Janez Valant

Hazardous potential of manufactured nanoparticles identified by in vivo assay

New products of nanotechnologies, including nanoparticles, need to be assessed according to their biological reactivity and toxic potential. Given the large number of diverse nanomaterials, a tiered approach is favoured. The aim of our work presented here is to elaborate an in vivo assay with terrestrial invertebrates (Porcellio scaber), which could serve as a first step of hazard identification of nanoparticles. We adapted the widely used acridine orange/ethidium bromide (AO/EB) assay to be applicable for cell membrane stability assessment of entire organ where the animal was exposed in vivo. The digestive glands (hepatopancreas) of terrestrial isopods were taken as a model test system. The assay was validated with Cu(NO(3))(2) and surfactants. The results showed that all tested nanoparticles, i.e. nanosized TiO(2), nanosized ZnO and fullerenes (C(60)) have cell membrane destabilization potential. As expected, C(60) is the most biologically potent. The AO/EB in vivo assay proved to be fast because response is recorded after 30 min of exposure, relatively simple because digestive glands are inspected immediately after isolation from exposed animals and promising approach because different types of nanoparticles could be tested for their biological potential. This assay provides data for the identification of hazardous potential of nanoparticles before subsequent steps in a tiered approach are decided.

Effect of ingested titanium dioxide nanoparticles on the digestive gland cell membrane of terrestrial isopods.

The aim of this study was to find out whether ingested titanium dioxide nanoparticles (nano-TiO(2)) cause cell membrane damage by direct contact or by lipid peroxidation. We assessed lipid peroxidation and digestive gland cell membrane stability of animals fed on food dosed with nano-TiO(2). Conventional toxicity measures were completed to determine if cellular effects are propagated to higher levels of biological complexity. An invertebrate model organism (Porcellio scaber, Isopoda, Crustacea) was fed with food containing nanosized TiO(2) and the result confirmed that at higher exposure concentrations after 3 d exposure, nano-TiO(2) destabilized cell membranes but lipid peroxidation was not detected. Oxidative stress as evidenced by lipid peroxidation was observed at longer exposure durations and high exposure doses. These data suggest that cell membranes are destabilized by direct interactions between nanoparticles and cell membrane, not solely via oxidative stress.

CELL MEMBRANE INTEGRITY AND INTERNALIZATION OF INGESTED TiO2 NANOPARTICLES BY DIGESTIVE GLAND CELLS OF A TERRESTRIAL ISOPOD

The present study was motivated by the paucity of reports on cellular internalization of ingested titanium dioxide (TiO(2)) nanoparticles (nano-TiO(2)). The model invertebrate (Porcellio scaber, Isopoda, Crustacea) was exposed to food dosed with nano-TiO(2) containing 100, 1,000, 3,000, or 5,000 µg nano-TiO(2) per gram of food. After 14 d of exposure, the amount of Ti in the entire body was analyzed by inductively coupled plasma-mass spectrometry, and elemental analyses of tissue cross sections were performed by particle induced X-ray emission. In addition, a series of toxicological markers including feeding parameters, weight change, and survival, as well as cytotoxic effects such as digestive gland cell membrane stability, were monitored. Internalization of ingested nano-TiO(2) by the isopods digestive gland epithelial cells was shown to depend on cell membrane integrity. Cell membranes were found to be destabilized by TiO(2) particles, and at higher extracellular concentrations of nano-TiO(2), the nanoparticles were internalized.

