Metal Oxide Nanoparticles Induce Unique Inflammatory Footprints in the Lung: Important Implications for Nanoparticle Testing

Background

Metal oxide nanoparticles (NPs) have been widely used in industry, cosmetics, and biomedicine.

Objectives

We examined hazards of several well-characterized high production volume NPs because of increasing concern about occupational exposure via inhalation.

Methods

A panel of well-characterized NPs [cerium oxide (CeO₂NP), titanium dioxide (TiO₂NP), carbon black (CBNP), silicon dioxide (SiO₂NP), nickel oxide (NiONP), zinc oxide (ZnONP), copper oxide (CuONP), and amine-modified polystyrene beads] was instilled into lungs of rats. We evaluated the inflammation potencies of these NPs 24 hr and 4 weeks postinstillation. For NPs that caused significant inflammation at 24 hr, we then investigated the characteristics of the inflammation. All exposures were carried out at equal-surface-area doses.

Results

Only CeO₂NP, NiONP, ZnONP, and CuONP were inflammogenic to the lungs of rats at the high doses used. Strikingly, each of these induced a unique inflammatory footprint both acutely (24 hr) and chronically (4 weeks). Acutely, patterns of neutrophil and eosinophil infiltrates differed after CeO₂NP, NiONP, ZnONP, and CuONP treatment. Chronic inflammatory responses also differed after 4 weeks, with neutrophilic, neutrophilic/lymphocytic, eosinophilic/fibrotic/granulomatous, and fibrotic/granulomatous inflammation being caused respectively by CeO₂NP, NiONP, ZnONP, and CuONP.

Conclusion

Different types of inflammation imply different hazards in terms of pathology, risks, and risk severity. In vitro testing could not have differentiated these complex hazard outcomes, and this has important implications for the global strategy for NP hazard assessment. Our results demonstrate that NPs cannot be viewed as a single hazard entity and that risk assessment should be performed separately and with caution for different NPs.     

Biochemical Responses of Duckweed (Spirodela polyrhiza) to Zinc Oxide Nanoparticles

The present study focuses on the biochemical responses of the aquatic plant duckweed (Spirodela polyrhiza L.) to zinc oxide nanoparticles (ZnO NPs). Laboratory experiments were performed using a 96-h exposure to 25-nm NPs at different concentrations (0, 1, 10, and 50 mg/L). Growth, chlorophyll-to-pheophytin ratio (D665/D665a) and activities of superoxide dismutase, catalase, peroxidase (POD), and Na⁺, K⁺-ATPase were determined as indices to evaluate the toxicity of NPs in the culture medium. To understand better whether the Zn²⁺ released from the ZnO NP suspensions plays a key role in toxicity of the NPs, we investigated particle aggregation and dissolution in the medium. Furthermore, two exposure treatments for the group with the highest concentration (50 mg/L) were performed: (1) exposure for the full 96 h (50a treatment) and (2) the medium being replaced with culture medium without NPs after 12 h (50b treatment). Our results indicate that ZnO NPs induced adverse effects in S. polyrhiza at the concentration of 50 mg/L in the culture medium. Zn²⁺ released from the NPs might be the main source of its toxicity to this species.     

Exposure to Cerium Oxide Nanoparticles Is Associated With Activation of Mitogen-activated Protein Kinases Signaling and Apoptosis in Rat Lungs

Objectives:

With recent advances in nanoparticle manufacturing and applications, potential exposure to nanoparticles in various settings is becoming increasing likely. No investigation has yet been performed to assess whether respiratory tract exposure to cerium oxide (CeO₂) nanoparticles is associated with alterations in protein signaling, inflammation, and apoptosis in rat lungs.

Methods:

Specific-pathogen-free male Sprague-Dawley rats were instilled with either vehicle (saline) or CeO₂ nanoparticles at a dosage of 7.0 mg/kg and euthanized 1, 3, 14, 28, 56, or 90 days after exposure. Lung tissues were collected and evaluated for the expression of proteins associated with inflammation and cellular apoptosis.

