Serum amyloid A in marine bivalves: An acute phase and innate immunity protein.

Serum amyloid A (SAA) is among the most potent acute phase proteins (APP) in vertebrates. After injury, its early expression can dramatically increase to promote the recruitment of immuno-competent cells, expression of pro-inflammatory proteins and the activation of the innate immune defences. Although APP have been studied in many vertebrates, only recently their search was extended to invertebrates and the finding of SAA-like molecules has opened new questions on the immune-regulatory functions of these soluble proteins in the animal kingdom. Taking advantage of the considerable amount of genomic and transcriptomic data currently available, we retrieved 51 SAA-like proteins in several protostome taxa comprising 21 marine bivalve species and basal metazoans. In addition to vertebrate-like SAAs, we identified a second protein type with peculiar features. In the bivalves Crassostrea gigas and Mytilus galloprovincialis, both digital expression analysis and qPCR data indicated an induction of the classical SAA after bacterial challenge.

Chaperone patterns in vernal keratoconjunctivitis are distinctive of cell and Hsp type and are modified by inflammatory stimuli.

BACKGROUND: Vernal keratoconjunctivitis (VKC) is a severe ocular allergy with pathogenic mechanism poorly understood and no efficacious treatment. The aims of the study were to determine quantities and distribution of Hsp chaperones in the conjunctiva of VKC patients and assess their levels in conjunctival epithelial and fibroblast cultures exposed to inflammatory stimuli. METHODS: Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, Hsp105, and Hsp110 were determined in conjunctiva biopsies from nine patients and nine healthy age-matched normal subjects, using immunomorphology and qPCR. Conjunctival epithelial cells and fibroblasts were cultured and stimulated with IL-1ß, histamine, IL-4, TNF-α, or UV-B irradiation, and changes in Hsp levels were determined by Western blotting. RESULTS: Hsp27, Hsp40, Hsp70, and Hsp90 levels increased in the patients' conjunctiva, whereas Hsp10, Hsp60, Hsp100, and Hsp105 did not. Double immunofluorescence demonstrated colocalization of Hsp27, Hsp40, Hsp70, and Hsp90 with CD68 and tryptase. Testing of cultured conjunctival cells revealed an increase in the levels of Hsp27 in fibroblasts stimulated with IL-4; Hsp40 in epithelial cells stimulated with IL-4 and TNF-α and in fibroblasts stimulated with IL-4, TNF-α, and IL-1ß; Hsp70 in epithelial cells stimulated with histamine and IL-4; and Hsp90 in fibroblasts stimulated with IL-1ß, TNF-α, and IL-4. UV-B did not induce changes. CONCLUSIONS: VKC conjunctiva displays distinctive quantitative patterns of Hsps as compared with healthy controls. Cultured conjunctival cells respond to cytokines and inflammatory stimuli with changes in the Hsps quantitative patterns. The data suggest that interaction between the chaperoning and the immune systems drives disease progression.

Dual analysis of host and pathogen transcriptomes in ostreid herpesvirus 1-positive Crassostrea gigas.

Ostreid herpesvirus type 1 (OsHV-1) has become a problematic infective agent for the Pacific oyster Crassostrea gigas. In particular, the OsHV-1 µVar subtype has been associated with severe mortality episodes in oyster spat and juvenile oysters in France and other regions of the world. Factors enhancing the infectivity of the virus and its interactions with susceptible and resistant bivalve hosts are still to be understood, and only few studies have explored the expression of oyster or viral genes during productive infections. In this work, we have performed a dual RNA sequencing analysis on an oyster sample with a high viral load. High sequence coverage allowed us to thoroughly explore the OsHV-1 transcriptome and identify the activated molecular pathways in C. gigas. The identification of several highly induced and defence-related oyster transcripts supports the crucial role played by the innate immune system against the virus and opportunistic microbes possibly contributing to subsequent spat mortality.

International round-robin study on the Ames fluctuation test.

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

DNA adducts in mussels and fish exposed to bulky genotoxic compounds.

Biological and procedural factors can influence DNA adduct detection in aquatic organisms. Among them, functional structure and metabolic traits represent major biological determinants for adducts formed by lipophilic pro-mutagenic contaminants. In detecting DNA adducts through the 32P-postlabelling assay, efficiency in DNA purification, digestion, labelling, as well as adduct enrichment and quantification may explain differences between independent studies. Reference DNA adducts have been used to verify some 32P-postlabelling aspects. Data obtained for mussels and fish treated with benzo[a]pyrene (B[a]P) and environmentally exposed to genotoxins confirm the above assertions. Although the 32P-postlabelling assay cannot be proposed for routine biomonitoring it appears a reliable and very sensitive index of exposure to genotoxic pollutants in both fish and mollusks.

