Expression of CYP4 and GSTr genes in Venerupis philippinarum exposed to benzo(a)pyrene

Autori: Boscolo Papo M, Maccatrozzo L, Bertotto D, Pascoli F, Negrato E, Poltronieri C, Binato G, Gallina A, Radaelli G

Riferimenti: Annals of Anatomy (2014) 196 (4), 241-6

Summary: Bivalve molluscs, such as Venerupis philippinarum, are often used as bioindicators of environmental pollution since they can bioaccumulate a large variety of pollutants because of their filter feeding. The Polycyclic Aromatic Hydrocarbon (PAH) benzo(a)pyrene (B(a)P) is an important contaminant, commonly present in the marine environment. Pollutants are generally metabolized by enzymes of phase I, mainly CYPs enzymes, and by conjugation enzymes of phase II like GST. In this study, we investigated by Real Time PCR the expression of CYP4 and GSTr (GST class rho) in the digestive gland of V. philippinarum exposed to different concentrations of B(a)P for 24 h and after a 24 h depuration period. Accumulation of B(a)P by clams has been confirmed by the HPLC-FLD analyses. Moreover, HPLC-FLD analyses evidenced that after depuration, B(a)P concentrations decreased in animals subjected to 0.03 mg/l and 0.5 mg/l exposures but did not decrease in animals subjected to 1 mg/l exposure. B(a)P exposure and depuration did not cause histopathological lesions in the different organs. The analysis of GSTr expression in the digestive gland showed a significant increase in mRNA in animals subjected to 1 mg/l exposure, whereas the analysis of CYP4 expression did not evidence differences among treatments. Moreover, the expression of both genes did not exhibit any differences after the purification treatment. The results demonstrate that B(a)P significantly affects the expression of GSTr mRNA in the digestive gland of V. philippinarum and suggest that GSTr gene could play an important role in the biotransformation of B(a)P.

Link: https://www.sciencedirect.com/science/article/pii/S0940960213001672?via%3Dihub

  Significance of the goby Zosterisessor ophiocephalus as a sentinel species for Venice Lagoon contamination: Combining biomarker responses and bioaccumulation

Autori: Pauletto M, Lopparelli RM, Pegolo S, Armani M, Zorzan E, Giantin M, Bertotto D, Gallocchio F, Zancanella V, Capolongo F, Binato G, Mutinelli F, Dacasto M

Riferimenti: Science of the Total Environment (2019) 660, pp. 959-973

Summary: The Venice Lagoon is an interesting example of an ecosystem suffering for a considerable anthropogenic impact, resulting in high concentrations of persistent organic pollutants (POPs) in lagoon sediments and seafood. In this context, biomonitoring is a crucially important task.
The present study aimed at evaluating the validity of a multiple biomarker approach in a benthic fish species. A total of 567 Zosterisessor ophiocephalus (Gobiidae) fish were collected in spring and autumn from three areas of Venice Lagoon (Porto Marghera, Val di Brenta, and Cà Roman) showing high, intermediate and low amounts of POPs, respectively. Aryl hydrocarbon receptor (AHR) and cytochrome P450 1A (CYP1A) mRNA levels, CYP1A protein amount and ethoxyresorufin O-deethylase activity (EROD) were measured in pooled liver and gills (mRNA levels only). Such biological data were then compared with polychlorinated biphenyls (PCBs) residues, measured in grass goby muscle by gas chromatography.
Aryl hydrocarbon receptor and CYP1A mRNAs, protein and EROD were upregulated in accordance with PCB amounts measured in Z. ophiocephalus muscles. In fact, the highest AHR and CYP1A induction was observed in fish sampled in close proximity of the industrial area of Porto Marghera.
Overall, the present study confirm the grass goby as a reliable sentinel species for Venice Lagoon, and AHR/CYP1A/EROD as a sensitive set of biomarkers of exposure for AHR ligands.

