Search for biomarkers used in toxicology and immunology combining in vitro and in vivo approaches-application to the asian catfish aquaculture (Pangasianodon hypophthalmus)
Friday seminar by Patrick Kestemont from the University of Namur, Belgium
In many fish species, intensive aquaculture has led to an increase of stocking density, with, as a corollary, an enhancement of interactions between individuals and degraded water quality due to excess feed supply and metabolite excretion. These conditions often enhance the sensitivity of fish to parasite and bacterial diseases, requiring increased use of chemicals and antibiotics. Most of these therapeutic treatments are nowadays forbidden because of the presence of chemical residues into the fish flesh and related food safety concerns for humans. As an alternative to the chemicals used to reduce the pathogen pressure, there is a growing interest to the reinforcement of the fish immune system, based on the supply of different types of immunostimulating substances. Analysis of chemical residues is complex and costly. Alternative approaches based on the development of specific biomarkers have been suggested, using the molecular or physiological responses of the exposed animals as an early signal of potential contamination by a xenobiotic compound. Immunostimulation approach has been tested on many species and compounds but the mechanisms of action of these immunostimulants are rather unknown. Search for biomarkers of contamination as well as mechanisms of action of immunostimulants have been investigated, combining in vitro and in vivo approaches, with the Asian catfish Pangasianodon hypophthalmus, one of the major aquaculture species in the world, as biological model. Malachite Green (MG) was used as antifungal therapeutic chemical, while Escherichia coli lipopolysaccharide was selected as immunostimulating agent against Edwardsiella ictaluri infection, one of the most frequent pathogens occurring in Asian catfish culture. In vitro and in vivo studies were based on the responses of peripheral blood mononuclear cells (PBMC) assessed by protein expression profiling, using the 2D-DIGE approach.
In the MG toxicity study, proteomic analyses suggested that this anti-fungal substance could lead to possible disturbances of DNA, including disruption of transcription and replication as well as repair processes, potential disruption of normal cell division, disorganization of the cytoskeleton, modification of the expression of several molecular chaperones as well as of proteins linked to the ubiquitin proteasome system. No proteins involved in the oxidative stress response were identified in any of the proteomic analyses performed. A Protein Expression Signature (PES) has been identified to detect the illegal use of MG with the final objective to apply this biomarker signature to an in situ monitoring in fish farm conditions. This PES would consist of 11 suggested proteins differentially expressed following MG treatment up to one month after depuration.
In the immune response study, proteomic analysis performed in vivo suggested that LPS could stimulate the over-expression of several immune proteins such as complement components, lysozyme precursor, transferrin and immunoglobulins. In addition, the results from the in vitro approach showed that the response of striped catfish to LPS involved an increase of many protein expression related to cytoskeleton, stress response, cell signaling, carbohydrate metabolic process as well as muscle contraction. All these protein responses result in an increase of the ability of striped catfish PBMC to produce and secrete pro-inflammatory mediator, which is fundamental to fight against a pathogen invasion.
In these studies (toxicity of MG or immunostimulation by LPS), both in vitro and in vivo approaches provided consistent responses, allowing the identification of a set of biomarkers. This highlights the interest to use a noninvasive in vitro screening method combined with the identification of sensitive biomarkers in order to get a better insight into the toxicity of specific chemicals or the ability of some compounds to strengthen the immune system of fish.
Research Unit in Environmental and Evolutionary Biology
University of Namur