Immunotoxicity of relevant mixtures of pesticides and metabolites on THP-1 cells.

Pesticides are used to combat agricultural pests but also trigger side effects on non-target organisms. Particularly, immune system dysregulation is a major concern due to the organism's increased vulnerability to diseases, including cancer development. Macrophages play essential roles in innate and adaptive immunity and can undergo classical (M1) or alternative (M2) activation. The M1 pro-inflammatory phenotype has an antitumor role, while M2 favors tumor promotion. Although previous studies have linked pesticide exposure to immune compromise, macrophage polarization is still poorly studied. Here, we investigated the effects of 72 h-long exposure to the mixture of four pesticides widely used in Brazil (glyphosate, 2,4-D, mancozeb, and atrazine), and their main metabolites (aminomethylphosphonic acid, 2,4-diclorophenol, ethylenethiourea, and desethylatrazine) on human leukemia monocytic THP-1 cell line at concentrations based on the Acceptable Daily Intake (ADI) value established in the country. The data revealed immunotoxicity related to impaired cell metabolism in all exposed groups, decreased cell attachment (Pes: 10-1; Met: 10-1; Mix: all concentrations), and disturbance in nitric oxide (NO) levels (Met: 10-1, 101; Mix: all concentrations). The polarization of macrophages towards a more pro-tumor M2-like phenotype was also supported by decreased secretion of the pro-inflammatory cytokine TNF-α (Pes 100, 101) and increased IL-8 (Pes 101). These outcomes alert about the risk of pesticide exposure in the Brazilian population.

Seasonal monitoring of cellular energy metabolism in a sentinel species, Dreissena polymorpha (bivalve): Effect of global change?

Aquatic organisms such as bivalves are particularly sensitive to seasonal fluctuations associated with climate changes. Energy metabolism management is also closely related to environmental fluctuations. Changes in both biotic and abiotic conditions, such as the reproduction status and temperature respectively, may affect the organism energy status. A bivalve sentinel species, Dreissena polymorpha was sampled along its one-year reproduction cycle in situ (2018-2019) to study natural modulations on several markers of energy metabolism regarding seasonal variations in situ. A panel of different processes involved in energy metabolism was monitored through different functions such as energy balance regulation, mitochondrial density, and aerobic/anaerobic metabolism. The typical schema expected was observed in a major part of measured responses. However, the monitored population of D. polymorpha showed signs of metabolism disturbances caused by an external stressor from April 2019. Targeting a major part of energy metabolism functions, a global analysis of responses suggested a putative impact on the mitochondrial respiratory chain due to potential pollution. This study highlighted also the particular relevance of in situ monitoring to investigate the impacts of environmental change on sentinel species.

A new protocol for the simultaneous flow cytometric analysis of cytotoxicity and immunotoxicity on zebra mussel (Dreissena polymorpha) hemocytes.

Immunotoxicity analysis receives a strong interest in environmental a priori and a posteriori risk assessment procedures considering the direct involvement of the immune system in the health status of organisms, populations and thus ecosystems. The freshwater mussel Dreissena polymorpha is an invasive species widely used in ecotoxicology studies and biomonitoring surveys to evaluate the impacts of contaminants on aquatic fauna. Bivalve hemocytes are the immunocompetent cells circulating in the open circulatory system of the organism. However, there is nowadays no consensus on a protocol to evaluate the immunocompetent state of this particular cell type using flow cytometry. Wild species such as D. polymorpha present several technical barriers complicating their analyze including (i) the quality and the purity of the hemolymph sample, (ii) the controversial characterization of hemocyte subpopulations and their diversity, (iii) the quantity of biological material, and (iv) the high inter-individual variability of hemocyte responses. The present work proposes several technical and analytical improvements to control the above-mentioned issues. The inclusion of sedimentation and cell detachment steps in the pre-analytical phase of the protocol substantially ameliorate the quality of the hemolymph sample as well as the accuracy of the cytometric measurements, by selecting the analyzed cells on their adhesion ability and by increasing the concentration of the analyzed events. The development of an effective triple-labeling procedure including the cellular probe Hoechst® 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity and phagocytosis activity in hemocytes. It also significantly enhanced the accuracy of hemocyte endpoint measurements by eliminating non-target events from the analysis and allowing relevant gating strategies. Finally, the use of pooled samples of hemolymph noticeably reduced inter-sample variability while providing more plasticity in the experimental design and improving the discriminating potency between treatments. The developed protocol is suitable for ex vivo exposure of hemocyte in a chemical/environmental toxicity assessment as well as for in vivo exposure in the laboratory or in situ biomonitoring surveys with few adaptations.

Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River.

