Alessandro Negri

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

BackgroundMixtures 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.ResultsCellular 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.ConclusionThe 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.

Gene Expression Rhythms in the Mussel Mytilus galloprovincialis (Lam.) across an Annual Cycle

Seasonal environmental changes may affect the physiology of Mytilus galloprovincialis (Lam.), an intertidal filter-feeder bivalve occurring commonly in Mediterranean and Atlantic coastal areas. We investigated seasonal variations in relative transcript abundance of the digestive gland and the mantle (gonads) of males and females. To identify gene expression trends – in terms of relative mRNA abundance- we used a medium-density cDNA microarray (1.7 K probes) in dual-color competitive hybridization analyses. Hierarchical clustering of digestive gland microarray data showed two main branches, distinguishing profiles associated with the “hot” months (May–August) from the other months. Genes involved in chitin metabolism, associated with mussel nutrition and digestion showed higher mRNA levels during summer. Moreover, we found different gene transcriptomic patterns in the digestive glands of males when compared to females, during the four stages of mussel gonadal development. Microarray data from gonadal transcripts also displayed clear patterns during the different developmental phases respect to the resting period (stage I) with peak relative mRNA abundance at the ripe phase (stage III) for both sexes. These data showed a clear temporal pattern in transcriptomic profiles of mussels sampled over an annual cycle. Physiological response to thermal variation, food availability, and reproductive status across months may contribute to variation in relative mRNA abundance.

Acute effects of benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel Mytilus galloprovincialis

In the present study, mussel (Mytilus galloprovincialis) digestive gland biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) were investigated. Mussels were exposed to a sublethal dose of B[a]P (75 nM; 19 microg/l per animal) for 24, 48 and 72h. The following biological responses were measured in the digestive gland tissues: (1) B[a]P hydroxylase (BPH) activity, as phase I biotransformation parameter; (2) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase (CAT) activity as potential biomarker of oxidative stress, (4) acetylcholinesterase (AChE) activity as an indication of possible neurotoxicity response. DNA damage was assessed over time using the single cell gel electrophoresis comet assay and the micronuclei test. BPH and GST activities showed an increasing trend over exposure period. CAT activity showed a symmetrical bell shape response with a maximum at 48h. AChE activity was significantly depressed after 48 and 72h exposure to B[a]P. Comet assay and micronuclei test in digestive gland cells suggest that B[a]P exposure induced significant DNA damage with a maximum response after 72h exposure.

Transcriptomic and proteomic effects of a neonicotinoid insecticide mixture in the marine mussel (Mytilus galloprovincialis, Lam.).

BACKGROUND Imidacloprid and Thiacloprid are two neonicotinoid insecticides whose use have been raising exponentially. Both act selectively as agonist of the insect nicotinic-Acetylcholine Receptor (nAChR) and therefore, by definition, they hold the same mode of action. Notwithstanding the growing attention to the ecotoxicological effects of neonicotinoids, there is a lack of information on their toxicodynamics and their mixture effects, in particular, in aquatic organisms. OBJECTIVES The main objectives of this work were: (i) assess sublethal effects of two neonicotinoids-Imidacloprid and Thiacloprid-in the tissues of the marine mussel (Mytilus galloprovincialis Lam.); (ii) identify the molecular dynamics elicited by the two chemicals through gene/protein expression profiling and a functional genomics approach; (iii) assess the effects of a neonicotinoid binary mixture. METHODS Sublethal effects were measured by means of digestive gland lysosomal membrane stability (LMS) and gill acetylcholinesterase (AChE) activity. Gene expression profiles were evaluated in the digestive gland using a 1.7K cDNA microarray and quantitative-PCR (Q-PCR). Proteome profiling was performed by means of two-dimensional electrophoresis of digestive gland cytosolic proteins. Functional genomics was based on the over-representation of Gene Ontology (GO) terms. RESULTS AND DISCUSSION Our results showed that (i) biomarkers responded in the micromolar range; (ii) Imidacloprid and Thiacloprid elicited distinct toxicodynamics as depicted by the different transcriptomic and proteomic profiles and the opposite trend of AChE activity; (iii) at biomarkers level, the joint effects of the two chemicals appeared to fulfill the principle of independence, but this was less evident at molecular level where a novel specific molecular signature took place. CONCLUSIONS These findings imply that different toxicodynamics may occur also as a response of chemicals with the same mode of action. Our results unveil also the incongruousness of the actual concept of pesticide mode of action in the context of ecological risk assessment analysis of chemical mixtures.

