Département Toxicologie de l’environnement

The objective of this study was to evaluate the suitability of the rainbow troutintestinal epithelial cell line (RTgutGC) as an in vitro model for studies of gut immunefunction and effects of functional feed ingredients. Effects of lipopolysaccharide(LPS) and three functional feed ingredients [nucleotides, mannanoligosaccharides(MOS), and beta-glucans] were evaluated in RTgutGC cells grown on conventionalculture plates and transwell membranes. Permeation of fluorescently-labeled albumin,transepithelial electrical resistance (TEER), and tight junction protein expressionconfirmed the barrier function of the cells. Brush border membrane enzyme activities[leucine aminopeptidase (LAP) and maltase] were detected in the RTgutGC cellsbut activity levels were not modulated by any of the exposures. Immune relatedgenes were expressed at comparable relative basal levels as these in rainbow troutdistal intestine. LPS produced markedly elevated gene expression levels of the proinflammatorycytokines il1b, il6, il8, and tnfa but had no effect on ROS production.Immunostaining demonstrated increased F-actin contents after LPS exposure. Amongthe functional feed ingredients, MOS seemed to be the most potent modulatorof RTgutGC immune and barrier function. MOS significantly increased albuminpermeation and il1b, il6, il8, tnfa, and tgfb expression, but suppressed ROS production,cell proliferation and myd88 expression. Induced levels of il1b and il8 were alsoobserved after treatment with nucleotides and beta-glucans. For barrier functionrelated genes, all treatments up-regulated the expression of cldn3 and suppressedcdh1 levels. Beta-glucans increased TEER levels and F-actin content. Collectively, thepresent study has provided new information on how functional ingredients commonlyapplied in aquafeeds can affect intestinal epithelial function in fish. Our findingssuggest that RTgutGC cells possess characteristic features of functional intestinalepithelial cells indicating a potential for use as an efficient in vitro model to evaluate effectsof bioactive feed ingredients on gut immune and barrier functions and their underlyingcellular mechanisms.

Nanomaterial risk governance requires models to estimate the material flow, fate and transport as well as uptake/bioavailability, hazard and risk in the environment. This study assesses the fit of such available models to different stages during the innovation of nano-enabled products. Through stakeholder consultations, criteria were identified for each innovation stage from idea conception to market launch and monitoring. In total, 38 models were scored against 41 criteria concerning model features, applicability, resource demands and outcome parameters. A scoring scheme was developed to determine how the models fit the criteria of each innovation stage. For each model, the individual criteria scores were added, yielding an overall fit score to each innovation stage. Three criteria were critical to stakeholders and incorporated as multipliers in the scoring scheme; the required time/costs and level of expertise needed to use the model, and for risk assessment models only, the option to compare PEC and PNEC. Regulatory compliance was also identified as critical, but could not be incorporated, as a nanomaterial risk assessment framework has yet to be developed and adopted by legislators. In conclusion, the scoring approach underlined similar scoring profiles across stages within model categories. As most models are research tools designed for use by experts, their score generally increased for later stages where most resources and expertise are committed. In contrast, stakeholders need relatively simple models to identify potential hazards and risk management measures at early product development stages to ensure safe use of nanomaterials without costs and resource needs hindering innovation.

