The diagnostic accuracy of truncated cardiovascular MR protocols for detecting non-ischemic cardiomyopathies.

Cardiovascular magnetic resonance imaging is one of the most important diagnostic modalities in the evaluation of cardiomyopathies. However, significant limitations are the complex and time-consuming workflows and the need of contrast agents. The aim of this multi-center retrospective study was to assess workflows and diagnostic value of a short, contrast agent-free cardiac magnetic resonance protocol. 160 patients from Heidelberg, Germany and 119 patients from Montreal, Canada with suspected cardiomyopathy and 20 healthy volunteers have been enrolled. Scans were performed at a 1.5Tesla or 3Tesla scanner in Heidelberg and at a 3Tesla scanner in Montreal. We used single-slice T1 map only. A stepwise analysis of images has been performed. The possible differential diagnosis after each step has been defined. T1-values and color-encoded T1 maps significantly contributed to the differential diagnosis in 54% of the cases (161/299); the final diagnosis has been done without late gadolinium enhancement images in 83% of healthy individuals, in 99% of patients with dilated cardiomyopathy, in 93% of amyloidosis patients, in 94% of patients with hypertrophic cardiomyopathy and in 85% of patients with hypertensive heart disease, respectively. Comparing the scan time with (48 ± 7 min) vs. without contrast agent (23 ± 5 min), significant time saving could be reached by the short protocol. Subgroup analysis showed the most additional diagnostic value of T1 maps in amyloidosis and hypertrophic cardiomyopathy or in confirmation of normal findings. In patients with unclear left ventricular hypertrophy, a short, non-contrast protocol can be used for diagnostic decision-making, if the quality of the T1 map is diagnostic, even if only one slice is available.

Metal bioaccumulation and physiological condition of the Pacific oyster (Crassostrea gigas) reared in two shellfish basins and a marina in Normandy (northwest France).

A 5-month experiment combining a geochemical survey of metals with a bioaccumulation study in batches of Crassostrea gigas was conducted in two shellfish farming areas and a marina in Normandy (France). Various endpoints at different levels of biological organization were studied. ROCCH data showed differences in biota contamination between the two shellfish areas but the present study revealed only slight differences in metallic contamination and biomarkers. By contrast, significantly different values were recorded in the marina in comparison with the two other sites. Indeed, higher levels of Cd, Cu and Zn were measured in the oysters from the marina, and these oysters also showed a poorer physiological condition (e.g., condition index, histopathological alterations and neutral lipid content). For coastal monitoring, the multi-biomarker approach coupled with an assessment of metallic contamination in biota appeared to be suitable for discriminating spatial differences in environmental quality after only a few months of exposure.

Toxicity of five antidepressant drugs on embryo-larval development and metamorphosis success in the Pacific oyster, Crassostrea gigas.

Unlike conventional pollutants, pharmaceutical residues are continuously discharged at low levels (low to mid ng l(-1) concentrations), which results in the chronic contamination of non-target organisms, but little is known about the effects of these residues. The purpose of this study was to provide the first assessment of the ecotoxicity of five antidepressants (selective serotonin reuptake inhibitors [SSRIs] fluoxetine and sertraline, tricyclic antidepressants [TCAs] clomipramine and amitriptyline, and serotonin norepinephrine reuptake inhibitor [SNRI] duloxetine) at a wide range of concentrations from 0.1 to 100,000 µg l(-1) on two early life stages in the Pacific oyster. The toxicity was quantified in D-shaped larvae after 36 h of exposure, and in 21-day-old pediveliger larvae after 24 h of exposure using the percentage of normal larval development and the metamorphosis rate as endpoints, respectively. The embryotoxicity assays reported that the EC50 values were within the same range of concentrations (67 to 192 µg l(-1)) for all of the tested molecules. The metamorphosis tests revealed that the antidepressants can be ranked along an increasing severity gradient: clomipramine < amitriptyline < duloxetine ~ fluoxetine. Sertraline appeared to be the less toxic molecule on this endpoint; however, a different concentration range was used. The embryotoxicity test was more sensitive than the metamorphosis bioassay for three of the five molecules tested, but the latter test showed more practical benefits. Overall, the obtained toxicity values were at least 10,000-fold higher than the reported environmental concentrations.