Quality assessment of environments under high anthropogenic pressures such as the Seine Basin, subjected to complex and chronic inputs, can only be based on combined chemical and biological analyses. The present study integrates and summarizes a multidisciplinary dataset acquired throughout a 1-year monitoring survey conducted at three workshop sites along the Seine River (PIREN-Seine program), upstream and downstream of the Paris conurbation, during four seasonal campaigns using a weight-of-evidence approach. Sediment and water column chemical analyses, bioaccumulation levels and biomarker responses in caged gammarids, and laboratory (eco)toxicity bioassays were integrated into four lines of evidence (LOEs). Results from each LOE clearly reflected an anthropogenic gradient, with contamination levels and biological effects increasing from upstream to downstream of Paris, in good agreement with the variations in the structure and composition of bacterial communities from the water column. Based on annual average data, the global hazard was summarized as "moderate" at the upstream station and as "major" at the two downstream ones. Seasonal variability was also highlighted; the winter campaign was least impacted. The model was notably improved using previously established reference and threshold values from national-scale studies. It undoubtedly represents a powerful practical tool to facilitate the decision-making processes of environment managers within the framework of an environmental risk assessment strategy.

A comprehensive study of the toxicity of natural multi-contaminated sediments: New insights brought by the use of a combined approach using the medaka embryo-larval assay and physico-chemical analyses.

Sediment compartment is a long term sink for pollutants and a secondary source of contamination for aquatic species. The abiotic factors controlling the bioavailability and thus the toxicity of complex mixtures of pollutants accumulated in sediments are poorly documented. To highlight the different factors influencing sediment toxicity, we identified and analyzed the physico-chemical properties, micro-pollutant contents, and toxicity level of six contrasted sediments in the Lot-Garonne continuum. Sediment toxicity was evaluated using the recently described Japanese medaka (Oryzias latipes) embryo-larval assay with direct exposure to whole sediment (MELAc). Multiple toxicity endpoints including embryotoxicity, developmental defects and DNA damage were analyzed in exposed embryos. Chemical analyses revealed significant variations in the nature and contamination profile of sediments, mainly impacted by metallic trace elements and, unexpectedly, polycyclic aromatic hydrocarbons. Exposure to sediments induced different toxic impacts on medaka early life stages when compared with the reference site. Principal component analysis showed that the toxic responses following exposure to sediments from the Lot River and its tributary were associated with micro-pollutant contamination: biometric measurements, hatching success, genotoxicity, craniofacial deformities and yolk sac malabsorption were specifically correlated to metallic and organic contaminants. Conversely, the main biological responses following exposure to the Garonne River sediments were more likely related to their physico-chemical properties than to their contamination level. Time to hatch, cardiovascular injuries and spinal deformities were correlated to organic matter content, fine particles and dissolved oxygen levels. These results emphasize the necessity of combining physico-chemical analysis of sediment with toxicity assessment to accurately evaluate the environmental risks associated with sediment contamination.

Molecular and phenotypic responses of Japanese medaka (Oryzias latipes) early life stages to environmental concentrations of cadmium in sediment.

Japanese medaka embryos were exposed to environmental concentrations of cadmium (Cd) to investigate adverse and adaptive responses in fish early life stages. Embryos were exposed during their whole development by static sediment-contact to environmental Cd concentrations (2 and 20 µg/g dry weight). Cd bioaccumulation, developmental defects, biochemical and biomolecular (qRT-PCR) responses were analyzed in embryos and hatchlings. A dose-dependent increase of Cd bioaccumulation and developmental defects was observed at hatching. Cd had clear impacts on heartbeat and cardiac morphogenesis and also induced to spinal deformities. The profile and the level of gene transcription were differentially modulated according to the Cd concentration, the duration of exposure and/or the developmental stage of fish. Pro-apoptotic bax and DNA repair rad51 transcripts were significantly repressed in embryos exposed to the highest Cd concentration. Repression of these genes was correlated to the increase of heart rate in 6-day-old embryos. NADH-dehydrogenase nd5 gene transcription was inhibited in larvae at the lowest concentration suggesting mitochondrial respiratory chain impairment, in association with Cd-induced teratogenicity. Finally, wnt1 gene was overexpressed indicating putative deregulation of Wnt signaling pathway, and suggested to be implied in the occurrence of some spinal and cardiac deformities. Results of this study permitted to propose some promising markers at the transcriptional and phenotypical level, responding to environmental concentrations of Cd. The present work also highlights the usefulness of the modified version of the medaka embryo-larval assay with sediment-contact exposure (MELAc) to investigate the toxicity and the modes of action of sediment-bound pollutants.

