Key publications
Boisseaux P, Noury P, Delorme N, Perrier L, Thomas-Guyon H, Garric J (2018). Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.
Environmental Science and Pollution Research,
25(17), 16720-16728.
Abstract:
Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters
Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.
Abstract.
Boisseaux P, Noury P, Thomas H, Garric J (2017). Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A.
Ecotoxicology and Environmental Safety,
139, 358-366.
Abstract:
Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A
This is a pioneering study in the ecotoxicological assessment of immunotoxic effects of the three selected drugs of concern to a freshwater gastropod species. Lymnaea stagnalis was exposed in the laboratory for 3 days to three drugs used for immune systems: diclofenac (nonsteroidal anti-inflammatory drug), cyclophosphamide (anti-cancer immunosuppressive drug) or cyclosporine a (anti-xenograft immunosuppressive drug). Exposure ranges included environmental realistic (1–10 μg L −1 ) and therapeutic concentrations (100–1000 μg L −1 ). At the end of exposure times, the immune parameters of individual snails were measured: hemocyte density and viability, hemocyte phagocytosis capacity and hemocyte-related oxidative activities (basal and NADPH-oxidase stimulated with zymosan particles). Diclofenac and cyclosporine a induced immune responses, although the effects were not strong. No immunosuppression was observed. Such subtle immunomodulations bring further interrogations regarding their long-term immunotoxicity and possible resulting tradeoffs with life-history traits. On the other hand, the prodrug cyclophosphamide did not induce significant immune responses. Since metabolism pathways differ greatly between vertebrates and invertebrates, this study also suggests that relevant vertebrate metabolites should be included in the immunotoxicity assessment of pharmaceuticals in non-target invertebrate species. Finally, the possible interactive effects of these pharmaceuticals sharing similar modes of action or effects features should also be explored.
Abstract.
Boisseaux P, Delignette-Muller ML, Abbaci K, Thomas H, Garric J (2016). Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections.
Fish and Shellfish Immunology,
57, 116-126.
Abstract:
Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections
The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly – regardless the time-intervals – at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight “day” effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the “day” effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails’ immunocompetence.
Abstract.
Boisseaux P, Noury P, Delignette-Muller ML, Thomas H, Garric J (2016). Recommendations for the analysis of hemocyte-related immunocompetent oxidative activity in the freshwater snail Lymnaea stagnalis. Journal of Xenobiotics, 6(2 2Special Issue), 25-27.
Lammel T, Boisseaux P, Navas JM (2015). Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1.
Environmental Toxicology,
30(10), 1192-1204.
Abstract:
Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1
Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (β-naphthoflavone (β-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on β-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard.
Abstract.
Lammel T, Boisseaux P, Fernández-Cruz ML, Navas JM (2013). Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2.
Particle and Fibre Toxicology,
10(1).
Abstract:
Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2
Background: Graphene and graphene derivative nanoplatelets represent a new generation of nanomaterials with unique physico-chemical properties and high potential for use in composite materials and biomedical devices. To date little is known about the impact graphene nanomaterials may have on human health in the case of accidental or intentional exposure. The objective of this study was to assess the cytotoxic potential of graphene nanoplatelets with different surface chemistry towards a human hepatoma cell line, Hep G2, and identify the underlying toxicity targets.Methods: Graphene oxide (GO) and carboxyl graphene (CXYG) nanoplatelet suspensions were obtained in water and culture medium. Size frequency distribution of the suspensions was determined by means of dynamic light scattering. Height, lateral dimension and shape of the nanoplatelets were determined using atomic force and electron microscopy. Cytotoxicity of GO and CXYG nanoplatelets was assessed in Hep G2 cells using a battery of assays covering different modes of action including alterations of metabolic activity, plasma membrane integrity and lysosomal function. Induction of oxidative stress was assessed by measuring intracellular reactive oxygen species levels. Interaction with the plasma membrane, internalization and intracellular fate of GO and CXYG nanoplatelets was studied by scanning and transmission electron microscopy.Results: Supplementing culture medium with serum was essential to obtain stable GO and CXYG suspensions. Both graphene derivatives had high affinity for the plasma membrane and caused structural damage of the latter at concentrations as low as 4 μg/ml. The nanoplatelets penetrated through the membrane into the cytosol, where they were concentrated and enclosed in vesicles. GO and CXYG accumulation in the cytosol was accompanied by an increase in intracellular reactive oxygen species (ROS) levels, alterations in cellular ultrastructure and changes in metabolic activity.Conclusions: GO and CXYG nanoplatelets caused dose- and time-dependent cytotoxicity in Hep G2 cells with plasma membrane damage and induction of oxidative stress being important modes of toxicity. Both graphene derivatives were internalized by Hep G2, a non-phagocytotic cell line. Moreover, they exerted no toxicity when applied at very low concentrations (< 4 μg/ml). GO and CXYG nanoplatelets may therefore represent an attractive material for biomedical applications. © 2013 Lammel et al.; licensee BioMed Central Ltd.