Molecular cloning of a new member of the p53 family from the Pacific oyster Crassostrea gigas and seasonal pattern of its transcriptional expression level.

Like other sessile filter-feeding molluscs, oysters may be exposed in the natural environment to a variety of contaminants. Long-term exposure to pollutants may be one factor affecting prevalence of cancerous-like disorders, such as neoplasia. Environmentally induced alterations in p53 protein expression, in relation to leukemia, have been reported in various mollusc species inhabiting polluted water, suggesting that p53 proteins can also be used as a marker for environmental research. This work reports the cloning and sequencing of a p53-like cDNA in the mollusc bivalve Crassostreagigas. The deduced amino acid sequences of p53 shared a high degree of homology with the homologues from other mollusc species, including typical eukaryotic p53 signature sequences. We examined the p53 transcription expression pattern during the annual cycle in oyster gills and whole soft tissues in four locations along the French coasts. Real-time PCR analysis suggested that strong variations at p53 mRNA level are probably synchronized with the seasonal cycle at the four locations investigated.

Shell repair process in the green ormer Haliotis tuberculata: a histological and microstructural study.

In the present paper, juvenile and adult shells of the green ormer Haliotis tuberculata ('Oreille de Saint-Pierre') were perforated in a zone close to the shell edge and the shell repair process was followed at two levels: (1) by observing the histology of the calcifying mantle in the repair zone and (2) by analyzing with SEM the microstructure of the shell repair zone. Histological data clearly show the presence of calcium carbonate granules into the connective tissues, but not in the epithelial cells. This suggests that calcium carbonate granules are synthesized by sub-epithelial cells and actively transported through the epithelium to the repair zone, via a process which may be similar to that described by Mount et al. [Mount, A.S., Wheeler, A.P., Paradkar, R.P., Snider, D., 2004. Hemocyte-mediated shell mineralization in the eastern oyster. Science 304, 297-300]. Furthermore, SEM observations show that the repair zone exhibits different stratified microstructures (spherulitic, thin prismatic, blocklike, sub-nacreous, nacreous, foliated-like), some of which are not continuous (i.e. lenticular) along the repair zone. This suggests a complex secreting regime of the calcifying mantle and an elaborate geometry of the epithelium involved in shell repair.

Preliminary study of the effects of commercial lactobacilli preparations on digestive metabolism of juvenile sea bass (Dicentrarchus labrax).

Two forms of the same commercial product (SORBIAL, Allonnes, France), one with live bacteria (PSA) and the other with heat-inactivated bacteria (PSI), containing a mixture of 2 strains of lactobacilli and their growth medium were tested as a diet complement for juvenile sea bass (Dicentrarchus labrax) during a 103-day experiment. In addition to zootechnical parameters (survival, growth, conformation), some effects on digestive metabolism were studied, including enzymatic, ultrastructural and microbial aspects. Microbial preparations improved survival rate. The ventral, dorsal and operculum malformations which usually occur in juveniles did not appear in those receiving PSA and PSI. Furthermore, they stimulated, but not constantly, trypsin and acid phosphatase activities. Intestinal ultrastructure showed an increase in the number of endocytosis vesicles at the apical pole of enterocytes in fishes receiving enrichments. Bacterial flora was not modified in terms of quantity, especially the lactic acid bacteria counts, which were not changed in fishes receiving live lactobacilli (PSA). The mode of action of these multiple beneficial effects appears complex and could be caused by different molecules inside the bacterial cell or excreted into their medium.

Influence of cryoprotective agent and cooling rate on frozen and thawed hemocytes from the Mollusk Haliotis tuberculata.