Transcriptional Response of the Mussel Mytilus galloprovincialis (Lam.) following Exposure to Heat Stress and Copper

Global warming is a major factor that may affect biological organization, especially in marine ecosystems and in coastal areas that are particularly subject to anthropogenic pollution. We evaluated the effects of simultaneous changes in temperature and copper concentrations on lysosomal membrane stability (N-acetyl-hexosaminidase activity) and malondialdehyde accumulation (MDA) in the gill of the blue mussel Mytilus galloprovincialis (Lam.). Temperature and copper exerted additive effects on lysosomal membrane stability, exacerbating the toxic effects of metal cations present in non-physiological concentrations. Mussel lysosomal membrane stability is known to be positively related to scope for growth, indicating possible effects of increasing temperature on mussel populations in metal-polluted areas. To clarify the molecular response to environmental stressors, we used a cDNA microarray with 1,673 sequences to measure the relative transcript abundances in the gills of mussels exposed to copper (40 µg/L) and a temperature gradient (16°C, 20°C, and 24°C). In animals exposed only to heat stress, hierarchical clustering of the microarray data revealed three main clusters, which were largely dominated by down-regulation of translation-related differentially expressed genes, drastic up-regulation of protein folding related genes, and genes involved in chitin metabolism. The response of mussels exposed to copper at 24°C was characterized by an opposite pattern of the genes involved in translation, most of which were up-regulated, as well as the down-regulation of genes encoding heat shock proteins and “microtubule-based movement” proteins. Our data provide novel information on the transcriptomic modulations in mussels facing temperature increases and high copper concentrations; these data highlight the risk of marine life exposed to toxic chemicals in the presence of temperature increases due to climate change.

Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations

In the present study, mussel (Mytilus galloprovincialis) digestive gland oxidative stress biomarkers and detoxification responses to acute exposure to nickel (Ni) were investigated. Mussels were exposed to two sublethal concentrations of Ni (135 μg/L per animal (2.5 μM) and 770 μg/L per animal (13 μM)) for 24, 48, 72, 96 h and 8 days. Following biological responses were measured: (1) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (2) catalase activity as antioxidant response, (3) malondialdehyde accumulation (MDA) as lipid peroxydation marker and metallothionein as specific response to metals exposure. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes during the exposure periods were assessed and the uptake rate constant determined. A correlation matrix (CM) between the investigated biomarkers and a principal component analysis (PCA) were achieved for the two tested concentrations. The Ni-uptake constant was higher in animals exposed to the lowest concentration. The CM and the PCA showed a time-dependent effect of the Ni exposure on the investigated biomarkers being more pronounced in animals exposed to the highest Ni concentration. While AChE showed a significant increase after 48 h and a further return to control values in the lowest concentration, it was drastically maintained inhibited in the highest concentration. Our data provided clues about the occurrence of different toxicokinetics and toxicodynamics of two Ni sublethal concentrations in an ecologically relevant organism.

The organophosphate Chlorpyrifos interferes with the responses to 17β-estradiol in the digestive gland of the marine mussel Mytilus galloprovincialis.

