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. Contribution to the field statement Unlike dyes, which are water-soluble, pigments are water-insoluble and thus viewed as inert coloring substances. However, historical pigments such as lead white or vermilion (mercuric sulfide) have been shown to be toxic, suggesting that pigments inertness may not be complete under biological conditions. Pigments being particulate (nano) materials, they are taken up by professional scavenger cells such as macrophages once they have penetrated into the body. One epitome of this situation is represented by tattooing: the pigments injected with the ink are taken up by dermal macrophages, which life cycle ensures the localized persistence of tattoos over time. Using an in vitro macrophage culture system adapted to study delayed effects, we have investigated the effects of a series of cobalt- and zinc-containing pigments on macrophages. First of all, the toxicity of the pigments correlated well with their solubility in acidic media, i.e. conditions prevailing in the phagolysosomes. Even when used at non-toxic doses, the cobalt and zinc pigments showed immediate and/or delayed effects on macrophage functions such as phagocytosis, adhesion, tissue repair or response to bacterial stimuli. Overall, these results show that some pigments may not be as inert as previously thought, and describe a system to investigate these effects.