Effects of magnetic cobalt ferrite nanoparticles on biological and artificial lipid membranes

Background The purpose of this work is to provide experimental evidence on the interactions of suspended nanoparticles with artificial or biological membranes and to assess the possibility of suspended nanoparticles interacting with the lipid component of biological membranes. Methods 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicles and human red blood cells were incubated in suspensions of magnetic bare cobalt ferrite (CoFe2O4) or citric acid (CA)-adsorbed CoFe2O4 nanoparticles dispersed in phosphate-buffered saline and glucose solution. The stability of POPC giant unilamellar vesicles after incubation in the tested nanoparticle suspensions was assessed by phase-contrast light microscopy and analyzed with computer-aided imaging. Structural changes in the POPC multilamellar vesicles were assessed by small angle X-ray scattering, and the shape transformation of red blood cells after incubation in tested suspensions of nanoparticles was observed using scanning electron microscopy and sedimentation, agglutination, and hemolysis assays. Results Artificial lipid membranes were disturbed more by CA-adsorbed CoFe2O4 nanoparticle suspensions than by bare CoFe2O4 nanoparticle suspensions. CA-adsorbed CoFe2O4-CA nanoparticles caused more significant shape transformation in red blood cells than bare CoFe2O4 nanoparticles. Conclusion Consistent with their smaller sized agglomerates, CA-adsorbed CoFe2O4 nanoparticles demonstrate more pronounced effects on artificial and biological membranes. Larger agglomerates of nanoparticles were confirmed to be reactive against lipid membranes and thus not acceptable for use with red blood cells. This finding is significant with respect to the efficient and safe application of nanoparticles as medicinal agents.

The importance of a validated standard methodology to define in vitro toxicity of nano-TiO 2

Several in vitro studies on the potential toxicity of nano-TiO2 have been published and recent reviews have summarised them. Most of these reports concluded that physicochemical properties of nanoparticles are fundamental to their toxicological effects. No published review has compared in vitro tests with similar test strategies in terms of exposure duration and measured endpoints and for this reason we have attempted to assess the degree of homogeneity among in vitro tests and to assess if they afford reliable data to support risk assessment. The responses in different in vitro tests appeared to be unrelated to primary particle size. The biologically effective concentrations in different tests can be seen to differ by as many as two orders of magnitude and such differences could be explained either by different sensitivities of cell lines to nanoparticles or by effect of the test media. Our review indicates that even when the in vitro tests measure the same biomarkers with the same exposure duration and known primary particle sizes, it is insufficient merely to use such data for risk assessment. In the future, validated standard methods should include a limited number of cell lines and an obligatory selection of biomarkers. For routine purposes, it is important that assays can be easily conducted, false negatives and false positives are excluded and unbiased interpretation of results is provided. Papers published to date provide an understanding of the mode on nano-TiO2 action but are not suitable for assessment and management of risk.

Lysosomal membrane stability in laboratory- and field-exposed terrestrial isopods Porcellio scaber (Isopoda, Crustacea)

Two established methods for assessment of the cytotoxicity of contaminants, the lysosomal latency (LL) assay and the neutral red retention (NRR) assay, were successfully applied to in toto digestive gland tubes (hepatopancreas) of the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). In vitro exposure of isolated gland tubes to copper was used as a positive control to determine the performance of the two methods. Lysosomal latency and the NRR assay were then used on in vivo (via food) laboratory-exposed animals and on field populations. Arbitrarily selected criteria for determination of the fitness of P. scaber were set on the basis of lysosomal membrane stability (LMS) as assessed with in toto digestive gland tubes. Decreased LMS was detected in animals from all polluted sites, but cytotoxicity data were not in agreement with concentrations of pollutants. Lysosomal membrane stability in the digestive gland tubes of animals from an environment in Idrija, Slovenia that was highly polluted with mercury (260 microg/g dry wt food and 1,600 microg/g dry wt soil) was less affected than LMS in laboratory animals fed with 5 and 50 microg Hg/g dry weight for 3 d. This probably indicates tolerance of P. scaber to mercury in the mercury-polluted environment and/or lower bioavailability of environmental mercury. In animals from the vicinity of a thermal power plant with environmental mercury concentrations three to four orders of magnitude lower than those in Idrija, LMS was severely affected. In general, the LL assay was more sensitive than the NRR assay. The LMS assay conducted on digestive gland tubes of terrestrial isopods is highly recommended for integrated biomarker studies.

