Mutagenicity of Blue Rayon Extracts of Fish Bile as a Biomarker in a Field Study

Blue rayon (BR) in combination with the Salmonella/microsome assay was used to evaluate the mutagenicity of fish bile samples. Specimens of Mugil curema from two sites were collected over a 1-year period. Piacaguera channel contains high concentrations of total polycyclic aromatic hydrocarbons (PAHs) and other contaminants, while Bertioga channel was considered the reference sites in this study. Bile was extracted with BR and tested with TA98, TA100, and YG1041 strains with and without S9 in dose response experiments. PAH metabolite equivalents were analyzed using reverse-phase high performance liquid chromatography /fluorescence. Higher mutagenic responses were observed for the contaminated site; YG1041 with S9 was the most sensitive strain/condition. Mutagenicity ranged from 3,900 to 14,000 rev./mg at the contaminated site and from 1,200 to 2,500 rev./mg of BR at the reference site. The responses of YG1041 were much higher in comparison with the TA98 indicating the presence of polycyclic compounds from the aromatic amine class that cause frameshift mutation. TA100 showed a positive mutagenic response that was enhanced following S9 treatment at both sites suggesting the presence of polycyclic compounds that require metabolic activation. benzo(a)pyrene, naphthalene, and phenanthrene metabolite equivalents were also higher in the bile of fish collected at the contaminated site. It was not possible to correlate the PAH metabolite quantities with the mutagenic potency. Thus, a combination of the Salmonella/microsome assay with YG1041 with S9 from BR bile extract seems to be an acceptable biomarker for monitoring the exposure of fish to mutagenic polycyclic compounds. Environ. Mal. Mutagen. 51:173-179, 2010. (C) 2009 Wiley-Liss, Inc.

Bioavailability of pharmaceuticals in waters close to wastewater treatment plants: use of fish bile for exposure assessment

Pharmaceuticals are ubiquitous in surface waters as a consequence of discharges from municipal wastewater treatment plants. However, few studies have assessed the bioavailability of pharmaceuticals to fish in natural waters. In the present study, passive samplers and rainbow trout were experimentally deployed next to three municipal wastewater treatment plants in Finland to evaluate the degree of animal exposure. Pharmaceuticals from several therapeutic classes (in total 15) were analyzed by liquid chromatography-tandem mass spectrometry in extracts of passive samplers and in bile and blood plasma of rainbow trout held at polluted sites for 10 d. Each approach indicated the highest exposure near wastewater treatment plant A and the lowest near that of plant C. Diclofenac, naproxen, and ibuprofen were found in rainbow trout, and their concentrations in bile were 10 to 400 times higher than in plasma. The phase I metabolite hydroxydiclofenac was also detected in bile. Hence, bile proved to be an excellent sample matrix for the exposure assessment of fish. Most of the monitored pharmaceuticals were found in passive samplers, implying that they may overestimate the actual exposure of fish in receiving waters. Two biomarkers, hepatic vitellogenin and cytochrome P4501A, did not reveal clear effects on fish, although a small induction of vitellogenin mRNA was observed in trout caged near wastewater treatment plants B and C.

Identification and application of bile metabolites to assess the exposure of fish to pharmaceuticals in the environment

Thousands of tons of pharmaceuticals are consumed yearly worldwide. Due to the continuous and increasing consumption and their incomplete elimination in wastewater treatment plants (WWTP), pharmaceuticals and their metabolites can be detected in receiving waters, although at low concentrations (ng to low μg/L). As bioactive molecules the presence of pharmaceuticals in the aquatic environment must be considered potentially hazardous for the aquatic organisms. In this thesis, the biotransformation and excretion of pharmaceuticals in fish was studied. The main biotransformation pathways of three anti‐inflammatory drugs, diclofenac, naproxen and ibuprofen, in rainbow trout were glucuronidation and taurine conjugation of the parent compounds and their phase I metabolites. The same metabolites were present in fish bile in aquatic exposures as in fish dosed with intraperitoneal injection. Higher bioconcentration factor in bile (BCFbile) was found for ibuprofen when compared to diclofenac and naproxen. Laboratory exposure studies were followed by a study of uptake of pharmaceuticals in a wild fish population living in lake contaminated with WWTP effluents. Of the analyzed 17 pharmaceuticals and six phase I metabolites, only diclofenac, naproxen and ibuprofen was present in bream and roach bile. It was shown, that diclofenac, naproxen and ibuprofen excreted by the liver can be found in rainbow trout and in two native fish species living in the receiving waters. In the bream and roach bile, the concentrations of diclofenac, naproxen and ibuprofen were roughly 1000 times higher than those found in the lake water, while in the laboratory exposures, the bioconcentration of the compounds and their metabolites in rainbow trout bile were at the same level as in wild fish or an order of magnitude higher. Thus, the parent compounds and their metabolites in fish bile can be used as a reliable biomarker to monitor the exposure of fish to environmental pharmaceuticals present in water receiving discharges from WWTPs.