Transcriptional responses and embryotoxic effects induced by pyrene and methylpyrene in Japanese medaka (Oryzias latipes) early life stages exposed to spiked sediments.

Japanese medaka (Oryzias latipes) embryos were exposed to sediments spiked with environmental concentrations (300 and 3,000 ng/g dry weight) of pyrene (Pyr) and methylpyrene (MePyr) throughout their development. Embryotoxicity, teratogenicity, and transcriptional responses (qRT-PCR) were analyzed in embryos and newly hatched larvae. The genotoxicity of the two polycyclic aromatic hydrocarbons (PAHs) was also tested in prolarvae using the comet assay. Exposure to each compound had a clear impact on embryonic development and resulted in several teratogenic effects, including cardiovascular injuries, reduced absorption of yolk sac reserves, and jaw and spinal deformities. Interestingly, the overall toxic effects of Pyr and MePyr considerably overlapped those induced following dioxin exposure. qRT-PCR analysis revealed the transcriptional induction of genes involved in mitochondrial energetic metabolism (coxI), xenobiotic biotransformation (cyp1a), and cell cycle regulation (wnt1) by the two PAHs. MePyr also activated cell cycle arrest (p53), oxidative DNA damage repair (ogg1), and retinoid-mediated (raldh2 and rarα1) gene transcription. DNA damage was not found to be significantly increased following Pyr and MePyr exposure. The lack of significant genotoxic effect in comparison to the control might be the consequence of the efficient onset of DNA damage repair mechanisms as suggested by ogg1 gene transcription upregulation. Results reported in the present study have brought new insights into the modes of action of Pyr, and the effects of MePyr exposure have been investigated in fish ELS for the first time.

Effects of copper and cadmium spiked-sediments on embryonic development of Japanese medaka (Oryzias latipes).

Because of their high capacity to accumulate contaminants such as persistent organic pollutants and heavy metals, aquatic sediments are considered as a long-term source of contamination for aquatic organisms. In compliance with the increasing interest both for sediment quality evaluation and the use of fish early life stage (ELS) toxicity assays, we proposed an embryo-larval test to evaluate embryotoxicity and genotoxicity of sediment-bound contaminants. Pre-blastula stage medaka (Oryzias latipes) embryos were exposed by static sediment contact to two model heavy metals (cadmium and copper) at environmental concentrations during the whole 10-day embryonic development. Lethal and sub-lethal effects were recorded in both embryos and larvae for 20 days post fertilisation (dpf) using several global toxicity and phenotypic endpoints. The comet assay was also performed on medaka prolarvae to evaluate genotoxic effects of the tested chemicals. Environmental concentrations of cadmium (Cd) and copper (Cu) did not affect embryo and larval survival. However, both heavy metals significantly induced morphological abnormalities, particularly spinal and cardiovascular deformities. Cd but not Cu induced tachycardia. Both heavy metals induced a significant increase in DNA damage at all tested concentrations. Resulting LOEC values for Cd and Cu corresponded to 1.9 and 8.5 µg/g d.w. sediment, respectively. Although metal bioavailability is probably lower for naturally contaminated sediments, the relatively low toxicity thresholds for both Cd and Cu raise the question of possible risk for fish embryos developing in direct contact to sediments. This study demonstrates the applicability, sensitivity and relevance of the Japanese medaka embryo-larval assay (MELA) to evaluate sediment hazardous potency at environmental concentrations of heavy metals.

Detection of DNA damage in yolk-sac larvae of the Japanese Medaka, Oryzias latipes, by the comet assay.

This study was set up to determine the suitability of the early life stage (ELS) alkaline comet assay for the detection of DNA strand breaks induced by genotoxicants in whole organism. This assay was performed on cells of medaka 2 days posthatch (dph). An efficient procedure for cell dissociation using enzymatic and mechanical digestion was developed. This protocol ensures 80% viability of cells and low DNA damage background. Cells from 2 dph medaka larvae were exposed in vitro to model genotoxicants, hydrogen peroxide, cadmium, and fluoranthene, followed by comet assay analysis. Results show a significant increase in the percentage of DNA damage of dissociated cells by all the tested compounds when compared to controls. The assay was also performed in vivo on medaka larvae (2 dph) exposed for 24 h to waterborne cadmium or fluoranthene. Significant induction of DNA damage levels were observed following larvae exposure to cadmium and fluoranthene at concentrations of 0.1 and 50 µM, respectively. This study demonstrates that cells of embryo life stage medaka respond to known DNA damaging agents and that the ELS comet assay may be a useful biomarker to detect DNA strand breakage in whole body of pluricellular organism induced by a range of agents. This technique may provide a sensitive, nonspecific endpoint of genotoxicity as part of ELS toxicity test.