Results:

No change in lung weight was detected over the course of the study; however, cerium accumulation in the lungs, gross histological changes, an increased Bax to Bcl-2 ratio, elevated cleaved caspase-3 protein levels, increased phosphorylation of p38 MAPK, and diminished phosphorylation of ERK-1/2-MAPK were detected after CeO₂ instillation (p<0.05).

Conclusions:

Taken together, these data suggest that high-dose respiratory exposure to CeO₂ nanoparticles is associated with lung inflammation, the activation of signaling protein kinases, and cellular apoptosis, which may be indicative of a long-term localized inflammatory response.     

Design and Development of Acetylthiocholine Electrochemical Biosensor Based on Zinc Oxide–Cerium Oxide Nanohybrid Modified Platinum Electrode

Acetylcholinesterase (AChE) enzyme has been predominantly used for the detection of pesticides and metal ions. But, these sensors respond to pesticides as well as metal ions at certain concentration, which results in poor selectivity. Hence in this work, the amount of thiocholine produced during AChE inhibition has been estimated to detect the residual activity of AChE enzyme in-turn to enhance the efficiency of the biosensor. In this context, Pt/ZnO–CeO₂/AChE/Chitosan based biosensor has been developed for sensitive voltammetric quantification of thiocholine in AChE. The sensor exhibited enhanced electron transfer rate, good conductivity and biocompatibility. Both the intrinsic and extrinsic parameters were simultaneously optimized using second order polynomial regression to get the best conditions for ATCh determination. Under optimized experimental conditions, the redox peak current was linear over the concentration range of 0.1–1.5 mM with detection and quantification limit of 0.05 and 0.15 μM respectively and the sensitivity of 1.47 μA mM^?1.     

Effects of Ce(III) and CeO2 Nanoparticles on Soil-Denitrification Kinetics

Cerium (Ce)-based compounds, such as CeO₂ nanoparticles (NPs), have received much attention in the last several years due to their popular applications in industrial and commercial uses. Understanding the impact of CeO₂ NPs on nutrient cycles, a subchronic toxicity study of CeO₂ NPs on soil-denitrification process was performed as a function of particle size (33 and 78 nm), total Ce concentration (50–500 mg L^?1), and speciation [Ce(IV) vs. Ce(III)]. The antimicrobial effect on the soil-denitrification process was evaluated in both steady-state and zero-order kinetic models to assess particle- and chemical-species specific toxicity. It was found that soluble Ce(III) was far more toxic than Ce(IV)O₂ NPs when an equal total concentration of Ce was evaluated. Particle size-dependent toxicity, species-dependent toxicity, and concentration-dependent toxicity were all observed in this study for both the steady-state and the kinetic evaluations. Changes in physicochemical properties of Ce(IV)O₂ NPs might be important in assessing the environmental fate and toxicity of NPs in aquatic and terrestrial environments.     

Acute and Chronic Response of Daphnia magna Exposed to TiO2 Nanoparticles in Agitation System

Aquatic toxicity of titanium dioxide nanoparticles (TiO₂ NPs) to Daphnia magna was characterized using a completely stirred bioassay system intended to keep particles in suspension thereby maintaining a consistent exposure. The 48-h LC₅₀ was 4.5 mg/L TiO₂ NPs, whereas LC₅₀ values for 7 and 14-days exposures were 2.7 and 1.9 mg/L, respectively. An exposure of 1.5 mg/L over a 21-days exposure resulted in significant reductions in fecundity. While reproduction was initially reduced in the 0.5 and 1.0 mg/L exposures, it recovered and was similar to the control by 21 days. For reproduction inhibition, NOEC was 1.0 mg/L. Exposure to 2.5 mg/L TiO₂ NPs resulted in 40 % of the organisms failing to become gravid; all surviving organisms exposed to 5.0 mg/L failed to become gravid. The increased sensitivity was due to the refinement in the bioassay system that kept NP in suspension resulting in consistent exposure concentrations.     