Link: https://www.sciencedirect.com/science/article/pii/S0048969719300397

  Histopathology and stress biomarkers in the clam Venerupis philippinarum from the Venice Lagoon (Italy)

Autori: Boscolo Papo M, Bertotto D, Quaglio F, Vascellari M, Pascoli F, Negrato E, Binato G, Radaelli G

Riferimenti: Fish and Shellfish Immunology (2014) 39 (1), pp. 42-50

Summary: The aim of this study was to evaluate the histomorphology and the stress response in the bivalve Venerupis philippinarum sampled in four differently polluted sites of the Venice Lagoon (Palude del Monte, Marghera, Ca' Roman and Val di Brenta). This species is often used as bioindicator of environmental pollution since it can bioaccumulate a large variety of pollutants because of its filter feeding. Chemical analyses for heavy metals (Cd, Cu, Hg and Pb) and polycyclic aromatic hydrocarbons (PAHs) were performed on whole soft tissues of V. philippinarum. The histological evaluation of clams revealed the presence of Perkinsus sp. infection in animals from all sites, although a very high prevalence of parasites was evidenced in clams from Ca' Roman. Perkinsus sp. were systemically distributed in the mantle, in the intestine and digestive gland, in gonads and gills. The trophozoites of Perkinsus sp. were found isolated or in cluster surrounded by a heavy hemocitical response.

Haemocytes always exhibited an immunopositivity to cytochrome P4501A (CYP1A), heat shock protein 70 (HSP70), 4-hydroxy-2-nonenal (HNE) and nitrotyrosine (NT) antibodies. The digestive gland of animals from Palude del Monte showed the highest malondialdehyde (MDA) concentration, whereas clams from Ca' Roman exhibited the highest quantity of metallothioneins.

Link: https://www.sciencedirect.com/science/article/pii/S1050464814001326

  Induction of brown cells in Venerupis philippinarum exposed to benzo(a)pyrene

Autori: Boscolo Papo M, Bertotto D, Pascoli F, Locatello L, Vascellari M, Poltronieri C, Quaglio F, Radaelli G

Riferimenti: Fish and Shellfish Immunology (2014) 40 (1), pp. 233-238

Summary: Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) commonly present in the marine environment and responsible for carcinogenic, teratogenic and mutagenic effects in various animal species. In the present study, we investigated by both histochemical and immunohistochemical approaches the effect of an acute exposure to different concentrations of B(a)P in the Manila clam Venerupis philippinarum. The general morphology of the different clam tissues, which was investigated histologically, evidenced a significant increase in the number of intestinal brown cells after B(a)P exposure. An increasing trend response to B(a)P was detected. The histochemical analysis for lipofuscin revealed the presence of lipofuscin-like substances inside the cytoplasm of intestinal brown cells. The same cells exhibited a PAS positivity and a reactivity to Schmorl's solution for melanin pigment. Moreover, intestinal brown cells exhibited an immunopositivity to HSP70 antibody confirming the increasing trend response to B(a)P detected by the histochemical analysis. Our results suggest that histological tissue changes resulting from exposure to B(a)P can be an useful marker in biomonitoring studies.

Link: https://www.sciencedirect.com/science/article/pii/S1050464814002472?via%3Dihub

  Can ecological history influence response to pollutants? Transcriptomic analysis of Manila clam collected in different Venice lagoon areas and exposed to heavy metal

Autori: Milan M, Matozzo V, Pauletto M, Di Camillo B, Giacomazzo M, Boffo L, Binao G, Marin MG, Patarnello T, Bargelloni L

Riferimenti: Aquatic Toxicology. Volume 174, May 2016, Pages 123–133. doi:10.1016/j.aquatox.2016.02.024.