Abstract.
Publications by category
Journal articles
Boisseaux P, Hopkinson P, Santillo D, Smith C, Garmulewicz A, Powell Z, Galloway T (2023). Environmental safety of second and third generation bioplastics in the context of the circular economy.
Ecotoxicol Environ Saf,
256Abstract:
Environmental safety of second and third generation bioplastics in the context of the circular economy.
Bioplastics derived from organic materials other than crude oil are often suggested as sustainable solutions for tackling end-of-life plastic waste, but little is known of their ecotoxicity to aquatic species. Here, we investigated the ecotoxicity of second and third generation bioplastics toward the freshwater zooplankton Daphnia magna. In acute toxicity tests (48 h), survival was impacted at high concentrations (g.L-1 range), within the range of salinity-induced toxicity. Macroalgae-derived bioplastic induced hormetic responses under chronic exposure (21 d). Most biological traits were enhanced from 0.06 to 0.25 g.L-1 (reproduction rate, body length, width, apical spine, protein concentration), while most of these traits returned to controls level at 0.5 g.L-1. Phenol-oxidase activity, indicative of immune function, was enhanced only at the lowest concentration (0.06 g.L-1). We hypothesise these suggested health benefits were due to assimilation of carbon derived from the macroalgae-based bioplastic as food. Polymer identity was confirmed by infra-red spectroscopy. Chemical analysis of each bioplastic revealed low metal abundance whilst non target exploration of organic compounds revealed trace amounts of phthalates and flame retardants. The macroalgae-bioplastic disintegrated completely in compost and biodegraded up to 86 % in aqueous medium. All bioplastics acidified the test medium. In conclusion, the tested bioplastics were classified as environmentally safe. Nonetheless, a reasonable end-of-life management of these safer-by-design materials is advised to ensure the absence of harmful effects at high concentrations, depending on the receiving environment.
Abstract.
Author URL.
Boisseaux P, Noury P, Delorme N, Perrier L, Thomas-Guyon H, Garric J (2018). Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.
Environmental Science and Pollution Research,
25(17), 16720-16728.
Abstract:
Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters
Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.
Abstract.
Tair-Abbaci K, Joachim S, Garric J, Boisseaux P, Exbrayat J-M, Porcher J-M, Geffard O (2017). Anatomical and histological characterization of the gametogenesis of Radix balthica (linnaeus, 1758) in comparison with Lymnaea stagnalis (linnaeus, 1758). Journal of Histology and Histopathology, 4(1).
Boisseaux P, Noury P, Thomas H, Garric J (2017). Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A.
Ecotoxicology and Environmental Safety,
139, 358-366.