Susceptibility to genetic damage and cell types in Mediterranean mussels.

Micronucleus (MN) frequency is generally accepted as a marker of chromosomal damage and has been studied in a variety of cells and species. In previous work, we detected significant dose-related MN increases in the epithelial-like gill cells and agranular haemocytes of Mytilus galloprovincialis treated with benzo[a]pyrene, a well-known mutagenic pollutant. In addition, we have studied micronuclei and other nuclear abnormalities in mussels collected from the Venice lagoon (Italy). Frequency changes, possibly related to genotoxic/toxic stress, in both granular and micronucleated cells from gills and haemolymph, were detected. Environmental data suggest the effect of genotoxic pollutants and the importance of cell replication in the interpretation of micronucleus frequencies.

Enzymatic biomarker measurement and study of DNA adduct formation in benzo

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.

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.

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.

Metabolic activation of benzo[a]pyrene in two fetal mouse hepatocyte lines: induction of DNA adducts and micronuclei.

We have studied the metabolic competence of two non-transformed epithelial-like cell lines derived from fetal mouse liver, C 6 and C 2.8, to activate the promutagen benzo[a]pyrene by measuring both the induction of DNA adducts through the nuclease P1-enhanced 32P-postlabeling assay and the formation of micronuclei. The pattern and level of DNA adducts detected in C 6 and C 2.8 cells treated with benzo[a]pyrene were compared with those obtained in human peripheral blood lymphocytes treated with the same compound and with [3H]anti-benzo[a]pyrene diolepoxide. In both the cell lines and in human lymphocytes we observed a consistent induction of distinct DNA adducts. In C 6 and C 2.8 cells, the most evident adduct showed a position similar to that of the main adduct induced by [3H]-anti-benzo[a]pyrene diolepoxide in human lymphocytes. In addition, benzo[a]pyrene caused a significant increase of micronucleated C 6 and C 2.8 cells, whereas the frequency of micronuclei did not increase in CHO cells treated, for comparison, in the same way.

Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins.

Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.

Analysis of microtubule rigidity using hydrodynamic flow and thermal fluctuations.

We report the use of two independent new methods to measure the flexural rigidity of microtubules. Microtubules were grown off axonemal pieces adhering to a glass coverslip. In the first method, a hydrodynamic flow was applied to microtubules and the flexural rigidity was derived from the analysis of the bending shape of the microtubules at equilibrium in the flow. In the second method, the flexural rigidity was derived from the thermal fluctuations of the free end of axoneme-bound microtubules. With both methods, the flexural rigidity of standard GDP microtubules was estimated to be 0.85 +/- 0.2 x 10(-23) newtons x m2 which corresponded to a persistence length of 2 +/- 0.2 mm. Binding of ligands known to affect the biochemical properties of microtubules affected their rigidity. The structural analogs of inorganic phosphate AlF4- and [BeF3-, H2O], which bind to the site of the gamma-phosphate of GTP on GDP microtubule and reconstitute the GDP-Pi microtubule intermediate state of GTP hydrolysis, cause an approximately 3-fold increase in microtubule flexural rigidity and persistence length. Taxol and taxotere, antitumoral microtubule-stabilizing drugs, in contrast cause a decrease in flexural rigidity and appear to affect the three-dimensional superstructure of microtubules, which can no longer be considered as semi-flexible rods. The relationship between the mechanical properties of microtubules and their biological function is discussed.

[The urinary mutagenicity test in monitoring exposure to aromatic polycyclic hydrocarbons in workers in the aluminum industry]. / Test di mutangenicità urinaria nel monitoraggio dell'esposizione ad idrocarburi policiclici aromatici di lavoratori dell'industria di alluminio.

The sensitivity of 3 urinary mutagenicity tests was assayed: the plate test, the fluctuation test and the micropreincubation test, in order to assess their possible use in monitoring human exposure to polycyclic aromatic hydrocarbons (PAH). Urine samples from workers of an anode production plant exposed to coal tar and from psoriatic patients undergoing treatment with coal-tar ointments were tested for mutagenic activity on strain TA98 Salmonella typhimurium, in the presence of the microsome fraction and deconjugating enzymes. Parallelly, the urinary concentration of PAH metabolites or one of their trace metabolites, 1-hydroxypyrene, was determined. Increased levels of PAH metabolites were observed in the urine of anode production workers after a work shift compared with controls. Results of the plate test and the fluctuation test performed on urine of exposed subjects, both smokers and nonsmokers, showed mutagenicity values similar to the controls. Much higher 1-hydroxypyrene concentrations were found in the urine of psoriatic patients treated with coal tar than in post-shift urine of anode production workers. The urine of the former was also mutagenic in the 3 mutagenicity tests used. The minimum mean dose of PAH metabolites was calculated, expressed as quantity of 1-hydroxypyrene, that would give a mutagenic response in the 3 tests: the micropreincubation test was found to be about 100 times more sensitive than the plate test and about 30 times more sensitive than the fluctuation test. The theoretical minimum urinary concentration of 1-hydroxypyrene detectable by each test was determined: the micropreincubation test was 15 times more sensitive than the plate test and 7 times more sensitive than the fluctuation test.(ABSTRACT TRUNCATED AT 250 WORDS)