Synchronous scanning phosphorimetry for the selective determination of chrysene: a metrological study

Room-temperature phosphorimetry was used to quantify trace levels of chrysene in sugar-cane spirits and in fish bile. A selective phosphorescence enhancer (AgNO3) and synchronous scanning allowed the detection of ng amounts of chrysene. Accuracy (113 ± 17%) and selectivity was evaluated using the CRM-NIST-1647d - Priority Pollutant Polycyclic Aromatic Hydrocarbons in acetonitrile. Analysis of sugar-cane spirit samples enabled recovery of 108 ± 18% which agreed with the one achieved using HPLC. Method's uncertainty was equivalent to 3.4 ng of the analyte, however, the analyte pre-concentration (SPE) improved sensibility and minimized the relative uncertainty. Characterization and homogeneity studies in fish bile were also performed.

Caracterização de hidrocarbonetos policíclicos aromáticos (HPA) em rios da bacia hidrográfica do Turvo/Grande

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Assessment of trophic transfer of benzo(a)pyrene genotoxicity from the post-larval pink shrimp F. brasiliensis to the juvenile Florida pompano T. carolinus

In the present study, the polycyclic aromatic hydrocarbon (PAH) genotoxicity was investigated in a one-step predator-prey relationship with the trophic-related marine species. Florida pompanos were fed for 5 and 10 days with pink shrimp post larvae previously exposed to benzo(a)pyrene (BaP) concentrations. Parent BaP body burden was measured in samples of Farfantepenaeus brasiliensis. BaP metabolites were determined in bile samples of Trachinotus carolinus and DNA damage was assessed through the comet and erythrocyte nuclear abnormalities (ENAs) assays in fish erythrocytes. BaP body burden increased significantly with the PAH concentration in pink shrimp PLs as well as the fish bile BaP metabolites. Both, comet and ENAs assays indicated significant increase on erythrocyte DNA damage of Florida pompanos fed with BaP-exposed pink shrimp on both feeding periods. The trophic route of BaP genotoxicity is discussed as well as the PAH biotransformation as the inducing mechanism for the DNA damages observed.

Bile And Liver Metallothionein Behavior In Copper-exposed Fish.

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.

A new page on the road book of inorganic mercury in fish body - tissue distribution and elimination following waterborne exposure and post-exposure periods

prova tipográfica / uncorrected proof

Hidrocarbonetos policíclicos aromáticos no meio ambiente: diferenciação de fontes em sedimentos e metabólitos em bile de peixes

Many studies on environmental ecosystems quality related to polycyclic aromatic hydrocarbons (PAH) have been carried out routinely due to their ubiquotus presence worldwide and to their potential toxicity after its biotransformation. PAH may be introduced into the environmet by natural and anthropogenic processes from direct runoff and discharges and indirect atmospheric deposition. Sources of naturally occurring PAHs include natural fires, natural oil seepage and recent biological or diagenetic processes. Anthropogenic sources of PAHs, acute or chronic, are combustion of organic matter (petroleum, coal, wood), waste and releases/spills of petroleum and derivatives (river runoff, sewage outfalls, maritime transport, pipelines). Besides the co-existence of multiples sources of PAH in the environmental samples, these compounds are subject to many processes that lead to geochemical fates (physical-chemical transformation, biodegradation and photo-oxidation), which leads to an alteration of their composition. All these facts make the identification of the hydrocarbons sources, if petrogenic, pyrolytic or natural, a challenge. One of the objectives of this study is to establish tools to identify the origin of hydrocarbons in environmental samples. PAH diagnostic ratios and PAH principal component analysis were tested on a critical area: Guanabara Bay sediments. Guanabara Bay is located in a complex urban area of Rio de Janeiro with a high anthropogenic influence, being an endpoint of chronic pollution from the Greater Rio and it was the scenario of an acute event of oil release in January 2000. It were quantified 38 compounds, parental and alkylated PAH, in 21 sediment samples collected in two surveys: 2000 and 2003. The PAH levels varied from 400 to 58439 ng g-1. Both tested techniques for origin identification of hydrocarbons have shown their applicability, being able to discriminate the PAH sources for the majority of the samples analysed. The bay sediments were separated into two big clusters: sediments with a clear pattern of petrogenic introduction of hydrocarbons (from intertidal area) and sediments with combustion characteristics (from subtidal region). Only a minority of the samples could not display a clear contribution of petrogenic or pyrolytic input. The diagnostic ratios that have exhibited high ability to distinguish combustion- and petroleum-derived PAH inputs for Guanabara Bay sediments were Phenanthrene+Anthracene/(Phenanthrene+Anthracene+C1Phenanthrene); Fluorantene/(Fluorantene+Pyrene); Σ (other 3-6 ring PAHs)/ Σ (5 alkylated PAH series). The PCA results prooved to be a useful tool for PAH source identification in the environment, corroborating the diagnostic indexes. In relation to the temporal evaluation carried out in this study, it was not verified significant changes on the class of predominant source of the samples. This result indicates that the hydrocarbons present in the Guanabara Bay sediments are mainly related to the long-term anthropogenic input and not directly related to acute events such as the oil spill of January 2000. This findings were similar to various international estuarine sites. Finally, this work had a complementary objective of evaluating the level of hydrocarbons exposure of the aquatic organisms of Guanabara Bay. It was a preliminary study in which a quantification of 12 individual biliar metabolites of PAH was performed in four demersal fish representing three different families. The analysed metabolites were 1-hydroxynaphtalene, 2-hidroxinaphtalene, 1hydroxyphenanthrene, 9-hydroxyphenanthrene, 2-hydroxyphenanthrene, 1hydroxypyrene, 3-hidroxibiphenil, 3- hydroxyphenanthrene, 1-hydroxychrysene, 9hydroxyfluorene, 4-hydroxyphenanthrene, 3-hydroxybenz(a)pyrene. The metabolites concentrations were found to be high, ranging from 13 to 177 µg g-1, however they were similar to worldwide regions under high anthropogenic input. Besides the metabolites established by the used protocol, it was possible to verified high concentrations of three other compounds not yet reported in the literature. They were related to pyrolytic PAH contribution to Guanabara Bay aquatic biota: 1-hydroxypyrine and 3-hydroxybenz(a)pyrine isomers