Abstract:
Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A
This is a pioneering study in the ecotoxicological assessment of immunotoxic effects of the three selected drugs of concern to a freshwater gastropod species. Lymnaea stagnalis was exposed in the laboratory for 3 days to three drugs used for immune systems: diclofenac (nonsteroidal anti-inflammatory drug), cyclophosphamide (anti-cancer immunosuppressive drug) or cyclosporine a (anti-xenograft immunosuppressive drug). Exposure ranges included environmental realistic (1–10 μg L −1 ) and therapeutic concentrations (100–1000 μg L −1 ). At the end of exposure times, the immune parameters of individual snails were measured: hemocyte density and viability, hemocyte phagocytosis capacity and hemocyte-related oxidative activities (basal and NADPH-oxidase stimulated with zymosan particles). Diclofenac and cyclosporine a induced immune responses, although the effects were not strong. No immunosuppression was observed. Such subtle immunomodulations bring further interrogations regarding their long-term immunotoxicity and possible resulting tradeoffs with life-history traits. On the other hand, the prodrug cyclophosphamide did not induce significant immune responses. Since metabolism pathways differ greatly between vertebrates and invertebrates, this study also suggests that relevant vertebrate metabolites should be included in the immunotoxicity assessment of pharmaceuticals in non-target invertebrate species. Finally, the possible interactive effects of these pharmaceuticals sharing similar modes of action or effects features should also be explored.
Abstract.
Boisseaux P, Delignette-Muller ML, Abbaci K, Thomas H, Garric J (2016). Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections.
Fish and Shellfish Immunology,
57, 116-126.
Abstract:
Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections
The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly – regardless the time-intervals – at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight “day” effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the “day” effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails’ immunocompetence.
Abstract.
Boisseaux P, Noury P, Delignette-Muller ML, Thomas H, Garric J (2016). Recommendations for the analysis of hemocyte-related immunocompetent oxidative activity in the freshwater snail Lymnaea stagnalis. Journal of Xenobiotics, 6(2 2Special Issue), 25-27.
Lammel T, Boisseaux P, Navas JM (2015). Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1.
Environmental Toxicology,
30(10), 1192-1204.
Abstract:
Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1
Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (β-naphthoflavone (β-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on β-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard.
Abstract.
Boisseaux P, Gust M, Betoulle S, Garric J (2014). Short-term immunotoxic effects of an anti-cancer drug (Etoposide) on the freshwater pondsnail Lymnaea stagnalis. Journal of Xenobiotics, 4(2).
Lammel T, Boisseaux P, Fernández-Cruz ML, Navas JM (2013). Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2.
Particle and Fibre Toxicology,
10(1).
Abstract:
Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2
Background: Graphene and graphene derivative nanoplatelets represent a new generation of nanomaterials with unique physico-chemical properties and high potential for use in composite materials and biomedical devices. To date little is known about the impact graphene nanomaterials may have on human health in the case of accidental or intentional exposure. The objective of this study was to assess the cytotoxic potential of graphene nanoplatelets with different surface chemistry towards a human hepatoma cell line, Hep G2, and identify the underlying toxicity targets.Methods: Graphene oxide (GO) and carboxyl graphene (CXYG) nanoplatelet suspensions were obtained in water and culture medium. Size frequency distribution of the suspensions was determined by means of dynamic light scattering. Height, lateral dimension and shape of the nanoplatelets were determined using atomic force and electron microscopy. Cytotoxicity of GO and CXYG nanoplatelets was assessed in Hep G2 cells using a battery of assays covering different modes of action including alterations of metabolic activity, plasma membrane integrity and lysosomal function. Induction of oxidative stress was assessed by measuring intracellular reactive oxygen species levels. Interaction with the plasma membrane, internalization and intracellular fate of GO and CXYG nanoplatelets was studied by scanning and transmission electron microscopy.Results: Supplementing culture medium with serum was essential to obtain stable GO and CXYG suspensions. Both graphene derivatives had high affinity for the plasma membrane and caused structural damage of the latter at concentrations as low as 4 μg/ml. The nanoplatelets penetrated through the membrane into the cytosol, where they were concentrated and enclosed in vesicles. GO and CXYG accumulation in the cytosol was accompanied by an increase in intracellular reactive oxygen species (ROS) levels, alterations in cellular ultrastructure and changes in metabolic activity.Conclusions: GO and CXYG nanoplatelets caused dose- and time-dependent cytotoxicity in Hep G2 cells with plasma membrane damage and induction of oxidative stress being important modes of toxicity. Both graphene derivatives were internalized by Hep G2, a non-phagocytotic cell line. Moreover, they exerted no toxicity when applied at very low concentrations (< 4 μg/ml). GO and CXYG nanoplatelets may therefore represent an attractive material for biomedical applications. © 2013 Lammel et al.; licensee BioMed Central Ltd.
