Evaluation of an in vitro assay to screen for the immunotoxic potential of chemicals to fish

Abstract

A wide variety of environmental contaminants has been shown to disrupt immune functions of fish and may compromise their defense capability against pathogens. Immunotoxic effects, however, are rarely considered in ecotoxicological testing strategies. The aim of this study was to systematically evaluate the suitability of an in vitro immuno-assay using selected fish immune parameters to screen for chemicals with known immunotoxic potential and to differentiate them from non-immunotoxicants. Non-stimulated and lipopolysaccharide-stimulated head kidney leukocytes of rainbow trout (Oncorhynchus mykiss) were exposed for 3 h or 19 h to chemicals with different modes of action. As immune parameters, phagocytosis activity, oxidative burst activity and cytokine transcription (IL-1β, TNFα, IL-10) were examined, accompanied by in silico modelling. The immunotoxicants dexamethasone, benzo(a)pyrene, ethinylestradiol and bisphenol A significantly altered the immune parameters at non-cytotoxic concentrations whereas diclofenac had only weak effects. However, the two baseline chemicals with no known immunotoxic potential, butanol and ethylene glycol, caused significant effects, too. From our results it appears that the in vitro fish leukocyte assay as performed in the present study has only a limited capacity for discriminating between immunotoxicants and non-immunotoxicants.

Assessing Fish Immunotoxicity by Means of In Vitro Assays: Are We There Yet?

“…This concentration represents no specific immunotoxic activity, but general baseline cytotoxicity ( 208 , 211 , 212 ). To test for specific immunotoxic effects in vitro , it is essential to apply non-cytotoxic concentrations in order to avoid false positive responses caused by the interference of the cytotoxic concentrations with cell viability ( 213 , 214 ).…”

“…Assay conditions such as incubation time or particle concentration, the analytical method as well as whether the phagocytes are activated or resting influence the results of phagocytosis assays ( 184 , 222 ). Also the use of appropriate controls to distinguish between particles adhered to the surface of the cells or ingested by the cells is mandatory ( 214 , 217 ). Importantly, toxicant effects on phagocytosis may be detectable only after activation of the cells by exposure to pathogenic signals such as bacterial lipopolysaccharide (LPS); the activation can lead to enhanced phagocytosis, enhanced production of reactive oxygen intermediates and nitric oxide, and enhanced secretion of pro-inflammatory cytokines (IL-1 TNFa, IL-6, IL-12, IL-1 [e.g., ( 141 , 214 , 223 , 224 )].…”

“…Also the use of appropriate controls to distinguish between particles adhered to the surface of the cells or ingested by the cells is mandatory ( 214 , 217 ). Importantly, toxicant effects on phagocytosis may be detectable only after activation of the cells by exposure to pathogenic signals such as bacterial lipopolysaccharide (LPS); the activation can lead to enhanced phagocytosis, enhanced production of reactive oxygen intermediates and nitric oxide, and enhanced secretion of pro-inflammatory cytokines (IL-1 TNFa, IL-6, IL-12, IL-1 [e.g., ( 141 , 214 , 223 , 224 )]. The results of the phagocytosis assay are presented as percentage of phagocytosing cells in the cell population, or as number of particles engulfed per cell ( 26 , 61 , 184 , 225 ).…”

“…if they produce false positives or false negatives. Rehberger et al ( 214 ) tested five immunotoxic chemicals and two non-immunotoxic chemicals at sub-cytotoxic concentrations using an in vitro assay with head kidney leukocytes of rainbow trout. The five immunotoxicants were correctly classified as immunotoxicants, although the pharmaceutical diclofenac elicited a rather weak response (pointing to the need of using a test battery, since depending on the mode of immunotoxic action, different in vitro assays will show different sensitivities).…”

“…The two cellular responses share a number of receptors and signalling pathways ( 276 ), many of them being located or associated with the cell membrane. Chemicals with narcotic mode of action interfere with the cell membrane organization and fluidity, and this activates stress pathways ( 277 ) which then may converge with immune-related receptors and signalling pathways ( 214 ). If the hypothesis of an interference between the cellular stress and immune responses is correct, this would represent a principal obstacle in using cell-based assays for in vitro immunotoxicity screening.…”