Cultures of circulating cells from abalone (Haliotis tuberculata) may be used in fundamental research or in biotechnology. This paper describes attempts to develop a cryoconservation method for these hemocytes in order to constitute a standardized cell stock. Among a panel of five distinct cryoprotective solutions, 10% v/v glycerol ('G solution') was the most effective and better post-thaw recovery was achieved after cooling at 1 degrees C/min than after more rapid cooling (3 degrees C or 9 degrees C/min). In 2-day-old cultures, cell viability, assessed by DNA or protein content, was 83 and 78%, respectively, and metabolic activity, measured by the MTT reduction assay, reached 96%. Viability rates were only slightly reduced after 6 days of culture, suggesting a low proportion of damaged cells among the surviving hemocytes. This study identified a cryoprotective solution and a freezing protocol that allow thawed hemocytes to recover a large part of their viability.

Cryopreservation of mantle dissociated cells from Haliotis tuberculata (Gastropoda) and postthawed primary cell cultures.

Dissociated mantle cells from the gastropod mollusc Haliotis tuberculata were cultured after a freezing-thawing procedure using either 10% dimethyl sulfoxide (Me(2)SO) or 10% glycerol (Gly) as a cryoprotector. The survival rate of 2-day-old cultured cryopreserved cells after thawing, based on analysis of DNA and protein contents, was nearly 80% in comparison with 2-day-old cultured fresh cells. Cells thawed after cryopreservation exhibited the maintenance of all tested physiological activities. Metabolic activity (measured by the MTT test) and the activity of alkaline phosphatase (a plasma membrane-bound enzyme) were not decreased in comparison to those in cultured fresh cells. In addition, cryopreserved cultured cells maintained a physiological stimulation ability in response to treatment with growth factors. These results taken together represent one of the most convincing demonstrations of the survival and of the recovery of intact functional activities of molluscan cells after a freeze-thawing procedure. Our results suggest that in the future primary cultures of cryopreserved mantle cells will be able to be used for fundamental research, in toxicity tests, or in the field of biotechnology.

Sequential analysis of hair mercury levels in relation to fish diet of an Amazonian population, Brazil.

Several studies in the Amazonian Basin have shown that riverine populations are exposed to methylmercury through fish consumption. It has been suggested that seasonal variations in hair mercury observed through sequential analyses may be related to the changes in fish species ingested by the local communities. The aim of the present study was to investigate the relationship between fish-eating practices and seasonal variation in mercury exposure. A group of 36 women from a village located on the banks of the Tapajós River, a major tributary of the Amazon, comprised the present study population. An interview-administered questionnaire was used to gather information on socio-demographic characteristics, fish-eating practices and other relevant information. The women also provided hair samples of at least 24 cm in length for mercury analysis. Hair total and inorganic mercury concentration was measured using a cold vapor atomic absorption analytical method. Trigonometric regression analysis was done to assess the seasonal variation of total mercury levels. Variations in inorganic mercury were examined by repeated measures analysis of variance, and analysis of contrast variable with a polynomial transformation. The results showed that hair mercury levels varied with the season. Higher levels were observed in months corresponding to the dry season, with lower levels in the rainy season. Herbivorous fish predominated the diet for 47.2% of the women during the dry season, but this rose to 72.2% during the rainy season. Those who reported eating fish daily had higher mercury levels in hair compared to those who only ate fish a few times per week. Retrospective mercury analyses, evaluated by the quantity of mercury present in each centimeter of hair, indicate that mean mercury level of the population decreased over the 2 years prior to the study. The percentage of inorganic mercury over the total mercury in hair increased towards the extremities of the hair strand. Higher percentages of inorganic mercury were found for the group who ate more fish (on a daily consumption basis). These results support the assumption that there are seasonal variations in methylmercury exposure and also a relationship between type of fish species consumed and the resulting hair mercury levels.

In Vitro Synthesis of Proteoglycans and Collagen in Primary Cultures of Mantle Cells from the Nacreous Mollusk, Haliotis tuberculata: A New Model for Study of Molluscan Extracellular Matrix.

