Background: There is a strong need for laboratory in vitro test systems for the toxicity of airborne particulate matter and nanoparticles. The measurement of oxidative stress potential offers a promising way forward. Objectives:Aworkshop was convened involving leading workers from the field in order to review the available test methods and to generate a Consensus Statement. Discussions: Workshop participants summarised their own research activities as well as discussion the relative merits of different test methods. Conclusions: In vitro test methods have an important role to play in the screening of toxicity in airborne particulate matter and nanoparticles. In vitro cell challenges were preferable to in vitro acellular systems but both have a potential major role to play and offer large cost advantages relative to human or animal inhalation studies and animal in vivo installation experiments. There remains a need to compare tests one with another on standardised samples and also to establish a correlation with the results of population-based epidemiology.
Compared with T lymphocytes from other organs, intestinal intraepithelial lymphocytes (IEL) proliferate weakly in response to CD3/TCR ligation, and they do not respond at all to treatment with other mitogenic stimuli. These signals also failed to induce expression of the IL-2R alpha-chain on the surface of most IEL. IEL from germ-free mice, from V gamma 1.1-transgenic mice, and from beta 2-microglobulin-deficient mice also gave a weak proliferative response. Therefore, the low proliferative response is not linked to the level of exposure to gut bacterial flora, the V gamma region expressed by the TCR-gamma delta + IEL, or the presence of class I molecules that may be recognized by CD8+ IEL. The relatively small amount of proliferation in response to TCR signaling, therefore, is not likely to be the result of induction of anergy caused by previous contact with Ag. In contrast, ligation of the CD3/TCR complex could elicit a rapid cytotoxic response and serine esterase release by IEL. The unusual functional capabilities and the activation state of IEL are independent of the TCR isotype expressed by these cells. Freshly isolated IEL have a high intracellular microtubule-associated protein kinase-2 (MAP-2K) activity level, further suggesting that these cells are activated despite their weak proliferative response. Consistent with this, MAP-2K is tyrosine-phosphorylated in both untreated and PMA-treated IEL. In contrast, MAP-2K activation and tyrosine phosphorylation occur in other T cells only when they are activated by PMA or other treatments. MAP-2K activity also is elevated in IEL from germ-free mice, demonstrating that activation does not depend on normal levels of exposure to bacterial flora. The activation of protein kinases such as MAP-2K could reflect the differentiation state of IEL or Ag receptor stimulation of some of these cells by epithelial cells in the preparation.
We demonstrate for 24 metal oxide (MOx) nanoparticles that it is possible to use conduction band energy levels to delineate their toxicological potential at cellular and whole animal levels. Among the materials, the overlap of conduction band energy (Ec) levels with the cellular redox potential (−4.12 to −4.84 eV) was strongly correlated to the ability of Co3O4, Cr2O3, Ni2O3, Mn2O3, and CoO nanoparticles to induce oxygen radicals, oxidative stress, and inflammation. This outcome is premised on permissible electron transfers from the biological redox couples that maintain the cellular redox equilibrium to the conduction band of the semiconductor particles. Both single-parameter cytotoxic as well as multi-parameter oxidative stress assays in cells showed excellent correlation to the generation of acute neutrophilic inflammation and cytokine responses in the lungs of C57 BL/6 mice. Co3O4, Ni2O3, Mn2O3, and CoO nanoparticles could also oxidize cytochrome c as a representative redox couple involved in redox homeostasis. While CuO and ZnO generated oxidative stress and acute pulmonary inflammation that is not predicted by Ec levels, the adverse biological effects of these materials could be explained by their solubility, as demonstrated by ICP-MS analysis. These results demonstrate that it is possible to predict the toxicity of a large series of MOx nanoparticles in the lung premised on semiconductor properties and an integrated in vitro/in vivo hazard ranking model premised on oxidative stress. This establishes a robust platform for modeling of MOx structure–activity relationships based on band gap energy levels and particle dissolution. This predictive toxicological paradigm is also of considerable importance for regulatory decision-making about this important class of engineered nanomaterials.
Although copper-containing nanoparticles are used in commercial products such as fungicides and bactericides, we presently do not understand the environmental impact on other organisms that may be inadvertently exposed. In this study, we used the zebrafish embryo as a screening tool to study the potential impact of two nano Cu-based materials, CuPRO and Kocide, in comparison to nanosized and micron-sized Cu and CuO particles in their pristine form (0-10 ppm) as well as following their transformation in an experimental wastewater treatment system. This was accomplished by construction of a modeled domestic septic tank system from which effluents could be retrieved at different stages following particle introduction (10 ppm). The Cu speciation in the effluent was identified as nondissolvable inorganic Cu(H2PO2)2 and nondiffusible organic Cu by X-ray diffraction, inductively coupled plasma mass spectrometry (ICP-MS), diffusive gradients in thin-films (DGT), and Visual MINTEQ software. While the nanoscale materials, including the commercial particles, were clearly more potent (showing 50% hatching interference above 0.5 ppm) than the micron-scale particulates with no effect on hatching up to 10 ppm, the Cu released from the particles in the septic tank underwent transformation into nonbioavailable species that failed to interfere with the function of the zebrafish embryo hatching enzyme. Moreover, we demonstrate that the addition of humic acid, as an organic carbon component, could lead to a dose-dependent decrease in Cu toxicity in our high content zebrafish embryo screening assay. Thus, the use of zebrafish embryo screening, in combination with the effluents obtained from a modeled exposure environment, enables a bioassay approach to follow the change in the speciation and hazard potential of Cu particles instead of difficult-to-perform direct particle tracking.
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