[Identification of genotoxic compounds used in leather processing industry]. / Identificazione di composti genotossici utilizzati nella lavorazione delle pelli.

The release of mutagens from 7 carbon black-based leather dyes and from leather samples at various stages of finishing was determined. After vigorous treatment with toluene, 4 commercial dyes yelded mutagenic extracts on Salmonella typhimurium in the presence of microsomal enzymes. Only in one case were the responsible chemicals identified as polycyclic aromatic hydrocarbons. The low bioavailability of mutagens contained in carbon black and their low mutagenic activity suggest that the risk associated with the use of these dyes is probably negligible. Soxhlet extracts with ethanol from finished leather were mutagenic on strain TA98 of Salmonella typhimurium in the absence of S9 mix. Analysis of extracts of leather samples at various intermediate stages of processing showed that mutagenic activity was detectable after the colouring process. The responsible compound was identified as a nitroazo dye (Colour Index: Acid Brown 83), with a mutagenic potential of about 4 revertant/micrograms. Eighteen commercial tannins containing mainly Cr(III) sulphates were assessed for genotoxicity. Most were contaminated with Cr(VI), a known mutagenic and carcinogenic agent, at levels sufficient to induce an increased frequency of SCE (sister chromatid exchanges) in mammalian cells (CHO, chinese hamster ovary) tested in vitro.

[Leather azo dyes: mutagenic and carcinogenic risks]. / I coloranti azoici per cuoio: rischio mutageno e cancerogeno.

The paper reviews the carcinogenicity and mutagenicity data on azo dyes used in the leather industry. Two water soluble benzidine-based dyes were classified as "probably carcinogenic to humans" by the International Agency for Research on Cancer (IARC). No other dyes have been evaluated by the IARC. Of the 48 azo dyes assayed in the Salmonella/microsome test, 20 gave positive results. Attention is drawn to the important role of the in vivo metabolism of azo compounds, which includes a preliminary reduction of the azo bonds and subsequent release of the aromatic amines of the dye. A useful assay (Prival test) for evaluating the mutagenic properties of azo dyes involves a reductive step that permits the release of any genotoxic agents present in the compounds. A list of leather azo dyes is furnished that are considered as potentially harmful due to the presence of a carcinogenic aromatic amine (benzidine, p-aminobenzene and derivatives) in their formulae.

[Urinary excretion of mutagens and cisplatin among the nursing staff at a medical oncology department exposed to cytostatic drugs]. / Escrezione urinaria di mutageni e cisplatino nel personale infermieristico dei reprti di oncologia media esposto a farmaci citostatici.

Exposure to cytostatic drugs was assessed in a group of 9 nurses employed in a hospital cancer therapy department by measuring the post-shift levels of urinary mutagens and cis-platinum. A slight but significant increase in urinary mutagenic activity compared to 11 controls was observed in the non-smokers: the mean values of mutagenic activity on the Ta100 strain in the presence of both microsomal and deconjugating enzymes were 4418 +/- 1186 and 2468 +/- 1681 respectively. Conversely, the urinary platinum concentration was below the detection limit of the analytical method (10 micrograms/l) in all samples. The increased urinary mutagenic activity in the exposed group can probably be attributed to the absorption of cyclophosphamide either during preparation and administration of the drug, or due to accidental contact with contaminated biological fluids, in view of the fact that the level of mutagens in urine samples from cyclophosphamide-treated patients is extremely high (up to 319,478 revertants/g creatinine in the case we examined).

Release of mutagens from finished leather.

Extracts of a leather widely used in the furniture and dress-making industries were tested for their mutagenic activity in the Salmonella/microsome assay. Extracts obtained after vigorous treatment of leather samples in a Soxhlet apparatus with toluene or ethanol were mutagenic in strain TA98 of S. typhimurium in the absence of S9 mix. The analysis of extracts of leather at various intermediate stages of processing showed that the mutagenic activity appeared after the coloration process. The responsible compound was identified to be an azo dye (Color Index: Acid Brown 83) whose mutagenic potency was about 4 revertants/micrograms.