Acclimation of Tetrahymena thermophila to bulk and nano-TiO2 particles by changes in membrane fatty acids saturation

We provide experimental evidence that changes in the membrane fatty acid profile of Tetrahymena thermophila incubated with nano- or bulk TiO(2) particle are not accompanied by ROS generation or lipid peroxidation. Consequently these changes are interpreted as acclimation to unfavorable conditions and not as toxic effects. T. thermophila cells were exposed to TiO(2) particles at different concentrations for 24h at 32°C. Treatment of cultures with nano- and bulk TiO(2) particles resulted in changes of membrane fatty acid profile, indicating increased membrane rigidity, but no lipid peroxidation or ROS generation was detected. There were no differences in membrane composition when T. thermophila was exposed to nanosized or bulk-TiO(2) particles. We also observed reversible filling of food vacuoles, but this was different in case of nano- or bulk TiO(2) exposure. Our results suggest that interactions of particles and cell membranes are independent of oxidative stress.

Internalization of consumed TiO 2 nanoparticles by a model invertebrate organism

There is little in vivo data concerning the fate of ingested TiO2 nanoparticles (nano-TiO2). We report here experiments aimed at assessing if ingested nano-TiO2 accumulates in the digestive gland epithelium or are internalized elsewhere in the body of the terrestrial isopod crustaceans. The animals (Porcellio scaber, Isopoda, Crustacea) fed for 3, 7, or 14 days on food dosed with 100 or 1000 µg nano-TiO2 showed no evidence of internalization of Ti measured by microparticle-induced X-ray emission method. The effect of ingested nanoparticles was measured by conventional toxicity measures such as feeding rate, weight change, andmortality and did not indicate any toxicity. However, cell membrane of digestive glands, measured with a modified method for assessing cell membrane stability, was affected already after 3 days of exposure to 1000 µg nano-TiO2 per gram dry weight of food indicating cytotoxic potential of ingested nanoparticles. Our results confirmed hypothesis on low toxic potential and no internalization of consumed TiO2 nanoparticles by a model invertebrate organism. However, cytological marker unequivocally indicated adverse effect of ingested nano-TiO2. We conclude that the isopod model system could be used for studying the fate and effect of ingested nanoparticles.

Biological reactivity of nanoparticles: mosaics from optical microscopy videos of giant lipid vesicles

Emerging fields such as nanomedicine and nanotoxicology, demand new information on the effects of nanoparticles on biological membranes and lipid vesicles are suitable as an experimental model for bio-nano interaction studies. This paper describes image processing algorithms which stitch video sequences into mosaics and recording the shapes of thousands of lipid vesicles, which were used to assess the effect of CoFe(2)O(4) nanoparticles on the population of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid vesicles. The applicability of this methodology for assessing the potential of engineered nanoparticles to affect morphological properties of lipid membranes is discussed.

A new approach to analyse effects of nanoparticles on lipid vesicles

Manufactured nanoparticles are potentially capable of inducing defects in lipid membranes. The effects of nanoparticles on cell membranes are one of the key issues in nanomedicine, nanotoxicology, food and pharmaceutical application of products of nanomaterials and others. Our aim is to demonstrate the nanoparticle - lipid vesicle interactions and to develop a controllable experimental setup for data acquisition. We studied interactions between nanoparticles (C60) and lipid vesicles (POPC), using ZnCl2 as a positive control. Light microscopy computer aided image segmentation was developed and population differences among vesicles incubated in different media were assessed. Data obtained by statistical image analysis methods revealed that nanoparticles (C60) caused changes in vesicle size distribution in the population of lipid vesicles as well as a burst of vesicles in time and in a concentration gradient. No significant changes in shape of vesicles were recorded. The advantage of the experimental set up presented here is that it employs statistical image analysis methods and direct microscopy observation of large populations of lipid vesicles. We discuss the applicability of this in vitro approach in analysing the effects of nanoparticles on simplified biological membranes.