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Interactions of a pesticide/heavy metal mixture in marine bivalves: a transcriptomic assessment.

Dondero F, Banni M, Negri A, Boatti L, Dagnino A, Viarengo A - BMC Genomics (2011)

Bottom Line: Finally, the relative mRNA abundance of twelve genes was followed by Q-PCR to either confirm or complement microarray data.These results, in general, were compatible with those from arrays and indeed confirmed the association of the relative abundance of two GM-2 ganglioside activator genes in the development of the hyperlipidosis syndrome observed in digestive gland lysosomes of single chemical exposed mussels.Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecologically relevant organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental and Life Sciences, Università del Piemonte Orientale Amedeo Avogadro, Alessandria, Italy. francesco.dondero@mfn.unipmn.it

ABSTRACT

Background: Mixtures of chemicals present in aquatic environments may elicit toxicity due to additive or synergistic effects among the constituents or, vice versa, the adverse outcome may be reduced by antagonistic interactions. Deviations from additivity should be explained either by the perturbations of toxicokinetic parameters and/or chemical toxicodynamics. We addressed this important question in marine mussels exposed subchronically to a binary mixture made of two wide-spread pollutants: the heavy metal nickel and the organic phosphorus pesticide Chlorpyrifos. To this aim, we carried out in tissues of Mytius galloprovincialis (Lam) a systems approach based on the evaluation and integration of different disciplines, i.e. high throughput gene expression profiling, functional genomics, stress biomakers and toxicokinetics.

Results: Cellular and tissue biomarkers, viz. digestive gland lysosomal membrane stability, lysosomal/cytosol volume ratio, neutral lipid content and gill acetylcholinesterase activity were, in general, altered by either the exposure to nickel and Chlorpyrifos. However, their joint action rendered (i) an overall decrease of the stress syndrome level, as evaluated through an expert system integrating biomarkers and (ii) statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further Gene Ontology-based functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray data (238 genes in total, representing about 14% of the whole microarray catalogue) showed two separate patterns for the single chemicals: the one belonging to the heavy metal -135 differentially expressed genes (DEGs) was characterized by the modulation of transcript levels involved in nucleic acid metabolism, cell proliferation and lipid metabolic processes. Chlorpyrifos exposure (43 DEGs) yielded a molecular signature which was biased towards carbohydrate catabolism (indeed, chitin metabolism) and developmental processes. The exposure to the mixture (103 DEGs) elicited a composite complex profile which encompassed the core properties of the pesticide but also a relevant set of unique features. Finally, the relative mRNA abundance of twelve genes was followed by Q-PCR to either confirm or complement microarray data. These results, in general, were compatible with those from arrays and indeed confirmed the association of the relative abundance of two GM-2 ganglioside activator genes in the development of the hyperlipidosis syndrome observed in digestive gland lysosomes of single chemical exposed mussels.

Conclusion: The transcriptomic assessment fitted with biological data to indicate the occurrence of different toxicodynamic events and, in general, a decrease of toxicity, driven by the mitigation or even abolition of lysosomal responses. Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecologically relevant organisms.

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Oil Red-O staining of lipids. cryostat sections of frozen mussel digestive gland were stained with the lysochrome Oil Red-O dye which is able to color neutral triglycerides and glycolipids. Panel A, control reference (vehicle treated); B, mixture 0.5 TU; C, mixture 1 TU; D, Chlorpyrifos 0.25 TU; E, Chlorpyrifos 0.5 TU; F, Chlorpyrifos 1 TU; G, Ni 0.25 TU; H, Ni 0.5 TU; I, Ni, 1 TU (see Table 1 for details on chemical concentrations).
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Figure 2: Oil Red-O staining of lipids. cryostat sections of frozen mussel digestive gland were stained with the lysochrome Oil Red-O dye which is able to color neutral triglycerides and glycolipids. Panel A, control reference (vehicle treated); B, mixture 0.5 TU; C, mixture 1 TU; D, Chlorpyrifos 0.25 TU; E, Chlorpyrifos 0.5 TU; F, Chlorpyrifos 1 TU; G, Ni 0.25 TU; H, Ni 0.5 TU; I, Ni, 1 TU (see Table 1 for details on chemical concentrations).

Mentions: The biological effects of exposure to the single chemicals and mixtures were evaluated through a battery of biological endpoints encompassing lysosomal responses in the digestive gland (LMS; lysosome/cytoplasm ratio; lipid accumulation) and acetylcholinesterase activity in the gills (Figure 1). As expected, the LMS assay showed a clear dose-response trend with a decrement of the hexosaminidase activity latency along with the increase of pollutant concentrations (Figure 1, panel A). An increased lysosomal/cytoplasm ratio was also observed (Figure 1, panel B). The lipid content rose dramatically at all Ni doses while the biocide elicited a significant response only at the highest tested concentration (Figure 1, panel C; Figure 2). Acetylcholinesterase activity was evaluated in the gills of bivalves as a specific biomarker of exposure to the organophosphate pesticide. About 80% inhibition and a typical threshold effect was ascertained in the case of Chlorpyrifos exposure. However, a significant effect and a similar trend was also observed in tissues from Ni exposures (Figure 1, panel D). Our results from mixtures indicated that n-acetyl-beta-hexosaminidase latency times in destabilized lysosome membranes were higher than the expected values calculated according to either the Concentration Addition or the Independent Action (IA) mixture toxicity reference system models (Table 2). This antagonistic outcome was also confirmed for the other lysosomal responses evaluated in the digestive tissue, viz. organelle enlargement, lipid accumulation and the inhibition of the acetylcholinesterase activity in gills (Table 2).


