spermatozoa in exposed and unexposed groups. At 24 h after injection, comet tail moments in male germ testicular cells were significantly enhanced in all groups exposed to AgNPs. One week later, lower levels of DNA damage compared to results of 24 h were noted. Four weeks after the exposure, DNA damage in all experimental groups was lower than in control. At all time points, the highest values of comet tail moment were observed after exposure to 5 mg/kg of 20 nm Ag.