Toxic Effects of Acetochlor on Mortality, Reproduction and Growth of Caenorhabditis elegans and Pristionchus pacificus

The effects of acetochlor on the mortality, growth and reproduction of two nematode species were assessed. The LC₅₀ values for Caenorhabditis elegans and Pristionchus pacificus were 1,296 and 210.7 mg/L at 24 h, and 540.0 and 126.4 mg/L at 48 h exposure, respectively. In three succession generations, reproductive capacity was more sensitive in P. pacificus than in C. elegans. Moreover, the sublethal test endpoint of final length was more sensitive with P. pacificus. This study suggested that acetochlor had no long-term effects on C. elegans at lower concentrations. The higher concentrations of acetochlor (from 40 to 160 mg/L) revealed sublethal toxicity to the two tested species, with P. pacificus being more sensitive than C. elegans.     

Acute Toxicity of Heavy Metals to Tetrahymena in an In Vitro Experiment and Envelope Damage Study

The toxicity of Cr³⁺, Cu²⁺ and Cd²⁺ to Tetrahymena growth metabolism was studied by microcalorimetry at 28°C, and the growth constant (k), peak time (T) and generation times (T _G) were calculated. The metal ion concentrations that resulted in 50 % inhibition (IC ₅₀) of population growth were obtained through the dynamic parameters. The results indicated that the order of toxicity was Cd²⁺ > Cr³⁺ > Cu²⁺. Inductively coupled plasma-atomic emission spectrometry results suggested that the metal ions affected the permeability of the cell membrane. Observations of the Cd-exposed organisms by scanning electron microscopy revealed damage to the cell membrane in the form of an altered surface appearance. The cells suffered serious damage after sufficient acting time. Attenuated total reflection Fourier transform infrared spectra revealed that amide groups and PO₂ ^? of the phospholipid phospho-diester, both located in the hydrophobic end of the outer layer of the cell membrane, were most readily affected.     

Toxicity of Silver and Titanium Dioxide Nanoparticle Suspensions to the Aquatic Invertebrate, Daphnia magna

The purpose of this study was to investigate the 48 h acute toxicity of capped silver nanoparticles (AgNPs), and capped and uncapped titanium dioxide (nTiO₂) to Daphnia magna neonates. In addition, a 24 days chronic toxicity study was performed for D. magna exposed to uncapped nTiO₂ to evaluate effects on growth, reproduction and survival. The 48 h median lethal concentrations (LC₅₀) for carboxy-functionalized capped AgNPs and uncapped nTiO₂ were 2.75 μg/L and 7.75 mg/L, respectively. In contrast, no mortalities were observed for Daphnia exposed to carboxy-functionalized capped nTiO₂ at concentrations up to 30 mg/L. In the chronic toxicity experiment with uncapped nTiO₂, the growth, reproduction and survival of D. magna were significantly (p < 0.05) reduced at concentrations ranging from 4.5 to 7.5 mg/L. Growth and reproduction were reduced by 35 % and 93 %, respectively in the treatments at the highest uncapped nTiO₂ concentration (7.5 mg/L). Time to first reproduction was delayed by 2–3 days in D. magna and the test organisms produced only 1–2 broods over 24 days exposure to the highest concentration of uncapped nTiO₂. Overall, the results from the present study indicate that exposures of aquatic invertebrates to nanoparticles could have important ecological effects on lower trophic levels in aquatic ecosystems.     

Ecotoxicological Evaluation of Sewage Sludge Contaminated with Zinc Oxide Nanoparticles

