Nadia Rachel Von Moos

Oxidative stress induced by inorganic nanoparticles in bacteria and aquatic microalgae – state of the art and knowledge gaps

Abstract Nanotechnology has revolutionised many areas of modern life, technology and research, which is reflected in the steadily increasing global demand for and consumption of engineered nanomaterials and the inevitable increase of their release into the environment by human activity. The overall long-term impact of engineered nanomaterials on ecosystems is still unknown. Various inorganic nanoparticles have been found to exhibit bactericidal properties and cause growth inhibition in model aquatic microalgae, but the mechanisms of toxicity are not yet fully understood. The causal link between particle properties and biological effects or reactive oxygen species generation is not well established and represents the most eminent quest of nanoecotoxicological investigation. In this review, the current mechanistic understanding of the toxicity of inorganic metal and metal oxide engineered nanomaterials towards bacterial and aquatic microalgal model organisms based on the paradigm of oxidative stress is presented along with a detailed compilation of available literature on the major toxicity factors and research methods.

Effects of copper-oxide nanoparticles, dissolved copper and ultraviolet radiation on copper bioaccumulation, photosynthesis and oxidative stress in the aquatic macrophyte Elodea nuttallii.

In this study, the uptake and sub-toxic effects of CuO nanoparticles (CuO-NPs), dissolved Cu(II) alone or in combination with UV radiation on the aquatic macrophyte Elodea nuttallii were studied. Emphasis was on Cu accumulation, growth, photosynthesis and the oxidative stress related enzymes peroxidase (POD) and superoxide dismutase (SOD). The results showed stronger Cu accumulation in plants exposed to 10 mg L(-1) CuO-NPs, corresponding to 1.4-2 mg L(-1) dissolved Cu(II), than to 256 μg L(-1) Cu(II). However, the ratio between the accumulated Cu and dissolved Cu in CuO treatments was lower than in Cu(II) treatments. Additional UV exposure increased accumulation in both treatments, with the effect being stronger for Cu accumulation from CuO-NPs than for dissolved Cu(II). Photosynthetic capacity was strongly reduced by UV treatment, whereas remained unaffected by Cu(II) or CuO-NP treatments. Similarly, the increase of SOD activity was more pronounced in the UV treatments. On the other hand, POD activity enhancement was strongest in the plants exposed to CuO-NPs for 24 h. Expression of the copper transporter COPT1 as revealed by RT-qPCR was inhibited by Cu(II) and CuO-NP treatment, limiting the uptake of excess Cu into the cells. Overall, the combined exposure of E. nuttallii to UV radiation with CuO-NPs or Cu(II) has a higher impact than exposure to CuO-NPs or Cu(II) alone. The results imply that heavy pollution of natural water with CuO-NPs or dissolved Cu might have stronger effects in combination with natural UV irradiation on organisms in situ.

Dynamics of sub-lethal effects of nano-CuO on the microalga Chlamydomonas reinhardtii during short-term exposure

Though nano-CuO has been classified as toxic toward aquatic microorganisms and its use in various applications is expected to increase in near future, its ecotoxicity is currently poorly understood. The aim of this study was to investigate the mechanisms of nano-CuO toxicity based on the paradigm of oxidative stress, the dynamics of response over 24h, and the modulating effect of exposure conditions on toxicity and responses. To this end, the model microalga Chlamydomonas reinhardtii was exposed to 10mgL(-1) nano-CuO in five different exposure media, including two different growth media, two Goods buffers, and natural lake water. The measured endpoints included cell growth, morphological aspect, chlorophyll autofluorescence, oxidative stress, and membrane permeabilization. The results suggest that agglomerated nano-CuO is toxic and that exposure media are decisive in whether or not particles or free Cu ions are the main mediators of toxicity. A significant particle effect was only observed in the Goods buffer 3-(N-morpholino) propanesulfonic acid. However, nano-CuO particles especially influenced the dynamics of response early in exposure, between 0h and 5h, suggesting that an adaptation to particle stress may occur or that particles modify the bioavailability of the free Cu ions in early exposure.

Portable oxidative stress sensor: Dynamic and non-invasive measurements of extracellular H2O2 released by algae