Interactions of a pesticide/heavy metal mixture in marine bivalves: a transcriptomic assessment.

Dondero F, Banni M, Negri A, Boatti L, Dagnino A, Viarengo A - BMC Genomics (2011)

Oil Red-O staining of lipids. cryostat sections of frozen mussel digestive gland were stained with the lysochrome Oil Red-O dye which is able to color neutral triglycerides and glycolipids. Panel A, control reference (vehicle treated); B, mixture 0.5 TU; C, mixture 1 TU; D, Chlorpyrifos 0.25 TU; E, Chlorpyrifos 0.5 TU; F, Chlorpyrifos 1 TU; G, Ni 0.25 TU; H, Ni 0.5 TU; I, Ni, 1 TU (see Table 1 for details on chemical concentrations).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094310&req=5

Figure 2: Oil Red-O staining of lipids. cryostat sections of frozen mussel digestive gland were stained with the lysochrome Oil Red-O dye which is able to color neutral triglycerides and glycolipids. Panel A, control reference (vehicle treated); B, mixture 0.5 TU; C, mixture 1 TU; D, Chlorpyrifos 0.25 TU; E, Chlorpyrifos 0.5 TU; F, Chlorpyrifos 1 TU; G, Ni 0.25 TU; H, Ni 0.5 TU; I, Ni, 1 TU (see Table 1 for details on chemical concentrations).
Mentions: The biological effects of exposure to the single chemicals and mixtures were evaluated through a battery of biological endpoints encompassing lysosomal responses in the digestive gland (LMS; lysosome/cytoplasm ratio; lipid accumulation) and acetylcholinesterase activity in the gills (Figure 1). As expected, the LMS assay showed a clear dose-response trend with a decrement of the hexosaminidase activity latency along with the increase of pollutant concentrations (Figure 1, panel A). An increased lysosomal/cytoplasm ratio was also observed (Figure 1, panel B). The lipid content rose dramatically at all Ni doses while the biocide elicited a significant response only at the highest tested concentration (Figure 1, panel C; Figure 2). Acetylcholinesterase activity was evaluated in the gills of bivalves as a specific biomarker of exposure to the organophosphate pesticide. About 80% inhibition and a typical threshold effect was ascertained in the case of Chlorpyrifos exposure. However, a significant effect and a similar trend was also observed in tissues from Ni exposures (Figure 1, panel D). Our results from mixtures indicated that n-acetyl-beta-hexosaminidase latency times in destabilized lysosome membranes were higher than the expected values calculated according to either the Concentration Addition or the Independent Action (IA) mixture toxicity reference system models (Table 2). This antagonistic outcome was also confirmed for the other lysosomal responses evaluated in the digestive tissue, viz. organelle enlargement, lipid accumulation and the inhibition of the acetylcholinesterase activity in gills (Table 2).

Bottom Line: Finally, the relative mRNA abundance of twelve genes was followed by Q-PCR to either confirm or complement microarray data.These results, in general, were compatible with those from arrays and indeed confirmed the association of the relative abundance of two GM-2 ganglioside activator genes in the development of the hyperlipidosis syndrome observed in digestive gland lysosomes of single chemical exposed mussels.Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecologically relevant organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental and Life Sciences, Università del Piemonte Orientale Amedeo Avogadro, Alessandria, Italy. francesco.dondero@mfn.unipmn.it

ABSTRACT

Background: Mixtures of chemicals present in aquatic environments may elicit toxicity due to additive or synergistic effects among the constituents or, vice versa, the adverse outcome may be reduced by antagonistic interactions. Deviations from additivity should be explained either by the perturbations of toxicokinetic parameters and/or chemical toxicodynamics. We addressed this important question in marine mussels exposed subchronically to a binary mixture made of two wide-spread pollutants: the heavy metal nickel and the organic phosphorus pesticide Chlorpyrifos. To this aim, we carried out in tissues of Mytius galloprovincialis (Lam) a systems approach based on the evaluation and integration of different disciplines, i.e. high throughput gene expression profiling, functional genomics, stress biomakers and toxicokinetics.

Results: Cellular and tissue biomarkers, viz. digestive gland lysosomal membrane stability, lysosomal/cytosol volume ratio, neutral lipid content and gill acetylcholinesterase activity were, in general, altered by either the exposure to nickel and Chlorpyrifos. However, their joint action rendered (i) an overall decrease of the stress syndrome level, as evaluated through an expert system integrating biomarkers and (ii) statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further Gene Ontology-based functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray data (238 genes in total, representing about 14% of the whole microarray catalogue) showed two separate patterns for the single chemicals: the one belonging to the heavy metal -135 differentially expressed genes (DEGs) was characterized by the modulation of transcript levels involved in nucleic acid metabolism, cell proliferation and lipid metabolic processes. Chlorpyrifos exposure (43 DEGs) yielded a molecular signature which was biased towards carbohydrate catabolism (indeed, chitin metabolism) and developmental processes. The exposure to the mixture (103 DEGs) elicited a composite complex profile which encompassed the core properties of the pesticide but also a relevant set of unique features. Finally, the relative mRNA abundance of twelve genes was followed by Q-PCR to either confirm or complement microarray data. These results, in general, were compatible with those from arrays and indeed confirmed the association of the relative abundance of two GM-2 ganglioside activator genes in the development of the hyperlipidosis syndrome observed in digestive gland lysosomes of single chemical exposed mussels.

Conclusion: The transcriptomic assessment fitted with biological data to indicate the occurrence of different toxicodynamic events and, in general, a decrease of toxicity, driven by the mitigation or even abolition of lysosomal responses. Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecologically relevant organisms.

Show MeSH
Related in: MedlinePlus