Establishment of toxicity thresholds in subpopulations of coelomocytes (amoebocytes vs. eleocytes) of Eisenia fetida exposed in vitro to a variety of metals: implications for biomarker measurements

Abstract

Coelomocytes comprise the immune system of earthworms and due to their sensitivity responding to a wide range of pollutants have been widely used as target cells in soil ecotoxicology. Recently, in vitro assays with primary cultures of coelomocytes based in the neutral red uptake (NRU) assay have been developed as promising tools for toxicity assessment chemical in a reproducible and cost-effective manner. However, NRU showed a bimodal dose-response curve previously described after in vivo and in vitro exposure of earthworm coelomocytes to pollutants. This response could be related with alterations in the relative proportion of coelomocyte subpopulations, amoebocytes and eleocytes. Thus, the aims of the present work were, first, to establish the toxicity thresholds that could be governed by different cell-specific sensitivities of coelomocytes subpopulations against a series of metals (Cu, Cd, Pb, Ni), and second to understand the implication that coelomocyte population dynamics (eleocytes vs. amoebocytes) after exposure to pollutants can have on the viability of coelomocytes (measured by NRU assay) as biomarker of general stress in soil health assessment. Complementarily flow cytometric analyses were applied to obtain correlative information about single cells (amoebocytes and eleocytes) in terms of size and complexity, changes in their relative proportion and mortality rates. The results indicated a clear difference in sensitivity of eleocytes and amoebocytes against metal exposure, being eleocytes more sensitive. The bimodal dose-response curve of NRU after in vitro exposure of primary cultures of coelomocytes to metals revealed an initial mortality of eleocytes (decreased NRU), followed by an increased complexity of amoebocytes (enhanced phagocytosis) and massive mortality of eleocytes (increased NRU), to give raise to a massive mortality of amoebocytes (decrease NRU). A synergistic effect on NRU was exerted by the exposure to high Cu concentrations and acidic pH (elicited by the metal itself), whereas the effects on NRU produced after exposure to Cd, Ni and Pb were due solely to the presence of metals, being the acidification of culture medium meaningless.

Effective activation of antioxidant system by immune-relevant factors reversely correlates with apoptosis of Eisenia andrei coelomocytes

“…Coelomocytes are the first line of cellular response of earthworm immune system and their number, type and activity can vary during immune responses (Homa et al 2013 ; Irizar et al 2015 ). In control animals (CTR) number and composition of coelomocytes were comparable at 24 and 72 h, but all of used stimulants (PMA, LPS, zymosan—Z, M. luteus —Ml) had statistically significantly decreased coelomocyte numbers in comparison to the control animals at either time point (Fig.…”

“…Such polarization was observed previously by Nacarelli and Fuller-Espie ( 2011 ) in coelomocytes from Eisenia hortensis incubated with zymosan. Although the coelomocyte polarization is clearly existent, it might differ between species and/or insults as just recently eleocytes of E. fetida were shown to be more sensitive to metal exposure (Irizar et al 2015 ).…”

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species

“…The most visible response difference is observed with metals, measured by neutral red uptake: after exposure, eleocytes started to die, while amebocytes showed enhanced phagocytosis. [21] Arthropods, including insects and crustaceans, use a range of cellular and humoral immune defensive strategies to protect themselves from damage. The main cellular defense mechanisms are phagocytosis, nodule formation (nodulation), and encapsulation.…”

Evaluation of Complex Toxicity of Canbon Nanotubes and Sodium Pentachlorophenol Based on Earthworm Coelomocytes Test

“…Coelomocytes, which are immunocompetent cells, play an essential role in the identification and elimination of xenobiotics [ 7 ], fighting microbial infections and healing wounds [ 8 ]. Due to their sensitivity to a wide range of pollutants, they have been widely used as one of the very sensitive biomarkers of environmental stress and are frequently used to assess genotoxicity [ 9 , 10 ]. The most used approach has consisted of in vivo exposures and subsequent extrusion of coelomocytes from earthworms for the measurement of a variety of biomarkers [ 11 , 12 ].…”

“…However, most of these studies have either focused on cell recognition or cell culture condition, and systematic research in the field of in vitro toxicity is scarce. Although coelomocytes are increasingly being used for metal exposure, studies on nanoparticles and pesticide toxicity are limited [ 7 , 9 , 15 ].…”