Reactive oxygen species (ROS) generated by aerobic organisms are essential for physiological processes such as cell signaling, apoptosis, immune defense and oxidative stress mechanisms. Unbalanced oxidant/antioxidant budgets are involved in many diseases and, therefore, the sensitive measurement of ROS is of great interest. Here, we present a new device for the real-time monitoring of oxidative stress by measuring one of the most stable ROS, namely hydrogen peroxide (H2O2). This portable oxidative stress sensor contains the heme protein cytochrome c (cyt c) as sensing element whose spectral response enables the detection of H2O2 down to a detection limit of 40 nM. This low detection limit is achieved by introducing cyt c in a random medium, enabling multiscattering that enhances the optical trajectory through the cyt c spot. A contact microspotting technique is used to produce reproducible and reusable cyt c spots which are stable for several days. Experiments in static and microfluidic regimes, as well as numerical simulations demonstrate the suitability of the cyt c/H2O2 reaction system for the real-time sensing of the kinetics of biological processes without H2O2 depletion in the measurement chamber. As an example, we detect the release of H2O2 from the green alga Chlamydomonas reinhardtii exposed to either 180 nM functionalized CdSe/ZnS core shell quantum dots, or to 10 mg/l TiO2 nanoparticles. The continuous measurement of extracellular H2O2 by this optical sensor with high sensitivity is a promising new means for real-time cytotoxicity tests, the investigation of oxidative stress and other physiological cell processes.

New insights into ROS dynamics: a multi-layered microfluidic chip for ecotoxicological studies on aquatic microorganisms

Abstract Reactive oxygen species (ROS) play an important role in the life of every cell, including cellular defense and signaling mechanisms. Continuous and quantitative ROS sensing can provide valuable information about the cell state, but it remains a challenge to measure. Here, we introduce a multi-layered microfluidic chip with an integrated optical sensor for the continuous sensitive detection of extracellular hydrogen peroxide (H2O2), one of the most stable ROS. This platform includes hydraulically controlled microvalves and microsieves, which enable the precise control of toxicants and complex exposure sequences. In particular, we use this platform to study the dynamics of toxicity-induced ROS generation in the green microalga Chlamydomonas reinhardtii during short-term exposures, recovery periods, and subsequent re-exposures. Two cadmium-based toxicants with distinct internalization mechanisms are used as stress inducers: CdSe/ZnS quantum dots (Qdots) and ionic cadmium (Cd2+). Our results show the quantitative dynamics of ROS generation by the model microalga, the recovery of cell homeostasis after stress events and the cumulative nature of two consecutive exposures. The dissolution of quantum dots and its possible influence on toxicity and H2O2 depletion is discussed. The obtained insights are relevant from ecotoxicological and physiological perspectives.

Pro-oxidant effects of nano-TiO2 on Chlamydomonas reinhardtii during short-term exposure

This study sheds light on the short-term dynamics of pro-oxidant processes related to the exposure of C. reinhardtii microalgae to nano-TiO2 using (a) conventional fluorescence probes for cellular pro-oxidant process and (b) a recently developed cytochrome c biosensor for the continuous quantification of extracellular H2O2. The main aims are to investigate nano-TiO2 toxicity and the modifying factors thereof based on the paradigm of oxidative stress and to explore the utility of extracellular H2O2 as a potential biomarker of the observed cellular responses. This is the first study to provide continuous quantitative data on abiotic and biotic nano-TiO2-driven H2O2 generation to systematically investigate the link between extracellular and cellular pro-oxidant responses. Acute exposures of 1 h were performed in two different exposure media (MOPS and lake water), with nominal particle concentrations from 10 mg L−1 to 200 mg L−1, with and without UV pre-illumination. Abiotic and biotic extracellular H2O2 were continuously measured with the biosensor and complemented with endpoints for abiotic ROS (H2DCF-DA), oxidative stress (CellROX® Green) and damage (propidium iodide) measured by flow cytometry at the beginning and end of exposure. Results showed that nano-TiO2 suspensions generated ROS under UV light (abiotic origin) and promoted ROS accumulation in C. reinhardtii (biotic origin). However, extracellular and intracellular pro-oxidant processes differed. Hence, extracellular H2O2 cannot per se serve as a predictor of cellular oxidative stress or damage. The main predictors best describing the cellular responses included “exposure medium”, “exposure time”, “UV treatment” as well as “exposure concentration”.

Beyond Unpredictability: The Importance of Reproducibility in Understanding the Protein Corona of Nanoparticles

While it is well established that the surface of a nanoparticle plays a pivotal role for the protein corona, the vast number of proteins present in biological media render general conclusions about affinities between nanoparticle surfaces and proteins nontrivial. Recently published articles increasingly reveal differences between systems and an ever increasing number of influencing factors for the protein corona. In contrast, the present study posits that the reported differences may, at least in part, be due to poor experimental design, which leads to biased results. The present study investigates protein adsorption onto silica nanoparticles with different chemical groups on the surface by the statistical analysis of triplicate measurements as well as control measurements. We demonstrate that 60% of the identified protein types did not have any significant affinities for the nanoparticles. Of the remaining 40%, 60% were driven by surface charges and only 40% preferentially adsorbed onto specific surface groups. Furthermore, we found that of the 20 most abundant proteins in the serum, only five bound to the nanoparticles studied here. We illustrate the importance of control replicate experiments to avoid exaggerated differences between systems and to properly quantify the differences and similarities between comparable systems.