Summary: Chronic exposure to environmental pollutants can exert strong selective pressures on natural populations, favoring the transmission over generations of traits that enable individuals to survive and thrive in highly impacted environments. The lagoon of Venice is an ecosystem subject to heavy anthropogenic impact, mainly due to the industrial activities of Porto Marghera (PM), which led to a severe chemical contamination of soil, groundwater, and sediments. Gene expression analysis on wild Manila clams collected in different Venice lagoon areas enabled to identify differences in gene expression profiles between clams collected in PM and those sampled in clean areas, and the definition of molecular signatures of chemical stress. However, it remains largely unexplored to which extent modifications of gene expression patterns persists after removing the source of contamination. It is also relatively unknown whether chronic exposure to xenobiotics affects the response to other chemical pollutants. To start exploring such issues, in the present study a common-garden experiment was coupled with transcriptomic analysis, to compare gene expression profiles of PM clams with those of clams collected in the less impacted area of Chioggia (CH) during a period under the same control conditions. Part of the two experimental groups were also exposed to copper for seven days to assess whether different “ecological history” does influence response to such pollutant.
The results obtained suggest that the chronic exposure to chemical pollution generated a response at the transcriptional level that persists after removal for the contaminated site. These transcriptional changes are centered on key biological processes, such as defense against either oxidative stress or tissue/protein damage, and detoxification, suggesting an adaptive strategy for surviving in the deeply impacted environment of Porto Marghera. On the other hand, CH clams appeared to respond more effectively to copper exposure than PM animals, proposing that chronic exposure to chemical toxicants either lowers the sensitivity to additional toxicants or blunts the capacity to respond to novel chemical challenges in PM clams.

Link: https://www.sciencedirect.com/science/article/pii/S0166445X16300534?via%3Dihub

  Ecotoxicological potential of non-steroidal anti-inflammatory drugs (NSAIDs) in marine organisms: Bioavailability, biomarkers and natural occurrence in Mytilus galloprovincialis

Autori: Mezzelani M, Gorbi S, Da Ros Z , Fattorini D, d'Errico G, Milan M, Bargelloni L,  Regoli F.

Riferimenti: Marine Environmental Research 121, pp. 31-39 (2016) 1e9.

Summary: Pharmaceuticals represent a major environmental concern since the knowledge on their occurrence, distribution and ecotoxicological potential is still limited particularly in coastal areas. In this study, bioaccumulation and cellular effects of various non steroidal anti-inflammatory drugs (NSAIDs) were investigated in mussels Mytilus galloprovincialis to reveal whether common molecules belonging to the same therapeutic class might cause different effects on non target organisms. Organisms exposed to environmental concentrations of acetaminophen (AMP), diclofenac (DIC), ibuprofen (IBU), ketoprofen (KET) and nimesulide (NIM) revealed a significant accumulation of DIC, IBU and NIM, while AMP and KET were always below detection limit. Nonetheless, for all tested NSAIDs, measurement of a large panel of ecotoxicological biomarkers highlighted impairment of immunological parameters, onset of genotoxicity and modulation of lipid metabolism, oxidative and neurotoxic effects. Laboratory results were integrated with a field study which provided the first evidence on the occurrence of DIC, IBU and NIM in tissues of wild mussels sampled during summer months from an unpolluted, touristic area of Central Adriatic Sea. Overall results demonstrated M. galloprovincialis as a good sentinel species for monitoring presence and ecotoxicological hazard of pharmaceuticals in the Mediterranean.

Link: https://www.sciencedirect.com/science/article/pii/S0141113616300307?via%3Dihub

  Transcriptomic profiling of Chamelea gallina from sites along the Abruzzo coast (Italy), subject to periodic localized mortality events

Autori: Milan M, Palazzo F, Papetti C, Grotta L, Marchetti S, ·Patarnello T, Bargelloni L, Martino G.

Riferimenti: Marine Biology (2016) 163:196. DOI 10.1007/s00227-016-2971-2.

Summary: The striped venus Chamelea gallina (Linnaeus 1758) is a species of substantial economic importance, distributed along the Mediterranean coast and the Eastern Atlantic coasts. Due to an intensive trade activity since the 1970’s, this species has experienced a rapid decline in the last 30 years, still exacerbated by irregular mortality events due to undetermined triggers. Global profiling of gene expression has the potential to disentangle the biological effects of various environmental stressors causing severe landings decline or mortality events. In this study, we report the development of a C. gallina-specific oligo-microarray, a first transcriptomic platform for global gene expression profiling in the striped venus. This tool was applied to compare gene expression profiles of clams collected in different periods along the Abruzzo coast in a reference site (T7) and in a site subject to mortality events (T4). Our results reveal substantial transcriptional modifications among the investigated sites and the significant up-regulation of several genes involved in the immune response in the T4 site. The transcriptomic differences between clams from the two investigated sites endorse distinct health and metabolic status under different environmental stressors. Overall, this approach provides a preliminary indication about the potential causes of periodic localized mortality affecting C. gallina.