The objective of this work was to evaluate the ecotoxicological qualitative risk associated with the use of sewage sludge containing Zn oxide nanoparticles (ZnO-NPs) as soil amendment. A sludge-untreated soil and two sludge-treated soils were spiked with ZnO-NPs (0–1,000 mg/kg soil). Soil ecotoxicity was assessed with Eisenia fetida (acute and sublethal end points), and the unfilterable and filterable (0.02 μm) soil leachates were tested with a battery of biomarkers using Chlorella vulgaris, Daphnia magna, and the fish cell line RTG-2 (Oncorhynchus mykiss). The production of E. fetida cocoons in sludge-treated soils was lower than that in sludge-untreated soils. The highest effect in the algal growth inhibition test was detected in sludge-untreated soil, most likely caused by the loss of organic matter in these samples. The D. magna results were always negative. Toxic effects (lysosomal cell function and production of reactive oxygen species) in RTG-2 cells were only observed in sludge-treated soils. In general, the toxicity of ZnO-NPs in sludge-treated soils was similar to that of sludge-untreated soil, and the filterable leachate fraction [Zn salt (Zn²⁺)] did not produce greater effects than the unfilterable fraction (ZnO-NPs). Thus, after the addition of ZnO-NP—enriched sewage sludge to agricultural soil, the risk of toxic effects for soil and aquatic organisms was shown to be low. These findings are important because repeated use of organic amendments such as sewage sludge may cause more and more increased concentrations of ZnO-NPs in soils over the long-term.     

Assessing the toxicity of contaminated soils using the nematode Caenorhabditis elegans as test organism

In this study, nine uncontaminated reference soils and 22 contaminated soils with different physico-chemical properties and contamination patterns were tested with a standardized toxicity test, using the nematode, Caenorhabditis elegans, as test organism. Fertility, growth and reproduction of C. elegans in the soils were compared with the exposure in standard soil Lufa St.2.2. C. elegans showed 100% fertility and a very low variability of growth in the reference soils. Although, reproduction varied considerably between the various reference soils, validity criteria (>30 offspring per test organism) were met in all reference soils. Moreover, Lufa St. 2.2 turned out to be a suitable and representative control soil. In order to clearly classify the effects of the polluted soils on C. elegans, toxicity thresholds were derived for nematode fertility (20% inhibition), growth (10% inhibition) and reproduction (40% inhibition) on the basis of the test inherent variability (MDD=minimal detectable difference), as well as their variability between the uncontaminated reference soils (MTI=maximal tolerable inhibition). The contaminated soils showed clear toxic effects on the nematodes, whereas the toxicity was better correlated to organic than to heavy metal contamination in bulk soil. Interestingly, the results of the nematode toxicity test were not well correlated with those of tests with oligochaetes, collembolans and plants, performed with the same soils, showing that the results are not redundant. The toxicity test using C. elegans turned out to be suitable for testing the toxicity of field collected soils and might by a valuable addition to soil test batteries.     

Regulation of Mitochondrial and Cytosol Antioxidant Systems of Fenugreek (<Emphasis Type="Italic">Trigonella foenum graecum</Emphasis> L.) Exposed to Nanosized Titanium Dioxide

In the present study, the interactions between nanoparticle (NP) exposure, root application and plants were examined. NPs are potentially responsible for conformational changes in polysaccharides, lipids, proteins, pectin, suberin and lignin molecules. 4 days of treatment with metal oxide caused a statistically significant increase in nicotinamide adénine dinucléotide oxidase activity in mitochondria and cytosol. Following exposure to TiO₂NP, even lipid peroxidation levels decreased in the mitochondria (leaves, stem and root) and in the cytosol (leaves and root), although it increased in the cytosol of the stem. Malondialdehyde accumulation was found to be higher in the cytosol compared to the mitochondria of stems, and in the cytosol of leaves and roots. NPs caused alterations in metabolism, antioxidant enzyme activities (guaiacol peroxidase, catalase and ascorbate peroxidase) and the generation of oxidative stress. Effects caused by exposures to NPs were influenced by differences in metabolic responses in plant parts, plant compartments, the period of exposure and the NP doses.     

Sensitivity of Danio rerio (Teleostei,Cyprinidae) During Two Stages of Development Based on Acute Toxicity Tests

The sensitivity of Danio rerio to three chemicals was compared at two growth stages [larval (10 ± 2 after hatching) and post-larval (60 ± 4 days after hatching)] based on acute toxicity tests. Thirty-nine 48 h acute toxicity tests were performed with the substances CuSO₄, NaCl and KCl. The 48 h LC₅₀ values at the two growth stages were compared by independent samples t-tests. The results showed a clear decrease in sensitivity when post-larval organisms were used. Since acute toxicity test methods for D. rerio that recommend using post-larval stage fish do not represent the most sensitive stage of the test organism, our study suggests a revision of the methods to use larval fish.     

