Paola Venier

Enzymatic biomarker measurement and study of DNA adduct formation in benzo[a]pyrene-contaminated mussels, Mytilus galloprovincialis

The aim of this study was to improve the knowledge on the metabolic pathways involved in benzo[a]pyrene (B[a]P) activation and on the relationship between adduct levels and enzymatic biomarker activities. With this purpose, a model to assess pollutant exposure via food supply has been developed for the sentinel organism, Mytilus galloprovincialis. Mussels were fed for 4 weeks with B[a]P-contaminated feed (50 mg/kg dry weight mussel). Bioaccumulation was studied by determination of B[a]P concentration in whole mussel by GC/MS analysis. Different biomarkers of pollutant exposure were measured to assess the metabolic state of the exposed organisms. CYP1A-like immunopositive protein titration and B[a]P hydroxylase (BPH) activity were assessed as indicators of phase I biotransformation. Glutathione-S-transferase (GST) activity was measured as an indicator of the conjugation activities. Catalase (CAT) and DT-diaphorase (DTD) activities were assessed as potential biomarkers of oxidative stress, whereas acetylthiocholine esterase (AChE) activity was measured as an indication of possible neurotoxicity of B[a]P exposure. DNA adduct levels were determined in digestive gland DNA by applying the 32P-postlabeling technique with nuclease P1 enhancement. For the developed conditions of exposure, B[a]P concentration reached in whole mussel tissues was very high (>500 mg/kg d.w. mussel) and significant B[a]P-induced changes were recorded for each enzymatic biomarkers. BPH and CAT activities were significantly increased by B[a]P exposure, whereas GST in the gills, DTD and AChE were significantly depressed. On the other hand, no change in CYP1A-like immunopositive protein content was observed. Induction and increase with time of bulky B[a]P-related DNA adducts were demonstrated in the digestive gland, although at low levels (0.269+/-0.082 adduct/10e8 dNps at maximum) by the 32P-postlabeling assay. DNA adduct level was significantly correlated with whole mussel tissue B[a]P concentration, so were all the enzymatic biomarkers measured except to GST activity in both gill and digestive gland tissues. BPH, DTD, CAT and AChE displayed a strong correlation with adduct levels. These results demonstrate the neurotoxicity and the genotoxicity of B[a]P exposure in the mussel. The induction of bulky DNA adducts in mussels demonstrates the existence of activation pathways already identified in vertebrates. It validates also the suitability of this model for further studies on B[a]P metabolism in mussels. Our results support the proposal of BPH, AChE, DTD and CAT activities as suitable biomarkers of PAH exposure for these sentinel species.

Detection of micronuclei in gill cells and haemocytes of mussels exposed to benzo[a]pyrene

Mediterranean mussels were exposed to benzo[a]pyrene for 2 days at doses which had previously caused the formation of specific adducts in gill DNA. Micronuclei and other nuclear abnormalities were detected in gill cells and haemocytes in order to ascertain the induction of cytogenetic damage in two different target cells in parallel. A number of procedural details were examined initially to improve the quality of slides obtained from mussel cells. Adequate cytological preparations were obtained when gill cells and haemocytes were suspended, respectively, in Alsever and sea water with EDTA, cytospun and fixed with absolute methanol. In the exposed mussels, micronuclei significantly increased in both the large gill cells (the main cell type) and the agranular haemocytes. Granular haemocytes, cells present in variable proportions between individual mussels, did not show cytogenetic damage except at the highest B[a]P doses. In the same slides, steady levels of binucleated cells were detected, whereas the incidence of other nuclear abnormalities was significantly higher in the exposed compared with control mussels. Precise knowledge of the replication kinetics of gill cells and haemocytes is still lacking.

Tissue dose, DNA adducts, oxidative DNA damage and CYP1A-immunopositive proteins in mussels exposed to waterborne benzo[a]pyrene

A collaborative study was performed on Mediterranean mussels (Mytilus galloprovincialis) exposed to a wide dose-range (0.5-1000 ppb) of benzo[a]pyrene (B[a]P). We selected this model polycyclic aromatic hydrocarbon in order to confirm the formation of a specific DNA adduct, previously detected in gill DNA, and to clarify the in vivo effects of this mutagenic chemical requiring host-metabolism in mussels. B[a]P concentration reached consistently higher values in the digestive gland than in other analyzed tissues of mussels exposed to B[a]P for 2 or 3 days. With the exception of some values at 1000 ppb of B[a]P. DNA adduct levels increased significantly with the dose in gills and digestive gland and ranged from 0.054 to 0.789 adducts per 10(8) nucleotides (mean values per dose-point). Conversely, more complex dose-response relationships were found by detecting in parallel the levels of an oxidative DNA lesion (8-OHdG) and of CYP1A-immunopositive proteins (the latter measured in the digestive gland only). Overall, the formation of DNA adducts, the evidence of oxidative DNA damage, and changes in CYP1A-immunopositive protein levels support the hypothesis that B[a]P can induce DNA damage in mussels through a number of different molecular mechanisms.

Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations

The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations. The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands. However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes. In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles. Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo[a]pyrene (B[a]P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated. In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons. A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected. Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA. Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences. Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations.

