Dongwook Kwon

Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

Background Titanium dioxide (TiO2) has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined. Methods and results In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70) together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation. Conclusion These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax. Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials.

Non-monotonic concentration–response relationship of TiO2 nanoparticles in freshwater cladocerans under environmentally relevant UV-A light

The effects of UV-A on the toxicity of TiO2 nano-particles (NPs) were evaluated using Moina macrocopa and Daphnia magna under environmentally relevant level of UV-A. The waterfleas were exposed to TiO2 NPs with different sizes of ~298nm, ~132nm, or ~72nm for up to 48h, with or without UV-A light. Whole body reactive oxygen species and transcription of antioxidant enzyme genes were measured, as well as the survival of the waterflea. In the presence of UV-A, the survival rates of M. macrocopa significantly decreased in concentration dependent way until ~1mg/L TiO2 NPs, but the survivals were reversed at greater concentrations. This peculiar non-monotonic trend of concentration-response relationship might be explained by changes of particle size under different light conditions. TiO2 NPs within a certain size range could be trapped in the filter apparatus and exert toxicity, and the NPs of greater size were subject to either precipitation or ingestion leading to no or little toxicity. Observed TiO2 toxicity was associated with oxidative stress in the filter apparatus. The results of this study showed that the size change due to UV-A irradiation should be considered in evaluation of ecological risks of TiO2 NP.

Dispersion, fractionation and characterization of sub-100nm P25 TiO2 nanoparticles in aqueous media

Despite of the widespread use of manufactured nanomaterials and increasing concerns on their potential risk, only limited information is available on their physicochemical properties in toxicologically relavant aqueous media. Here, P25 TiO2 (Evonik GmbH), one of the well-known and widely-used photocatalytic TiO2, was dispersed and fractionated in aqueous media, and its physicochemical properties, especially for its sub-100 nm fraction, was carefullly studied. The colloidal properties of TiO2 nanoparticles, such as ag]glomeration and sedimentation, were found strongly dependent on the physicochemical characteristics of nanoparticles (e.g., hydrodynamic size distribution, type of capping ligands and surface charge) as well as those of the aqueous media used (e.g., ionic strength and chemical compositions). This study has shown the importance of standardized dispersion and characterization protocol for toxicity tests, which is urgently needed for reliable, reproducible and impartial toxicity tests of manufactured nanomaterials.

Effects of surface-modifying ligands on the colloidal stability of ZnO nanoparticle dispersions in in vitro cytotoxicity test media

The extrinsic physicochemical properties of nanoparticles (NPs), such as hydrodynamic size, surface charge, surface functional group, and colloidal stabilities, in toxicity testing media are known to have a significant influence on in vitro toxicity assessments. Therefore, interpretation of nanotoxicity test results should be based on reliable characterization of the NPs’ extrinsic properties in actual toxicity testing media. Here, we present a set of physicochemical characterization results for commercially available ZnO NPs, including core diameter, hydrodynamic diameter, surface charges, and colloidal stabilities, in two in vitro toxicity testing media (Roswell Park Memorial Institute [RPMI] and Dulbecco’s Modified Eagle’s Medium [DMEM]), as well as simple cell viability assay results for selected ZnO NPs. Four commercially available and manufactured ZnO NPs, with different core sizes, were used in this study, and their surface charge was modified with five different surface coating materials (sodium citrate, tris(2-aminoethyl)amine, poly(acrylic acid), poly(allylamine hydrochloride), and poly-L-lysine hydrochloride). The results showed that ZnO NPs were better dispersed in cell culture media via surface modification with positively or negatively charged molecules. Moreover, in the presence of fetal bovine serum (FBS) in RPMI and DMEM media, ZnO NPs were found even better dispersed for a longer period (at least 48 hours). For the HeLa cells exposed to ZnO NPs in DMEM media without FBS, surface charge-dependent cytotoxicity trends were observed, while these trends were not observed for those cells cultured in FBS-containing media. This confirmed the important roles of surface-modifying compounds and of surface charge on the resultant cytotoxicities of NPs.

In vivo biodegradation of colloidal quantum dots by a freshwater invertebrate, Daphnia magna

Impacts of planktonic invertebrate, Daphnia magna, on the speciation of colloidal quantum dots (QD) were investigated using fluorescence spectromicroscopic technique. Well-dispersed (GA/TOPO)QD were prepared by forming a supramolecular assembly of hydrophobic (TOPO)QD with biomacromolecules (i.e., Gum Arabic, GA). Biological degradation of this nanomaterial was monitored by fluorescence spectromicroscopic methods. Our study confirmed the major uptake pathway of manufactured nanomaterials and in vivo biodegradation processes in a well-known toxicity test organism, D. magna. In addition, we also found that D. magna can induce significant deterioration of aquatic media by releasing fragments of partially degraded QD colloids. These biological processes may significantly change the predicted toxicities of nanomaterials in aquatic environments. Thus, we propose that the impacts of aquatic living organisms on the environmental fate of manufactured nanomaterials (MNs) should be carefully taken into account when assessing the risk of MNs to the environment and human health.