Comparative Responses to Metal Oxide Nanoparticles in Marine Phytoplankton

A series of experiments was undertaken on three different marine microalgae to compare the effect of two metal oxide nanoparticles (NPs) on different physiological responses to stress: zinc oxide (ZnO), a known toxic compound for microalgae, and the never before tested yttrium oxide (Y₂O₃). The effect of these potential pollutants was estimated for different physiological variables and temporal scales: Growth, carbon content, carbon-to-nitrogen (C:N) ratio, and chlorophyll fluorescence were evaluated in long-term assays, and reactive oxygen species (ROS) production was evaluated in a short-term assay. Population growth was the most susceptible variable to the acute toxic effects of both NPs as measured in terms of number of cells and of biomass. Although Phaeodactylum tricornutum and Alexandrium minutum were negatively affected by ZnO NPs, this effect was not detected in Tetraselmis suecica, in which cell growth was significantly decreased by Y₂O₃ NPs. Biomass per cell was negatively affected in the most toxic treatments in T. suecica but was positively affected in A. minutum. ZnO treatments induced a sharper decrease in chlorophyll fluorescence and higher ROS than did Y₂O₃ treatments. The pronounced differences observed in the responses between the species and the physiological variables tested highlight the importance of analyzing diverse groups of microalgae and various physiological levels to determine the potential effects of environmental pollutants.     

Sublethal Toxic Effects of Nonylphenol Ethoxylate and Nonylphenol to Moina macrocopa

The aim of this paper was to examine the sublethal toxic effects of nonylphenol ethoxylate (NP₁₀EO), its primary degradation product nonylphenol (NP), and their mixture on Moina macrocopa. Chronic toxicity tests were carried out by using sublethal chemical concentrations. Results showed that all treatments reduced the survivorship, body length, and reproduction of M. macrocopa with NP being 10 %–20 % more toxic to M. macrocopa than NP₁₀EO. Results also indicated that the toxic effects of NP₁₀EO and NP mixture on M. macrocopa were more severe than that of any single chemical alone. At the highest concentration in this experiment, 0.337 mg L^?1 NP₁₀EO plus 0.0154 mg L^?1 NP treatment caused the survivorship of M. macrocopa to zero, neonates number of reproductions to zero, 45.5 % reduction in the body length, and 88 % reduction in the total neonates number.     

Enhanced sensitivity of skeletal muscle growth in offspring of mice long-term selected for high body mass in response to a maternal high-protein/low-carbohydrate diet during lactation

Aim

To investigate the effects of a high-protein/low-carbohydrate diet fed to mice of different genotypes during pregnancy and/or lactation on offspring skeletal muscle growth and metabolism.

Methods

Pregnant mice from strains selected for high body mass (DU6) or endurance running performance (DUhLB) and from an unselected control strain (DUK) were fed iso-energetic diets containing 20 % (C) or 40 % protein and low carbohydrate (HP) from mating to weaning at day 21 of age. At birth, offspring were cross-fostered resulting in different exposure to maternal prenatal-preweaning diets (C–C, HP–C, C–HP, HP–HP). Rectus femoris muscle of male mice (n = 291) was examined at day 23, 44, 181 and 396 of age for cellular growth and metabolism.

Results

At day 23 of age, body and muscle growth was retarded by 30–40 % (P < 0.0001) in response to the C–HP and HP–HP, but not to the HP–C diet, due to reduced fibre size (P < 0.0001) but not fibre number. DNA was highly reduced in DU6, less in DUhLB, but not in DUK muscle (strain × diet; P < 0.0001). Despite some compensation, muscle growth was still impaired (P < 0.001) in adulthood (day 44; day 181), but at senescence only in DU6 mice (strain × diet; P < 0.05). Only at weaning, isocitrate and lactate dehydrogenase activities were increased or decreased (P < 0.0001), respectively, without influence on fibre type composition.

