Immediate and Sustained Effects of Cobalt and Zinc-Containing Pigments on Macrophages.

Pigments are among the oldest nanoparticulate products known to mankind, and their use in tattoos is also very old. Nowadays, 25% of American people aged 18 to 50 are tattooed, which poses the question of the delayed effects of tattoos. In this article, we investigated three cobalt [Pigment Violet 14 (purple color)] or cobalt alloy pigments [Pigment Blue 28 (blue color), Pigment Green 14 (green color)], and one zinc pigment [Pigment White 4 (white color)] which constitute a wide range of colors found in tattoos. These pigments contain microparticles and a significant proportion of submicroparticles or nanoparticles (in either aggregate or free form). Because of the key role of macrophages in the scavenging of particulate materials, we tested the effects of cobalt- and zinc-based pigments on the J774A.1 macrophage cell line. In order to detect delayed effects, we compared two exposure schemes: acute exposure for 24 hours and an exposure for 24 hours followed by a 3-day post-exposure recovery period. The conjunction of these two schemes allowed for the investigation of the delayed or sustained effects of pigments. All pigments induced functional effects on macrophages, most of which were pigment-dependent. For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4 showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigments tested induced a slight but significant increase in tumor necrosis factor secretion. This effect, however, was transitory. Conversely, only Pigment Blue 28 induced both a short and sustained increase in interleukin 6 secretion. Results showed that in response to bacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declined after exposure to pigments followed by a recovery period. For chemoattractant cytokines (MCP-1 or MIP-1α), delayed effects were observed with a secretion decreased in presence of Pigment Blue 28 and Pigment violet 14, both with or without LPS stimuli. The pigments also induced persisting changes in some important macrophage membrane markers such as CD11b, an integrin contributing to cell adhesion and immunological tolerance. In conclusion, the pigments induced functional disorders in macrophages, which, in some cases, persist long after exposure, even at non-toxic doses.

A Low-Serum Culture System for Prolonged in Vitro Toxicology Experiments on a Macrophage System.

Immunotoxicology sensu lato comprises not only toxicity toward immune cells, but also biological reactions from immune cells exposed to toxicants, reactions that may have deleterious effects at the organismal level. Within this wide frame, a specific case of interest is represented by the response of macrophages to particulate materials, with the epitome examples of asbestos and crystalline silica. For such toxicants that are both persistent and often encountered in an occupational setting, i.e. at low but repeated doses, there is a need for in vitro systems that can take into account these two parameters. Currently, most in vitro systems are used in an acute exposure mode, i.e., with a single dose and a readout made shortly if not immediately after exposure. We describe here how adequate changes of the culture methods applied to the murine macrophage cell line J774A.1 enable longer periods of culture (several days), which represents a first opportunity to address the persistence and dose-rate issues. To respond to this, the protocol uses a reduction in the concentration of the animal serum used for cell culture, as well as a switch from fetal to adult serum, which is less rich in proliferation factors. By doing so, we have considerably reduced cell proliferation, which is a problem with cell lines when they are supposed to represent slowly-dividing cells such as resident macrophages. We also succeeded in maintaining the differentiated functions of macrophages, such as phagocytosis or inflammatory responses, over the whole culture period. Furthermore, the presence of serum, even at low concentrations, provides excellent cell viability and keeps the presence of a protein corona on particulate materials, a feature that is known to strongly modulate their effects on cells and is lost in serum-free culture. Besides data showing the impact of these conditions on macrophages cell line cultures, illustrative examples are shown on silica- and cobalt-based pigments.

A proteomic view of cellular responses of macrophages to copper when added as ion or as copper-polyacrylate complex.

Copper is an essential metal for life, but is toxic at high concentrations. In mammalian cells, two copper transporters are known, CTR1 and CTR2. In order to gain insights on the possible influence of the import pathway on cellular responses to copper, two copper challenges were compared: one with copper ion, which is likely to use preferentially CTR1, and one with a copper-polyacrylate complex, which will be internalized via the endosomal pathway and is likely to use preferentially CTR2. A model system consisting in the J774A1 mouse macrophage system, with a strong endosomal/lysosomal pathway, was used. In order to gain wide insights into the cellular responses to copper, a proteomic approach was used. The proteomic results were validated by targeted experiments, and showed differential effects of the import mode on cellular physiology parameters. While the mitochondrial transmembrane potential was kept constant, a depletion in the free glutahione content was observed with copper (ion and polylacrylate complex). Both copper-polyacrylate and polyacrylate induced perturbations in the cytoskeleton and in phagocytosis. Inflammatory responses were also differently altered by copper ion and copper-polyacrylate. Copper-polyacrylate also perturbed several metabolic enzymes. Lastly, enzymes were used as a test set to assess the predictive value of proteomics. SIGNIFICANCE: Proteomic profiling provides an in depth analysis of the alterations induced on cells by copper under two different exposure modes to this metal, namely as the free ion or as a complex with polyacrylate. The cellular responses were substantially different between the two exposure modes, although some cellular effects are shared, such as the depletion in free glutathione. Targeted experiments were used to confirm the proteomic results. Some metabolic enzymes showed altered activities after exposure to the copper-polyacrylate complex. The basal inflammatory responses were different for copper ion and for the copper-polyacrylate complex, while the two forms of copper inhibited lipopolysaccharide-induced inflammatory responses.

Impact of Physico-Chemical Properties of Cellulose Nanocrystal/Silver Nanoparticle Hybrid Suspensions on Their Biocidal and Toxicological Effects.

There is a demand for nanoparticles that are environmentally acceptable, but simultaneously efficient and low cost. We prepared silver nanoparticles (AgNPs) grafted on a native bio-based substrate (cellulose nanocrystals, CNCs) with high biocidal activity and no toxicological impact. AgNPs of 10 nm are nucleated on CNCs in aqueous suspension with content from 0.4 to 24.7 wt%. XANES experiments show that varying the NaBH4/AgNO3 molar ratio affects the AgNP oxidation state, while maintaining an fcc structure. AgNPs transition from 10 nm spherical NPs to 300 nm triangular-shaped AgNPrisms induced by H2O2 post-treatment. The 48 h biocidal activity of the hybrid tested on B. Subtilis is intensified with the increase of AgNP content irrespective of the Ag+/Ag0 ratio in AgNPs, while the AgNSphere-AgNPrism transition induces a significant reduction of biocidal activity. A very low minimum inhibitory concentration of 0.016 mg AgNP/mL is determined. A new long-term biocidal activity test (up to 168 h) proved efficiency favorable to the smaller AgNPs. Finally, it is shown that AgNPs have no impact on the phagocytic capacity of mammalian cells.