In Mollusca, the mantle produces an organic matrix that mineralizes in time to make shell. Primary mantle cell cultures from the nacreous gastropod Haliotis tuberculata have been established as useful experimental model to investigate in vitro synthesis of both proteoglycans/glycosaminoglycans (PGs/GAGs) and collagen. First, we tested different enzymatic digestion procedures to find the method that gives the highest percentage of viable and adherent cultured cells. Enzymatic digestion with 0.1% pronase plus 0.1% collagenase was routinely used. Six days after the initiation of culture, about 80% of cells were viable, among which 20% were adherent as quantified by the MTT reduction assay. In addition, the protein synthesis estimated by [(3)H]leucine incorporation remained constant during this period. For the first time, we demonstrated a de novo synthesis of PGs/GAGs and collagen in primary cultures of mantle cells. After 48 hours of labeling, among the [(3)H]-d-glucosamine macromolecules synthesized, [(3)H]PGs/GAGs represented 43%, divided into 45% heparan sulfate, 37% chondroitin/dermatan sulfate, and 6% hyaluronic acid. Early elution on anion-exchange chromatography of these PGs/GAGs indicated that most of them appeared as undersulfated GAG molecules. De novo synthesis of collagen represents 4.52% +/- 0.84% (SD) with respect to the total protein synthesis. Such a model will facilitate studies on the synthesis of PGs/GAGs and collagen as components of the extracellular matrix and its regulation in Mollusca. Both PGs/GAGs and collagen participate in molecular events that regulate cell adhesion, migration, and proliferation. Further studies with this type of in vitro model should provide knowledge about novel aspects of molluscan cell signaling, in relation to extracellular matrix components.

Collagen study and regulation of the de novo synthesis by IGF-I in hemocytes from the gastropod mollusc, Haliotis tuberculata.

To evidence a collagen synthesis and identify which type(s) of collagen is present in hemocytes from the mollusc Haliotis tuberculata, we have performed three separate approaches, namely, de novo synthesis by cultured cells, immunological approaches, and northern blot analysis. We demonstrated first that after 40-hr labeling, the de novo synthesis of collagen in the cell layer of cultured hemocytes represents 9.48 +/- 1.25% with respect to the total [(3)H]proline-labeled protein synthesis. In addition, IGF-I elicited a significant stimulation of collagen synthesis in cultured hemocytes in a dose-dependent manner from 10(-10) to 10(-8) M. The maximal stimulation (10(-9) M) induced an increase of 286 +/- 56% with respect to 100% control. By immunocytochemistry and immunoblotting, we showed that hemocytes present immunoreactive molecules to antibodies directed against the type I fibrillar collagen. In addition, using as a probe Hf 677 corresponding to a human pro alpha1(I) collagen cDNA and which encompasses the (Gly-X-Y) repeated sequence found in all Metazoa, four collagen transcripts of approximately 6.4, 5, 2.2, and 2 kb in length have been detected. These data suggest the presence of fibrillar type I collagen in hemocytes and are compatible with the concept that these cells are involved in the extracellular matrix deposition, a cardinal function in tissue repair as well as in developmental processes. Our model may appear as an excellent system to study the role of growth factors on the regulation of collagen synthesis by molluscan hemocytes. J. Exp. Zool. 287:275-284, 2000.

Methylmercury exposure affects motor performance of a riverine population of the Tapajós river, Brazilian Amazon.