Conclusion

A high-protein/low-carbohydrate diet fed to dams during lactation, but not during pregnancy, retards skeletal muscle growth in offspring with greater response of a heavy, obese compared with a physically fit and a control genotype and causes a transient shift towards oxidative versus glycolytic muscle metabolism.     

Effects of Physiochemical Properties of Test Media on Nanoparticle Toxicity to Daphnia magna Straus

The physicochemical property of standard test media significantly influenced the aggregation and dissolution of Ag, CuO and ZnO nanoparticles (NPs) and the toxicity of the NPs to Daphnia magna. For all the NPs, the highest amount of metal ions was released from the ISO medium, whereas acute toxicity to D. magna was highest in the moderately hard water medium (EC₅₀ = 4.94, 980, and 1,950 μg L^?1 for Ag, CuO, and ZnO, respectively). By comparing EC₅₀ values based on the total and dissolved concentrations of NPs with those of metal salt solutions, we found that both particulate and dissolved fractions were likely responsible for the toxicity of Ag NPs, whereas the dissolved fraction mostly contributed to the toxicity of CuO and ZnO NPs.     

Toxicity of ZnO Nanoparticles,ZnO Bulk,and ZnCl2 on Earthworms in a Spiked Natural Soil and Toxicological Effects of Leachates on Aquatic Organisms

The present study assessed the uptake and toxicity of ZnO nanoparticles (NPs), ZnO bulk, and ZnCl₂ salt in earthworms in spiked agricultural soils. In addition, the toxicity of aqueous extracts to Daphnia magna and Chlorella vulgaris was analyzed to determine the risk of these soils to the aquatic compartment. We then investigated the distribution of Zn in soil fractions to interpret the nature of toxicity. Neither mortality nor differences in earthworm body weight were observed compared with the control. The most sensitive end point was reproduction. ZnCl₂ was notably toxic in eliminating the production of cocoons. The effects induced by ZnO-NPs and bulk ZnO on fecundity were similar and lower than those of the salt. In contrast to ZnO bulk, ZnO-NPs adversely affected fertility. The internal concentrations of Zn in earthworms in the NP group were greater than those in the salt and bulk groups, although bioconcentration factors were consistently <1. No relationship was found between toxicity and internal Zn amounts in earthworms. The results from the sequential extraction of soil showed that ZnCl₂ displayed the highest availability compared with both ZnO. Zn distribution was consistent with the greatest toxicity showed by the salt but not with Zn body concentrations. The soil extracts from both ZnO-NPs and bulk ZnO did not show effects on aquatic organisms (Daphnia and algae) after short-term exposure. However, ZnCl₂ extracts (total and 0.45-μm filtered) were toxic to Daphnia.     

Effects of Five Antifouling Biocides on Settlement and Growth of Zoospores from the Marine Macroalga Ulva lactuca L.

Antifouling biocides are found in the marine ecosystem were they can affect non-target organisms. In this study the effects of five antifouling biocides on the settlement and growth of Ulva lactuca zoospores were investigated. The biocides investigated were copper (Cu²⁺), 4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (DCOIT), triphenylborane pyridine (TPBP), tolylfluanid and medetomidine. Full concentration–response curves where determined for each compound. EC₅₀ values were determined for copper, DCOIT, TPBP and tolylfluanid, all of which inhibited settlement and growth in a concentration dependent manner with the following toxicity ranking; tolylfluanid (EC₅₀ 80 nmol L^?1) ~ DCOIT (EC₅₀ 83 nmol L^?1) > TPBP (EC₅₀ 400 nmol L^?1) > Cu²⁺ (EC₅₀ 2,000 nmol L^?1). Medetomidine inhibited settlement and growth only at the extreme concentration of 100,000 nmol L^?1 (93 % effect). The low toxicity is possibly a consequence of a lack of receptors that medetomidine can bind to in the U. lactuca zoospores.