Background Many pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists are low compared with those of natural ligands, their ability to act via multiple mechanisms might enhance the biological effect. The organophosphate Chlorpyrifos (CHP) has been shown to be weakly estrogenic and cause adverse neurodevelopmental effects in mammals. However, no information is available on the endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue of both estrogens and pesticides, the possible effects of CHP on the responses to the natural estrogen 17β-estradiol (E2) were investigated. Methodology/Principal Findings Mussels were exposed to CHP (4.5 mg/l, 72 hrs) and subsequently injected with E2 (6.75 ng/g dw). Responses were evaluated in CHP, E2 and CHP/E2 treatment groups at 24 h p.i. by a biomarker/transcriptomic approach. CHP and E2 induced additive, synergistic, and antagonistic effects on lysosomal biomarkers (lysosomal membrane stability, lysosome/cytoplasm volume ratio, lipofuscin and neutral lipid accumulation). Additive and synergistic effects were also observed on the expression of estrogen-responsive genes (GSTπ, catalase, 5-HTR) evaluated by RT-Q-PCR. The use of a 1.7K cDNA Mytilus microarray showed that CHP, E2 and CHP/E2, induced 81, 44, and 65 Differentially Expressed Genes (DEGs), respectively. 24 genes were exclusively shared between CHP and CHP/E2, only 2 genes between E2 and CHP/E2. Moreover, 36 genes were uniquely modulated by CHP/E2. Gene ontology annotation was used to elucidate the putative mechanisms involved in the responses elicited by different treatments. Conclusions The results show complex interactions between CHP and E2 in the digestive gland, indicating that the combination of certain pesticides and hormones may give rise to unexpected effects at the molecular/cellular level. Overall, these data demonstrate that CHP can interfere with the mussel responses to natural estrogens.

Expression analysis of the molluscan p53 protein family mRNA in mussels (Mytilus spp.) exposed to organic contaminants

In this study, we report the tissue expression analysis of the p53 protein family mRNA in mussels (Mytilus spp.) by means of quantitative RT-PCR. The tissue specific response was evaluated after 24 h exposure to a sublethal benzo[a]pyrene (B[a]P) concentration (75 nM), showing a 2.6 fold induction in digestive gland cells and a dramatic gene down regulation in circulating hemocytes. The comet assay and DNA gel diffusion tests showed significant effects in hemocytes and negligible differences in the digestive gland nuclei, implicating p53 in DNA damage of molluscan hemocytes. Finally, the kinetics of p53 protein family mRNA expression in the digestive gland of animals exposed to B[a]P and crude oil (0.5 ppm) showed partially overlapping trends, characterised by a common down regulation after 1 week exposure. These data should be carefully considered in view of the biological effects of organic pollutants and particularly following spills.

Use of highly sensitive sublethal stress responses in the social amoeba Dictyostelium discoideum for an assessment of freshwater quality

In this work, the sensitivity of a battery of tests on the social amoeba Dictyostelium discoideum has been assessed within a freshwater toxicity study. The results obtained from the evaluation of survival and replication rate of D. discoideum were compared to those derived with a series of widely used tests for freshwater toxicity assessment, i. e. bioassays using Vibrio fischeri, Daphnia magna and Pseudokirchneriella subcapitata. The effects on sublethal endpoints, i.e. lysosomal membrane stability (LMS) and endocytotic rate, were analysed in conjunction with high-level endpoints to verify the potential to make a typical bioassay more sensitive. The field ecotoxicological investigation employing D. discoideum is part of a monitoring study assessing environmental quality of the Bormida River (Italy), subjected until recently to a chronic industrial pollution. The survey was carried out at several stations (upstream and downstream of a chemical factory outlet) in two different periods. In 2002, the results of chemical analyses performed on river water indicated no contamination. The ecotoxicological data obtained in this period showed that no evidence of biological effects was observed using V. fischeri and D. magna bioassays. In spite of the previous classical acute toxicity tests, significant differences in cell viability of D. discoideum were found. By analysing the effects measured on LMS and endocytotic rate, more relevant changes were observed for these sublethal stress biomarkers compared to survival. The chronic toxicity data showed significant changes in cell growth both of P. subcapitata and D. discoideum. Nevertheless, more sensitive and rapid responses were obtained when assessing the effects of exposure on D. discoideum. The chemical and ecotoxicological data obtained in 2006 indicated a full recovery of the quality of the river water (neither contamination nor toxicity found). Altogether, the results reported in this study underline that the use of a battery of biomarkers in conjunction with high-level endpoints may help follow the pollutant-induced stress syndrome in the organisms from early sublethal effects to starting mortality.