An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

Un contexte réglementaire de plus en plus strict en évaluation du risque écotoxicologique des milieux aquatiques exige de renforcer les outils d’évaluation. A ce titre, l’étude des biomarqueurs de génotoxicité doit être privilégiée, compte tenu du rôle central de l’ADN dans le fonctionnement du vivant. L’exposition à des agents génotoxiques peut générer des dommages par interaction directe avec l’ADN, mais aussi indirectement, en modulant l’efficacité des mécanismes de réparation de l’ADN, ou la régulation épigénétique de l’expression des gènes. Aujourd’hui, la plupart des biomarqueurs de génotoxicité visent les dommages primaires à l’ADN ou la mutagènicité mais les effets indirects sur sa fonctionnalité sont encore peu étudiés. Dans ce contexte, à l’issue d’une analyse bibliographique comprenant la rédaction d’une revue sur les mécanismes de réparation des dommages à l’ADN chez le poisson, ce travail visait au développement méthodologique de plusieurs biomarqueurs de génotoxicité à l’aide de trois lignées cellulaires pisciaires (RTL-W1, RTGill-W1 et PLHC-1). Pour ce faire, l’essai des comètes en conditions alcalines a été décliné sous plusieurs versions dans l’objectif d’utiliser une technique de base unique permettant la mesure complémentaire de plusieurs biomarqueurs de génotoxicité : les dommages primaires à l’ADN, les activités de réparation et le niveau de méthylation des cytosines du génome. Les résultats soulignent l’intérêt des trois lignées en évaluation de la génotoxicité 1) pour détecter in vitro de manière sensible des atteintes primaires à l’ADN de natures variées à de faibles concentrations grâce à un essai des comètes modifié par une étape de digestion enzymatique avec une glycosylase (Fpg), 2) pour évaluer l’influence des contaminants sur l’activité de réparation par excision de bases (BER) via la mesure de la capacité d’incision d’un ADN substrat porteur de lésions de type 8-oxoGua par des extraits cellulaires (essai BERc). Le niveau de méthylation des cytosines (5-meCyt) des lignées RTgill-W1et RTL-W1 a été mesuré par HPLC-MS-MS, leur valeur élevée permet d’envisager le paramètre méthylation comme biomarqueur potentiel. Ce volet nécessitera cependant des étapes de validations ultérieures car il n’a pas été techniquement possible de mettre au point un essai des comètes modifié pour la mesure du niveau de méthylation de l’ADN. Plusieurs activités de réparation des lignées RTgill-W1 et RTL-W1 ont été caractérisées et révèlent de bonnes aptitudes de réparation de type « Base Excision Repair » (BER) et « Photo Enzymatic Repair » (PER) et une plus faible capacité au « Nucleotide Excision Repair » (NER), soit un profil proche de celui décrit in vivo et sans différence marquée entre les deux lignées. Les biomarqueurs développés sur les lignées cellulaires de poisson au cours de ce travail ont également été appliqués à la mesure des effets génotoxiques d’effluents issus du lessivage de revêtements routiers. / In a context of growing awareness of aquatic pollution impacts, there is an increasing need to develop methods for hazards and risk assessment of pollutants. In this context, genotoxicity endpoints are of a great concern since even when evaluated at a sub-cellular or cellular level, impaired DNA structure, repair and/or functions can have delayed (long term) consequences at higher level of organization such as individual and population. Some genotoxicant can have direct effect on DNA, but they can also interact indirectly, by modulating the repair mechanism efficiency or by acting on epigenetic mechanism such as DNA méthylation level. An unrepaired DNA damage and epigenetic modification can both lead to functional alteration and/or genetic structure at the population level. However, most of the existing genotoxicity test only measure primary DNA damage induces by genotoxicant; thus there is a real need to develop new tools to investigate those different kinds of genotoxicity. For this purpose, this work aims at developing knowledge in DNA repair capacities of to fish cell lines, RTL-W1 and RTG-W1, in order to develop new genotoxicity biomarker, measuring primary DNA damage by means of modified version of the comet assay. The results highlights the interest of in vitro biological models such as fish cell lines for the assessment of environmental genotoxicity, especially using a Fpg-modified comet assay allowing a sufficient increase of the assay sensibility to detect genotoxicity at environmentally relevant concentration. Results also characterize BER and PER capacities as being efficient repair mechanism in those fish cell lines, whereas NER, although also present, seems less efficient. A new biomarker based on the BER incision capacities of cellular extracts has also been developed and used to assess the genotoxicity of environemental effluent.

5 Abstract Five substances from the group of so-called personal care products, known for their low degradability and regular environmental detection, were tested for toxicity using two fish tissue lines (RTgill-W1 a RTG-2) isolated from rainbow trout (Oncorhynchus miykiss). The tested substances were hexadecylpyridinium chloride (HDP), chlorhexidine (CHX), octenidine (OCT), thymol (THM) and triclosan (TCS). A cell viability assay was performed with each of these compounds using Alamar Blue ™ (AB), 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and neutral red (NR) protocols. The results were used to construct dose-response curves along with an EC50 value for each of these substances. The EC50 values ranged from 0,51 (HDP) to 33,75 µg.ml-1 (THM) for RTgill-W1 and from 0,31 (HDP) to 33,37 µg.ml-1 (THM) for RTG-2. The theoretical LC50 estimation was calculated according to Tanneberger et al. (2013). For all substances, cytochrome P450 1A activity was monitored using 7-ethoxyresorufin-o-deethylase (EROD), four out of five tested chemicals were statistically positive for EROD, the highest EROD response was observed for the most toxic compound - HDP. Only TCS did not show statistically significant cytochrome P450 1A activity. In addition, oxidative stress was measured with the fluorescent dye...