Marine mammals, such as whales, have a high proportion of body fat and so are susceptible to the accumulation, and associated detrimental health effects, of lipophilic environmental contaminants. Recently, we developed a wild-type cell line from humpback whale fibroblasts (HuWa). Extensive molecular assessments with mammalian wild-type cells are typically constrained by a finite life span, with cells eventually becoming senescent. Thus, the present work explored the possibility of preventing senescence in the HuWa cell line by transfection with plasmids encoding the simian virus large T antigen (SV40T) or telomerase reverse transcriptase (TERT). No stable expression was achieved upon SV40 transfection. Transfection with TERT, on the other hand, activated the expression of telomerase in HuWa cells. At the time of manuscript preparation, the transfected HuWa cells (HuWa_TERT) have been stable for at least 59 passages post-transfection. HuWa_TERT proliferate rapidly and maintain initial cell characteristics, such as morphology and chromosomal stability. The response of HuWa_TERT cells to an immune stimulant (lipopolysaccharide (LPS)) and an immunotoxicant (Aroclor1254) was assessed by measurement of intracellular levels of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β and tumour necrosis factor (TNF)-α. HuWa_TERT cells constitutively express IL-6, IL-1β and TNFα. Exposure to neither LPS nor Aroclor1254 had an effect on the levels of these cytokines. Overall, this work supports the diverse applicability of HuWa cell lines in that they display reliable long-term preservation, susceptibility to exogenous gene transfer and enable the study of humpback whale-specific cellular response mechanisms.

Water pollution is ubiquitous globally, yet how the effects of pollutants propagate through natural ecosystems remains poorly understood. This is because the interactive effects of multiple stressors are generally hard to predict. Agriculture and municipal wastewater treatment plants (WWTPs) are often major sources of contaminants for streams, but their relative importance and the role of different pollutants (e.g. nutrients or pesticides) are largely unknown. Using a 'real world experiment' with sampling locations up- and downstream of WWTPs, we studied how effluent discharges affected water quality and macroinvertebrate communities in 23 Swiss streams across a broad land-use gradient.
Variation partitioning of community composition revealed that overall water quality explained approximately 30% of community variability, whereby nutrients and pesticides each independently explained 10% and 2%, respectively. Excluding oligochaetes (which were highly abundant downstream of the WWTPs) from the analyses, resulted in a relatively stronger influence (3%) of pesticides on the macroinvertebrate community composition, whereas nutrients had no influence. Generally, the macroinvertebrate community composition downstream of the WWTPs strongly reflected the upstream conditions, likely due to a combination of efficient treatment processes, environmental filtering and organismal dispersal. Wastewater impacts were most prominently by the Saprobic index, whereas the SPEAR index (a trait-based macroinvertebrate metrics reflecting sensitivity to pesticides) revealed a strong impact of arable cropping but only a weak impact of wastewater.
Overall, our results indicate that agriculture can have a stronger impact on headwater stream macroinvertebrate communities than discharges from WWTP. Yet, effects of wastewater-born micropollutants were clearly quantifiable among all other influence factors. Improving our ability to further quantify the impacts of micropollutants requires highly-resolved water quality and taxonomic data with adequate spatial and temporal sampling. These improvements would help to better account for the underlying causal pathways that drive observed biological responses, such as episodic contaminant peaks and dispersal-related processes.

Stream biofilms have been shown to be among the most sensitive indicators of environmental stress in aquatic ecosystems and several endpoints have been developed to measure biofilm adverse effects caused by environmental stressors. Here, we compare the effects of long-term exposure of stream biofilms to diuron, a commonly used herbicide, on several traditional ecotoxicological endpoints (biomass growth, photosynthetic efficiency, chlorophyll-a content, and taxonomic composition), with the effects measured by recently developed methods [community structure assessed by flow cytometry (FC-CS) and measurement of extracellular polymeric substances (EPS)]. Biofilms grown from local stream water in recirculating microcosms were exposed to a constant concentration of 20 μg/L diuron over a period of 3 weeks. During the experiment, we observed temporal variation in photosynthetic efficiency, biomass, cell size, presence of decaying cells and in the EPS protein fraction. While biomass growth, photosynthetic efficiency, and chlorophyll-a content were treatment independent, the effects of diuron were detectable with both FC and EPS measurements. This demonstrates that, at least for our experimental setup, a combination of different ecotoxicological endpoints can be important for evaluating biofilm environmental stress and suggests that the more recent ecotoxicological endpoints (FC-CS, EPS protein content and humic substances) can be a useful addition for stream biofilm ecotoxicological assessment.