Abstract.
Publications by year
2023
Boisseaux P, Hopkinson P, Santillo D, Smith C, Garmulewicz A, Powell Z, Galloway T (2023). Environmental safety of second and third generation bioplastics in the context of the circular economy.
Ecotoxicol Environ Saf,
256Abstract:
Environmental safety of second and third generation bioplastics in the context of the circular economy.
Bioplastics derived from organic materials other than crude oil are often suggested as sustainable solutions for tackling end-of-life plastic waste, but little is known of their ecotoxicity to aquatic species. Here, we investigated the ecotoxicity of second and third generation bioplastics toward the freshwater zooplankton Daphnia magna. In acute toxicity tests (48 h), survival was impacted at high concentrations (g.L-1 range), within the range of salinity-induced toxicity. Macroalgae-derived bioplastic induced hormetic responses under chronic exposure (21 d). Most biological traits were enhanced from 0.06 to 0.25 g.L-1 (reproduction rate, body length, width, apical spine, protein concentration), while most of these traits returned to controls level at 0.5 g.L-1. Phenol-oxidase activity, indicative of immune function, was enhanced only at the lowest concentration (0.06 g.L-1). We hypothesise these suggested health benefits were due to assimilation of carbon derived from the macroalgae-based bioplastic as food. Polymer identity was confirmed by infra-red spectroscopy. Chemical analysis of each bioplastic revealed low metal abundance whilst non target exploration of organic compounds revealed trace amounts of phthalates and flame retardants. The macroalgae-bioplastic disintegrated completely in compost and biodegraded up to 86 % in aqueous medium. All bioplastics acidified the test medium. In conclusion, the tested bioplastics were classified as environmentally safe. Nonetheless, a reasonable end-of-life management of these safer-by-design materials is advised to ensure the absence of harmful effects at high concentrations, depending on the receiving environment.
Abstract.
Author URL.
2018
Boisseaux P, Noury P, Delorme N, Perrier L, Thomas-Guyon H, Garric J (2018). Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.
Environmental Science and Pollution Research,
25(17), 16720-16728.
Abstract:
Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters
Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.
Abstract.
2017
Tair-Abbaci K, Joachim S, Garric J, Boisseaux P, Exbrayat J-M, Porcher J-M, Geffard O (2017). Anatomical and histological characterization of the gametogenesis of Radix balthica (linnaeus, 1758) in comparison with Lymnaea stagnalis (linnaeus, 1758). Journal of Histology and Histopathology, 4(1).
Boisseaux P, Noury P, Thomas H, Garric J (2017). Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A.
Ecotoxicology and Environmental Safety,
139, 358-366.
Abstract:
Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A
This is a pioneering study in the ecotoxicological assessment of immunotoxic effects of the three selected drugs of concern to a freshwater gastropod species. Lymnaea stagnalis was exposed in the laboratory for 3 days to three drugs used for immune systems: diclofenac (nonsteroidal anti-inflammatory drug), cyclophosphamide (anti-cancer immunosuppressive drug) or cyclosporine a (anti-xenograft immunosuppressive drug). Exposure ranges included environmental realistic (1–10 μg L −1 ) and therapeutic concentrations (100–1000 μg L −1 ). At the end of exposure times, the immune parameters of individual snails were measured: hemocyte density and viability, hemocyte phagocytosis capacity and hemocyte-related oxidative activities (basal and NADPH-oxidase stimulated with zymosan particles). Diclofenac and cyclosporine a induced immune responses, although the effects were not strong. No immunosuppression was observed. Such subtle immunomodulations bring further interrogations regarding their long-term immunotoxicity and possible resulting tradeoffs with life-history traits. On the other hand, the prodrug cyclophosphamide did not induce significant immune responses. Since metabolism pathways differ greatly between vertebrates and invertebrates, this study also suggests that relevant vertebrate metabolites should be included in the immunotoxicity assessment of pharmaceuticals in non-target invertebrate species. Finally, the possible interactive effects of these pharmaceuticals sharing similar modes of action or effects features should also be explored.