Gold mining and deforestation in the Brazilian Amazon are increasing mercury pollution of the extensive water system, exposing riverine populations to organic mercury through fish-eating. The aim of the present study was to evaluate the effect of such exposure on motor performance. This cross-sectional study was carried out in May 1996, in a village located on the banks of the Tapajós river in the Amazonian Basin, Brazil. Information concerning sociodemographics, health, smoking habits, alcohol drinking, dietary habits and work history were collected using an interview-administered questionnaire. Mercury concentrations were measured by cold vapor atomic absorption in blood and hair of each participant, of whom those aged between 15 and 79 years were assessed for motor performance (n = 84). Psychomotor performance was evaluated using the Santa Ana manual dexterity test, the Grooved Pegboard Fine motor test and the fingertapping motor speed test. Motor strength was measured by dynamometry for grip and pinch strength. Following the exclusion of 16 persons for previous head injury, working with mercury in the goldmining sites, or for diabetes, the relationship between performance and bioindicators of mercury was examined using multivariate statistical analyses, taking into account covariables. All participants in the study reported eating fish, which comprised 61.8% of the total meals eaten during the preceding week. The median hair total mercury concentration was 9 microg/g. Organic mercury accounted for 94.4 = 1.9% of the total mercury levels. Multivariate analysis of variance indicated that hair mercury was inversely associated with overall performance on the psychomotor tests, while a tendency was observed with blood mercury. Semipartial regression analyses showed that hair total mercury accounted for 8% to 16% of the variance of psychomotor performance. Neither hair nor blood total mercury was associated with the results of the strength tests in women and men. Although dose-effect relationships were observed in this cross-sectional study, they may reflect higher exposure levels in the past. The findings of this study demonstrated neurobehavioral manifestations of subtle neurotoxic effects on motor functions, associated with low-level methylmercury exposure.

Cytogenetic damage related to low levels of methyl mercury contamination in the Brazilian Amazon.

The mercury rejected in the water system, from mining operations and lixiviation of soils after deforestation, is considered to be the main contributors to the contamination of the ecosystem in the Amazon Basin. The objectives of the present study were to examine cytogenetic functions in peripheral lymphocytes within a population living on the banks of the Tapajós River with respect to methylmercury (MeHg) contamination, using hair mercury as a biological indicator of exposure. Our investigation shows a clear relation between methylmercury contamination and cytogenetic damage in lymphocytes at levels well below 50 micrograms/gram, the level at which initial clinical signs and symptoms of mercury poisoning occur. The first apparent biological effect with increasing MeHg hair level was the impairment of lymphocyte proliferation measured as mitotic index (MI). The relation between mercury concentration in hair and MI suggests that this parameter, an indicator of changes in lymphocytes and their ability to respond to culture conditions, may be an early marker of cytotoxicity and genotoxicity in humans and should be taken into account in the preliminary evaluation of the risks to populations exposed in vivo. This is the first report showing clear cytotoxic effects of long-term exposure to MeHg. Although the results strongly suggest that, under the conditions examined here, MeHg is both a spindle poison and a clastogen, the biological significance of these observations are as yet unknown. A long-term follow-up of these subjects should be undertaken.

Neurotoxic effects of low-level methylmercury contamination in the Amazonian Basin.

Many studies have demonstrated mercury contamination in the Amazonian ecosystem, particularly in fish, a dietary mainstay of populations in this region. The present study focused on potential health effects of this low-level methylmercury exposure. The study was carried out in a village on the Tapajós River, a tributary of the Amazon, on 91 adults inhabitants (15-81 years), whose hair mercury levels were inferior to 50 mu/g. Performance on a neurofunctional test battery and clinical manifestations of nervous system dysfunction were examined in relation to hair mercury concentrations. Near visual contrast sensitivity and manual dexterity, adjusted for age, decreased significantly with hair mercury levels (P < 0.05), while there was a tendency for muscular fatigue to increase and muscular strength to decrease in women. For the most part, clinical examinations were normal, however, hair mercury levels were significantly higher (P < 0.05) for persons who presented disorganized movements on an alternating movement task and for persons with restricted visual fields. These results suggest dose-dependent nervous system alterations at hair mercury levels below 50 micrograms/g, previously considered a threshold for clinical effects. The profile of dysfunction in this adult population is consistent with the current knowledge on methyl-mercury poisoning. The long-term implications of these findings are unknown and need to be addressed.

Preliminary evidence of neurotoxicity associated with eating fish from the Upper St. Lawrence River Lakes.