Bioaccumulation of butyltins and liver damage in the demersal fish Cathorops spixii (Siluriformes, Ariidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bile acids as potential pheromones in pintado catfish Pseudoplatystoma corruscans (Spix &Agassiz, 1829): eletrophysiological and behavioral studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphology and Histochemistry of the Liver of Carnivorous Fish Hemisorubim platyrhynchos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An Estuarine Low Temperature Fish Kill in Mississippi, with Remarks on Restricted Necropsies

In January 1973, large numbers of Mugil cephalus (striped mullet), weighing approximately 250 gm each, died in two freshwater localities in tidewater bayous of Jackson County, Mississippi. Fish identified as Mugil curema, M. cephalus, Megalops atlantica, Dormitator maculatus, and Fundulus grandis were found dead in other low saline estuarine areas. Fish-kills during cold periods are less commonly encountered in Mississippi than in Texas or Florida. This particular incident is attributed to conditions of stress for fishes incompletely acclimated to the encountered low temperatures. The most deleterious stress was the low saline water which probably allowed a breakdown in the fishes' ion-osmoregulatory mechanisms. Striped mullet and other euryhaline fishes in salinities greater than 6 ppt survived, as did freshwater centrarchids and ictalurids in areas with dying mullet. Other stresses thought to contribute to the weakening of striped mullet in Paige Bayou during the period of rapidly decreasing temperatures include starvation and high levels of pesticide residues. In examined fish, the alimentary tracts were devoid of food, the gall bladders were distended and leaking bile, the livers contained excess lipid material and were often stained throughout with bile pigments, and the levels of DDT metabolites and endrin residues in the liver were higher than in control fish. Stress caused by low levels of dissolved oxygen, toxic substances in the water, or disease was discounted as a cause of death.

Linking Ah receptor mediated effects of sediments and impacts on fish to key pollutants in the Yangtze Three Gorges Reservoir, China – A comprehensive perspective

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River's impoundment by the Three Gorges Dam, faces numerous anthropogenic impacts that challenge its unique ecosystem. Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, have been widely detected in the Yangtze River, but only little research was yet done on AhR-mediated activities. Hence, in order to assess effects of organic pollution, with particular focus on AhR-mediated activities, several sites in the TGR area were examined applying the "triad approach". It combines chemical analysis, in vitro, in vivo and in situ investigations to a holistic assessment. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011/2012, respectively, to identify relevant endpoints. Sediment was tested in vitro with the ethoxyresorufin-O-deethylase (EROD) induction assay, and in vivo with the Fish Embryo Toxicity Test and Sediment Contact Assay with Danio rerio. Activities of phase I (EROD) and phase II (glutathione-S-transferase) biotransformation enzymes, pollutant metabolites and histopathological alterations were studied in situ in P. vachellii. EROD induction was tested in vitro and in situ to evaluate possible relationships. Two sites, near Chongqing and Kaixian city, were identified as regional hot-spots and further investigated in 2013. The sediments induced in the in vitro/in vivo bioassays AhR-mediated activities and embryotoxic/teratogenic effects - particularly on the cardiovascular system. These endpoints could be significantly correlated to each other and respective chemical data. However, particle-bound pollutants showed only low bioavailability. The in situ investigations suggested a rather poor condition of P. vachellii, with histopathological alterations in liver and excretory kidney. Fish from Chongqing city exhibited significant hepatic EROD induction and obvious parasitic infestations. The polycyclic aromatic hydrocarbon (PAH) metabolite 1-hydroxypyrene was detected in bile of fish from all sites. All endpoints in combination with the chemical data suggest a pivotal role of PAHs in the observed ecotoxicological impacts.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.