Abstract.
2016
Boisseaux P, Delignette-Muller ML, Abbaci K, Thomas H, Garric J (2016). Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections.
Fish and Shellfish Immunology,
57, 116-126.
Abstract:
Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections
The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly – regardless the time-intervals – at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight “day” effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the “day” effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails’ immunocompetence.
Abstract.
Boisseaux P, Noury P, Delignette-Muller ML, Thomas H, Garric J (2016). Recommendations for the analysis of hemocyte-related immunocompetent oxidative activity in the freshwater snail Lymnaea stagnalis. Journal of Xenobiotics, 6(2 2Special Issue), 25-27.
2015
Lammel T, Boisseaux P, Navas JM (2015). Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1.
Environmental Toxicology,
30(10), 1192-1204.
Abstract:
Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1
Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (β-naphthoflavone (β-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on β-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard.
Abstract.
2014
Boisseaux P, Gust M, Betoulle S, Garric J (2014). Short-term immunotoxic effects of an anti-cancer drug (Etoposide) on the freshwater pondsnail Lymnaea stagnalis. Journal of Xenobiotics, 4(2).
2013
Lammel T, Boisseaux P, Fernández-Cruz ML, Navas JM (2013). Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2.
Particle and Fibre Toxicology,
10(1).
Abstract:
Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2
Background: Graphene and graphene derivative nanoplatelets represent a new generation of nanomaterials with unique physico-chemical properties and high potential for use in composite materials and biomedical devices. To date little is known about the impact graphene nanomaterials may have on human health in the case of accidental or intentional exposure. The objective of this study was to assess the cytotoxic potential of graphene nanoplatelets with different surface chemistry towards a human hepatoma cell line, Hep G2, and identify the underlying toxicity targets.Methods: Graphene oxide (GO) and carboxyl graphene (CXYG) nanoplatelet suspensions were obtained in water and culture medium. Size frequency distribution of the suspensions was determined by means of dynamic light scattering. Height, lateral dimension and shape of the nanoplatelets were determined using atomic force and electron microscopy. Cytotoxicity of GO and CXYG nanoplatelets was assessed in Hep G2 cells using a battery of assays covering different modes of action including alterations of metabolic activity, plasma membrane integrity and lysosomal function. Induction of oxidative stress was assessed by measuring intracellular reactive oxygen species levels. Interaction with the plasma membrane, internalization and intracellular fate of GO and CXYG nanoplatelets was studied by scanning and transmission electron microscopy.Results: Supplementing culture medium with serum was essential to obtain stable GO and CXYG suspensions. Both graphene derivatives had high affinity for the plasma membrane and caused structural damage of the latter at concentrations as low as 4 μg/ml. The nanoplatelets penetrated through the membrane into the cytosol, where they were concentrated and enclosed in vesicles. GO and CXYG accumulation in the cytosol was accompanied by an increase in intracellular reactive oxygen species (ROS) levels, alterations in cellular ultrastructure and changes in metabolic activity.Conclusions: GO and CXYG nanoplatelets caused dose- and time-dependent cytotoxicity in Hep G2 cells with plasma membrane damage and induction of oxidative stress being important modes of toxicity. Both graphene derivatives were internalized by Hep G2, a non-phagocytotic cell line. Moreover, they exerted no toxicity when applied at very low concentrations (< 4 μg/ml). GO and CXYG nanoplatelets may therefore represent an attractive material for biomedical applications. © 2013 Lammel et al.; licensee BioMed Central Ltd.
Abstract.
