Effects of Carbon Nanotube Environmental Dispersion on an Aquatic Invertebrate, Hirudo medicinalis

Abstract

The recent widespread applications of nanomaterials, because of their properties, opens new scenarios that affect their dispersal in the environment. In particular multiwall carbon nanotubes (MWCNTs), despite their qualities, seem to be harmful for animals and humans. To evaluate possible toxic effects caused by carbon nanotube environmental dispersion, with regard to aquatic compartment, we proposed as experimental model a freshwater invertebrate: Hirudo medicinalis. In the present study we analyse acute and chronic immune responses over a short (1, 3, 6 and 12 hours) and long time (from 1 to 5 weeks) exposure to MWCNTs by optical, electron and immunohistochemical approaches. In the exposed leeches angiogenesis and fibroplasia accompanied by massive cellular migration occur. Immunocytochemical characterization using specific markers shows that in these inflammatory processes the monocyte-macrophages (CD45+, CD68+) are the most involved cells. These immunocompetent cells are characterized by sequence of events starting from the expression of pro-inflammatory cytokines (in particular IL-18), and amyloidogenensis. Our combined experimental approaches, basing on high sensitive inflammatory response can highlight adverse effects of nanomaterials on aquatic organisms and could be useful to assess the MWCNTs impact on aquatic, terrestrial animal and human health.

Amyloidogenesis and Responses to Stress

“…MWCNTs in the leech body wall provoke nonspecific responses characterized by proliferation and migration of macrophages toward the stimulated area. Girardello et al [32] show that these immune cells, recruited in the area of inflammation, produce amyloid material, as demonstrated by Thioflavin-T staining. In addition, the authors correlated this synthesis with a concomitant overexpression of IL-18, confirming the importance of this cytokine in the process.…”

“…In addition, the authors correlated this synthesis with a concomitant overexpression of IL-18, confirming the importance of this cytokine in the process. Extracellular amyloid fibrils entrap and isolate the nanotube aggregation but, forming a kind of spongy coating, can have an additional function in concentrating the cytokines produced by neighbor activated cells thus attracting and driving the macrophages engaged in immune responses [32] (Figure 3, panels A-E). Amyloids are also synthesized by the cells that are recruited in the lesioned body wall of diverse invertebrates to work as a "collision mat," the strategic solution patterned to close a boat leak, by forming a clot.…”

Systemic distribution of single-walled carbon nanotubes in a novel model: alteration of biochemical parameters, metabolic functions, liver accumulation, and inflammation in vivo

“…Following standard serial ethanol dehydration, specimens were embedded in an Epon-Araldite 812 mixture. Semithin sections, obtained using a Reichert Ultracut S ultratome (Leica Microsystems, Wetzlar, Germany), were stained by the Feulgen staining according to Girardello et al 27 and observed under a light microscope (Olympus Corporation). Data were recorded using a DS5-M-L1 digital camera system.…”

“…We isolated SWCNTs from excised tissues as described in Albini et al 21 and Girardello et al 27 Livers from mice 3 weeks after the third SWCNT injection were excised, digested in 5 N KOH (Sigma-Aldrich Co.) for 2 hours at room temperature, washed, suspended in 100 µL of distilled water to avoid potassium salt formation, and dried on a copper grid (formvar carbon film) for TEM analysis (JEM-1010; JEOL, Tokyo, Japan). Images were acquired using a Morada digital camera (Olympus Corporation).…”

Cellular responses induced by multi-walled carbon nanotubes: in vivo and in vitro studies on the medicinal leech macrophages

“…It is well known that invertebrates orchestrate rapid and sensitive responses upon the presence of pollution. Our previous works 17 , 18 have demonstrated that leech activates a rapid immune response in presence of MWCNTs. As already largely described, this model presents multiple advantages, being cost-effective, easily manipulable and devoid of significant ethical considerations and regulatory restrictions in relation to its use 23 , 24 .…”