Link: https://link.springer.com/article/10.1007/s00227-016-2971-2

  Transcriptional and cellular effects of Non-SteroidalAnti-Inflammatory Drugs (NSAIDs) in experimentally exposed mussels, Mytilus galloprovincialis

Autori: Mezzelani M, Gorbi S,  Fattorini D,  d’Errico G.,  Benedetti M, Milan M., Bargelloni L, Regoli F.

Riferimenti: Aquatic Toxicology 180 (2016) 306–319. 

Summary: The aim of the present investigation was to provide new insights on accumulation and possible adverse effects of various non-steroidal anti-inflammatory drugs (NSAIDs) in mussels, Mytilus galloprovincialis, exposed to an environmentally realistic concentration (0.5 μg/L) of individual compounds, Acetaminophen (AMP), Diclofenac (DIC), Ibuprofen (IBU), Ketoprofen (KET) or Nimesulide (NIM). The measurement of drugs in mussel tissues was integrated with both functional alterations at cellular level and transcriptomic responses. Results indicated the capability of mussels to accumulate DIC and NIM, while AMP, IBU and KET were always below detection limit. A large panel of ecotoxicological biomarkers revealed the early onset of alterations induced by tested NSAIDs on immunological responses, lipid metabolism and DNA integrity. The gene transcription analysis through DNA microarrays, supported cellular biomarker results, with clear modulation of a large number of genes involved in the arachidonic acid and lipid metabolism, immune responses, cell cycle and DNA repair. The overall results indicated an ecotoxicological concern for pharmaceuticals in M. galloprovincialis, with transcriptional responses appearing as sensitive exposure biomarkers at low levels of exposure: such changes, however, are not always paralleled by corresponding functional effects, suggesting caution when interpreting observed effects in terms of perturbed cellular pathways. Fascinating similarities can also be proposed in the mode of action of NSAIDs between bivalves and vertebrate species.

Link: https://www.sciencedirect.com/science/article/pii/S0166445X16302831?via%3Dihub

  Transcriptome analysis of the brain of the sea bream (Sparus aurata) after exposure to human pharmaceuticals at realistic environmental concentrations

Autori: Hampel M, Blasco J, Babbucci M, Ferraresso S, Bargelloni L, Milan M.

Riferimenti: Marine Environmental Research. S0141-1136(17)30084-3. 10.1016/j.marenvres.2017.04.012. 

Summary: The aim of the present investigation was to provide new insights on accumulation and possible adverse effects of various non-steroidal anti-inflammatory drugs (NSAIDs) in mussels, Mytilus galloprovincialis, exposed to an environmentally realistic concentration (0.5 μg/L) of individual compounds, Acetaminophen (AMP), Diclofenac (DIC), Ibuprofen (IBU), Ketoprofen (KET) or Nimesulide (NIM). The measurement of drugs in mussel tissues was integrated with both functional alterations at cellular level and transcriptomic responses. Results indicated the capability of mussels to accumulate DIC and NIM, while AMP, IBU and KET were always below detection limit. A large panel of ecotoxicological biomarkers revealed the early onset of alterations induced by tested NSAIDs on immunological responses, lipid metabolism and DNA integrity. The gene transcription analysis through DNA microarrays, supported cellular biomarker results, with clear modulation of a large number of genes involved in the arachidonic acid and lipid metabolism, immune responses, cell cycle and DNA repair. The overall results indicated an ecotoxicological concern for pharmaceuticals in M. galloprovincialis, with transcriptional responses appearing as sensitive exposure biomarkers at low levels of exposure: such changes, however, are not always paralleled by corresponding functional effects, suggesting caution when interpreting observed effects in terms of perturbed cellular pathways. Fascinating similarities can also be proposed in the mode of action of NSAIDs between bivalves and vertebrate species.