Pollution of hydrographic basins has affected the flora and fauna that thrive in these aquatic ecosystems, and fish, which constitute an important food resource, often contain a plethora of potentially toxic chemicals. In a major research project on early neurotoxic effects of environmental exposure to manganese among residents in Southwest Quebec, fish consumption from 2 lakes of the Upper St. Lawrence River System, was surveyed as a potential confounding factor. Participants were selected using a random, stratified sampling strategy from lists of the Quebec Health Plan. Following exclusions, 273 men and women between 20-69 years were retained for the present analysis. A total of 103 (37.7%) reported eating fish from the Upper St. Lawrence. Although fisheaters and non-fisheaters were similar for most socio-demographic variables, significantly more fisheaters (65.2%) reported consuming alcoholic beverages as compared to non-fisheaters (42.4%) (Chi Sq. <0.01). To eliminate this possible bias, fisheaters were matched to non-fisheaters for the variables sex, alcohol consumption (never or occasionally vs. regularly), age (+/-5y) and education (+/-2y). A total of 63 matched pairs were thus created. Paired analyses (t-test or Signed Rank) showed that fisheaters had higher levels of blood organic mercury and lead. Analysis of nervous system functions revealed that both groups performed similarly on tests of sensory function, visual memory and recognition, fine motor performance and some motor tests, but fisheaters performed significantly more poorly (p<0.05) on tests requiring cognitive flexibility, word naming, auditory recall, and more complex motor tasks. The profile of deficits is consistent with diminished capacity for information processing. These observations were made within a study that was not specifically designed to examine the effects of fish eating from these two lakes, and the characterization of fish dietary habits has many limitations. Nevertheless, the findings are sufficiently compelling to warrant further studies, since fish from the Upper St. Lawrence Lakes are known to contain multiple neurotoxic substances.

Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study.

In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors. A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus. All these vertebrate peptides induced a dose-dependent increased incorporation of 3H-leucine and 14C-uridine in whole digestive gland cell suspensions. However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides. In addition, insulin and IGF-I, but not EGF and bFGF, stimulated 3H-leucine incorporation in control dispersed mantle edge cells. These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs. On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs.

Evidence of early nervous system dysfunction in Amazonian populations exposed to low-levels of methylmercury.

There is increasing concern about the potential neurotoxic effects of exposure to methylmercury in Amazonian populations due to mercury (Hg) release from gold-mining activities. A preliminary study was undertaken in two villages on the Tapajos River, an effluent of the Amazon, situated over 200 km downstream from the extraction areas. The study population included 29 young adults (< or = 35 years), 14 women and 15 men, randomly chosen from a previous survey. Hair analyses were conducted with cold vapor atomic fluorescence spectrophotometry. Total hair Hg (THg) varied between 5.6 micrograms/g and 38.4 micrograms/gl, with MeHg levels from 72.2% to 93.3% of the THg. A quantitative behavioural neurophysiological test battery, designed for use under standard conditions, in an area without electricity and for persons with minimal education was administered to all participants. The results of visual testing showed that although all participants had good near and far visual acuity, color discrimination capacity (Lanthony D-15 desaturated panel) decreased with increasing THg (F = 4.1; p = 0.05); near visual contrast sensitivity profiles (Vistech 6000) and peripheral visual field profiles (Goldman Perimetry with Targets I and V) were reduced for those with the highest levels of THg. For the women, manual dexterity (Santa Ana, Helsinki version) decreased with increasing THg (F = 16.7; p < 0.01); this was not the case for the men. Although the women showed a tendency towards reduced grip strength, muscular fatigue did not vary with THg for either sex. The findings of this study demonstrate that it is possible, using a sensitive test battery, to detect alterations in nervous system functions, consistent with knowledge on Hg toxicity, at levels below the currently recognized threshold of 50 micrograms/g THg.

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc, Haliotis tuberculata.

A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [3H]-leucine and [3H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [3H]-leucine or for [3H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.