An optimized dispersion of manufactured nanomaterials forin vitro cytotoxicity assays

Colloidal characteristics of manufactured nanomaterials (e.g., hydrodynamic size distributions, surface charges, agglomeration, and sedimentation) in the actual toxicity test media have significant effects on the results ofin vitro toxicity assessments. Here, colloidal properties of widely used photocatalytic TiO2 (P25, Degussa Evonik GmbH) in commonly-usedin vitro cell culture media were carefully studied as a function of media types and their components (e.g., fetal bovine serum concentration). Temporal changes of optical densities at 340 nm and corresponding hydrodynamic size distributions were monitored for more than 48 hours by using UV-vis absorbance spectrometer and dynamic light scattering instruments. The colloidal stabilities of TiO2 dispersions were found strongly dependent on the added FBS concentrations as well as the type of the cell culture media used. Additionally, optimized dispersion and characterization protocol were proposed for easy and efficient way to monitor appropriate nanoparticle dosimetryin vitro.

Potential risks of the natural nanoparticles from the acid mine drainage and a novel approach for their toxicity assessment

Acid mine drainage (AMD) is a serious environmental problem due to its acidic pH and high contents of heavy metal ions. Thus, assessment of AMD toxicity has been widely investigated at individual, physiological and molecular levels using various test organisms. However, most studies focused on the toxicity of whole AMD while very few studies identified the toxicity of natural nanoparticles (NPs) originated from AMD. In AMD systems, natural NPs such as amorphous hydroxides, ferrihydrites, schwertmannite and goethite seemed to be formed, which could induce letahl and sublethal toxicity toward aquatic organisms. In this review, we summerized the toxicity of whole AMD and manufactured NPs, and suggested a novel approach for toxicity assessment of natural NPs from AMD.

Effects of Size, Impurities, and Citrate Capping on the Toxicity of Manufactured Silver Nano-particles to Larval Zebrafish (Danio rerio)

Objectives: This study was conducted to identify factors determining the toxicity of manufactured silver nanoparticles (AgNPs) on aquatic organisms. Methods: For this purpose, we prepared several AgNPs with varied characteristics, including hydrodynamic size (nano-ABCAgCit vs μ-sized-ABCAgCit), impurities (ABCAg stock vs ABCAg), and citrate capping (ABCAgCit), using a commercially available manufactured AgNP (ABCAg stock). Acute tests were conducted using larval zebrafish (Danio rerioI). In addition, in order to determine the ecotoxicological potentials of various capping agents, toxicity tests were conducted with microbes, waterfleas, and fish for eight different capping agents that are used for NPs. Results: The toxicity of AgNPs in terms of 96 h fish LC50 increased in the following order: ABCAg stock Conclusions: In this study, we found that for acute lethality, the contribution of impurities and particle size was significant, but that of citrate was negligible.

Preparation and characterization of stable nano-Ag dispersions for nanotoxicological studies

In spite of the increasing importance of manufactured nano materials, only limited information is available on the behavior of their colloidal suspensions (e.g., agglomeration, aggregation and precipitation) in the media. In this study, we investigated the effects of capping ligand concentrations (e.g. citrate and fetal bovine serum) on the colloidal stability of silver nanoparticle (nano-Ag) in relevant toxicity test media. By monitoring their sedimentation and agglomeration behavior, the optimum conditions for stable colloidal nano-Ag suspensions were found for nanotoxicological studies. The agglomeration and sedimentation kinetics were slowed down as the ligand concentration increased. When citrate concentration in the MHW was increased from 4.54 to 45.45 ([citrate]/[nano-Ag]), the size of the agglomerates was significantly reduced from 178.0 ± 7.0 to 14.7 ± 1.4 nm. On the other hand, reduction of hydrodynamic sizes from 330.3 ± 18.6 to 75.7 ± 9.3 nm were observed when the concentration of FBS in RPMI-1640 media increased from 0.01 to 10%.

Current limitations and challenges of nanoparticle toxicity assessments

Due to the increasing concerns on the potential risk of manufactured nanoniaterials. understanding their toxicity mechanism, environmental fates, and impacts on human health and ecosystems became one of the urgent scientific issues to be addressed. Here, based on our recent studies on the P25 Ti02 (Evonik GmbH) dispersion, current limitations and challenges in nanoparticle toxicity assessments will be discussed. Particularly, among the many issues of nanoparticle toxicology, we will focus on the issues of the appropriate dosing metrics of NPs and effects of various physicochemical properties on the dosing metrics as well as the limitations of current cytotoxicity assay protocols for the assessment of NP toxicity.