Link: https://www.sciencedirect.com/science/article/pii/S0166445X16302831?via%3Dihub

  Microbiota and environmental stress: how pollution affects microbial communities in Manila clams

Autori: Milan M, Carraro L, Fariselli P, Martino ME, Cavalieri D, Vitali F, Boffo L, Patarnello T, Bargelloni L, Cardazzo B

Riferimenti: Aquatic Toxicology  2018 Jan;194:195-207. doi: 10.1016/j.aquatox.2017.11.019.

Summary: Given the crucial role of microbiota in host development, health, and environmental interactions, genomic analyses focusing on host-microbiota interactions should certainly be considered in the investigation of the adaptive mechanisms to environmental stress. Recently, several studies suggested that microbiota associated to digestive tract is a key, although still not fully understood, player that must be considered to assess the toxicity of environmental contaminants. Bacteria-dependent metabolism of xenobiotics may indeed modulate the host toxicity. Conversely, environmental variables (including pollution) may alter the microbial community and/or its metabolic activity leading to host physiological alterations that may contribute to their toxicity. Here, 16s rRNA gene amplicon sequencing has been applied to characterize the hepatopancreas microbiota composition of the Manila clam, Ruditapes philippinarum. The animals were collected in the Venice lagoon area, which is subject to different anthropogenic pressures, mainly represented by the industrial activities of Porto Marghera (PM). Seasonal and geographic differences in clam microbiotas were explored and linked to host response to chemical stress identified in a previous study at the transcriptome level, establishing potential interactions among hosts, microbes, and environmental parameters. The obtained results showed the recurrent presence of putatively detoxifying bacterial taxa in PM clams during winter and over-representation of several metabolic pathways involved in xenobiotic degradation, which suggested the potential for host-microbial synergistic detoxifying actions. Strong interaction between seasonal and chemically-induced responses was also observed, which partially obscured such potentially synergistic actions. Seasonal variables and exposure to toxicants were therefore shown to interact and substantially affect clam microbiota, which appeared to mirror host response to environmental variation. It is clear that understanding how animals respond to chemical stress cannot ignore a key component of such response, the microbiota.

Link: https://www.sciencedirect.com/science/article/pii/S0166445X17303478?via%3Dihub

  Ecotoxicological effects of the herbicide glyphosate in non-target aquatic species: Transcriptional responses in the mussel Mytilus galloprovincialis

Autori: Milan M, Dalla Rovere G, Smits M, Ferraresso S, Pastore P, Marin MG, Bogialli S, Patarnello T, Bargelloni L, Matozzo V

Riferimenti: Environmental Pollution 2018 Mar 2;237:442-451.

Summary: Glyphosate has been the most widely used herbicide worldwide over the last three decades, raising increasing concerns for its potential impacts on environmental and human health. Recent studies revealed that glyphosate occurs in soil, surface water, and groundwater, and residues are found at all levels of the food chain, such as drinking water, plants, animals, and even in humans. While research has demonstrated that glyphosate can induce a broad range of biological effects in exposed organisms, the global molecular mechanisms of action still need to be elucidated, in particular for marine species. In this study, we characterized for the first time the molecular mechanisms of action of glyphosate in a marine bivalve species after exposure to environmentally realistic concentrations. To reach such a goal, Mediterranean mussels Mytilus galloprovincialis, an ecologically and economically relevant species, were exposed for 21 days to 10, 100, and 1000 μg/L and digestive gland transcriptional profiles were investigated through RNA-seq. Differential expression analysis identified a total of 111, 124, and 211 differentially regulated transcripts at glyphosate concentrations of 10, 100, and 1000 μg/L, respectively. Five genes were found consistently differentially expressed at all investigated concentrations, including SERP2, which plays a role in the protection of unfolded target proteins against degradation, the antiapoptotic protein GIMAP5, and MTMR14, which is involved in macroautophagy. Functional analysis of differentially expressed genes reveals the disruption of several key biological processes, such as energy metabolism and Ca2+ homeostasis, cell signalling, and endoplasmic reticulum stress response. Together, the results obtained suggest that the presence of glyphosate in the marine ecosystem should raise particular concern because of its significant effects even at the lowest concentration.

Link: https://www.sciencedirect.com/science/article/pii/S0269749117348364?via%3Dihub