Genetic effects of chromium compounds

Seven different test systems were utilized to investigate the genetic activity of chromium compounds: infidelity of DNA replication in vitro by DNA pol alpha from calf thymus, damage of DNA detected by alkaline elution in treated mammalian cells or in DNA purified and treated in vitro, DNA repair synthesis in mammalian cells in vitro detected by autoradiography or scintillation counting after labelling with [3H]dThd, gene mutations in the Salmonella typhimurium Ames test, gene mutations (6TG resistance) in cultured hamster cells, sister-chromatid exchanges in different rodent cell cultures, and transformation to anchorage-independent growth of hamster cells in vitro (soft-agar assay). Potassium dichromate and chromium chloride were used as water-soluble Cr(VI) and Cr(III) salts. Several reference mutagens (EMS, MMS, MMC, 4NQO) were included in the single tests as positive controls. Cr(VI) was active in all the tested systems, except in the induction of DNA damage and DNA repair synthesis in cultured cells. Cr(III), on the other hand, was absolutely inactive unless a direct interaction with purified DNA was permitted by the test conditions. The relevance of data from the various tests to the understanding of the mechanisms of the genotoxic activity of chromium is discussed. Effects other than the direct interaction of Cr(III) with DNA are inferred, which can cause infidelity of the DNA polymerase functions.

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

BackgroundSessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes.ResultsWe have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways.ConclusionsThe Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world.

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences

BackgroundAlthough Bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria.ResultsWe have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant M. galloprovincialis sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database http://mussel.cribi.unipd.it.ConclusionWe defined the first species-specific catalogue of M. galloprovincialis ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels.

The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: A widespread and diverse family of immune-related molecules

The key component of the classical complement pathway C1q is regarded as a major connecting link between innate and acquired immunity due to the highly adaptive binding properties of its trimeric globular domain gC1q. The gC1q domain also characterizes many non-complement proteins involved in a broad range of biological processes including apoptosis, inflammation, cell adhesion and cell differentiation. In molluscs and many other invertebrates lacking of adaptive immunity, C1q domain containing (C1qDC) proteins are abundant, they most probably emerged as lectins and subsequently evolved in a specialized class of pattern recognition molecules through the expanding interaction properties of gC1q. Here we report the identification of 168 C1qDC transcript sequences of Mytilus galloprovincialis. The remarkable abundance of C1qDC transcripts in the Mediterranean mussel suggests an evolutionary strategy of gene duplication, functional diversification and selection of many specific C1qDC variants. A comprehensive transcript sequence survey in Protostomia also revealed that the C1qDC family expansion observed in mussel could have occurred in some specific taxa independently from the events leading to the establishment of a large complement of C1qDC genes in the Chordates lineage.

Formation of DNA adducts in the gill tissue of Mytilus galloprovincialis treated with benzo[a]pyrene.

Abstract The formation of DNA adducts was evident after treatment of spawned or resting Mediterranean mussels (Mytilus galloprovincialis Lmk.) with 0.5–100 μg/l of benzo[a]pyrene for 2 and 3 days. Reference DNA samples, in vitro radiolabelled with 0.5, 5, 50μM 3H-anti (±)-B[a]P-diol-epoxide, were initially used to compare two DNA purification procedures. Following a standard four-step extraction starting with phenol, in comparison to a simplified single-step extraction without phenol, we obtained lower yields of bound radioactivity in the reference DNA samples. After simplified DNA purification and nuclease PI enhanced 32P-postlabelling assay we detected a reproducible dose-dependent increase of a specific spot in gills of mussels treated with B[a]P, although this spot was present in low amounts. Short (2 days) and prolonged (27 days) pretreatment of mussels with a polychlorinated biphenyl mixture, Aroclor 1254, did not increase the levels of B[a]P-related adducts. On the whole, these results indicate the formation of detectable amounts of DNA reactive intermediates in gills of mussels treated with B[a]P. Although the pathway of formation and the molecular identity of the specific adducts remain unclear, their presence suggests that polycyclic aromatic hydrocarbons may cause genetic damage in marine mussels.

Toll-like receptors and MyD88 adaptors in Mytilus: complete cds and gene expression levels.

TLR- and MyD88-related sequences have been previously investigated in Mytibase and then in new transcript reads obtained by Illumina technology from the mussel, Mytilus galloprovincialis. Based on full cds and domain organizations of virtual translations, we identified 23 Toll-like receptors (TLRs) and 3 MyD88 adaptors. MgTLRs can be arranged in 4 clusters according to extra-cellular LRR domain content. MgTLR-b, -i and -k were the only ones containing a multiple cysteine cluster (mccTLR), a domain composition also found in Drosophila Toll-1 and 18-wheeler. The 3 MyD88 we identified in M. galloprovincialis were also retrieved from Mytilus edulis, as well as MgTLR-b and -i. All MgTLRs were constitutively expressed in digestive gland whereas only 4 of them were also present in hemocytes. On the opposite, the 3 MgMyD88s were constitutively expressed in all the tissues. In vivo challenge of M. galloprovincialis with bacteria caused the up regulation of only MgTLR-i, but of all the 3 MgMyD88s. Highest response was induced by Gram-negative Vibrio anguillarum at 9h p.i. Injection of filamentous fungus, Fusarium oxysporum, resulted in up regulation of MgTLR-i and MgMyD88-c at 9h p.i. Such similar pattern of responses suggested MgMyD88-c represents the intra cytoplasm partner of MgTLR-i. Their interaction constituted the first cellular event revealing the existence of a Toll-signaling pathway in Lophotrochozoa.