Exposure of Polycyclic Aromatic Hydrocarbons (PAHs) and Crude Oil to Atlantic Haddock (Melanogrammus aeglefinus): A Unique Snapshot of the Mercapturic Acid Pathway.

Fish exposed to xenobiotics like petroleum-derived polycyclic aromatic hydrocarbons (PAHs) will immediately initiate detoxification systems through effective biotransformation reactions. Yet, there is a discrepancy between recognized metabolic pathways and the actual metabolites detected in fish following PAH exposure like oil pollution. To deepen our understanding of PAH detoxification, we conducted experiments exposing Atlantic haddock (Melanogrammus aeglefinus) to individual PAHs or complex oil mixtures. Bile extracts, analyzed by using an ion mobility quadrupole time-of-flight mass spectrometer, revealed novel metabolites associated with the mercapturic acid pathway. A dominant spectral feature recognized as PAH thiols set the basis for a screening strategy targeting (i) glutathione-, (ii) cysteinylglycine-, (iii) cysteine-, and (iv) mercapturic acid S-conjugates. Based on controlled single-exposure experiments, we constructed an interactive library of 33 metabolites originating from 8 PAHs (anthracene, phenanthrene, 1-methylphenanthrene, 1,4-dimethylphenanthrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene). By incorporation of the library in the analysis of samples from crude oil exposed fish, PAHs conjugated with glutathione and cysteinylglycine were uncovered. This qualitative study offers an exclusive glimpse into the rarely acknowledged mercapturic acid detoxification pathway in fish. Furthermore, this furnishes evidence that this metabolic pathway also succeeds for PAHs in complex pollution sources, a notable discovery not previously reported.

Discovery of polycyclic aromatic acid metabolites in fish exposed to the petroleum compounds 1-methylphenanthrene and 1,4-dimethylphenanthrene.

Most of the polycyclic aromatic hydrocarbons (PAHs) in petroleum are alkylated (alkyl PAHs), still the metabolism of these alkyl PAHs to the expected acid products (polycyclic aromatic acids; PAAs) has yet to be demonstrated in oil-exposed fish. Should these compounds be discovered in fish as they have in ragworm, rodents, and humans, they could present an indicative biomarker for assessing oil pollution. In this study, the ability to biotransform alkyl PAHs to PAAs was examined on Atlantic haddock (Melanogrammus aeglefinus). Exposure to phenanthrene, 1-methyphenanthrene or 1,4-dimethylphenanthrene was performed via intraperitoneal injection. An Ion Mobility Quadrupole Time-Of-Flight Mass Spectrometer (IMS-Q-TOF MS) was used in exploratory analysis of extracted bile samples. Acquisition of four-dimensional information by coupling liquid chromatography with the IMS-Q-TOF MS and in-silico prediction for feature prioritization in the data processing workflow allowed several tentative identifications with high degree of confidence. This work presents the first detection of PAAs in fish and suggests the importance of investigating alkyl PAHs in ecotoxicological studies of oil-polluted fish environments.

Multibiomarker approach in fish to assess a heavily polluted Brazilian estuary, Guanabara Bay.

Brazil's Guanabara Bay (GB), located in Rio De Janeiro, is a deeply contaminated, eutrophic waterbody that challenges the understanding of the effects of pollutants on the biota. This paper presents a strategy to evaluate the impact of contamination utilizing a multibiomarker approach in two fish species: corvine (Micropogonias furnieri) and burrfish (Chilomycterus spinosus). The strategy is comprised of a general biomarker of fish' physical condition, the condition factor (CF), and specific biomarkers of pollutant exposure such as acetylcholinesterase (AChE), metallothionein (MT) activity and biliary polycyclic aromatic hydrocarbons (PAH) metabolites. Our results indicate that fish from GB are greatly affected by environmental pollution. CF values were lower in fishes from GB than in the reference site indicating that these fishes were under higher environmental stress. Lower AChE activity level in both species showed vulnerability to the presence of pesticide residues. Higher levels of MT in both species in GB reflect the consequences of heavy metal exposure in the bay, in spite of their bioavailability being restricted specially by the high organic matter content of GB. The levels of PAHs were higher in GB for both fish species, indicating exposure to these substances. However, the fish species showed different behavior regarding the origin of the PAHs. The multibiomarker approach used in this study evidently depicted effects on the health of fish in a waterbody with a complex polluted situation and further categorized the effects of anthropogenic activities in this aquatic system.

Biliary polycyclic aromatic hydrocarbons and enzymatic biomarkers in Eugerres brasilianus along four tropical estuaries.

Polycyclic aromatic hydrocarbons (PAH) in bile and biochemical biomarkers were evaluated in Brazilian mojarra Eugerres brasilianus along four estuaries in northeastern Brazil. Bile PAHs naphthalene, phenanthrene chrysene, pyrene and benzo[a]pyrene were lowest at Formoso River Estuarine System (FRES), an area with low population density focused on tourism. Fish sampled in Suape Estuarine Complex (SEC), where a growing industrial port complex is established indicated higher naphthalene and pyrene concentrations compared with FRES. Fish sampled in highly urbanized and populated Bacia do Pina Estuarine Complex (BPEC) and Barra de Jangada Estuarine System (BJES) indicated an increase in all PAHs compared to FRES. Activities of phase 1 Ethoxyresorufin-O-deethylase, phase 2 glutathione-S-transferase and antioxidant defense catalase were induced up to 20, 2 and 2-fold in BJES and BPEC compared to FRES. This study confirms E. brasilianus as an important sentinel species, providing baseline information for these tropical estuaries with different degrees of anthropogenic pressure.

Biotransformation in the fish Prochilodus lineatus: An organ-specific approach to cyp1a gene expression and biochemical activity.

The biotransformation ability of the organism is the result of organ-specific responses. This paper presents a molecular and biochemical approach to elucidate the biotransformation mechanisms in different organs of Prochilodus lineatus induced at 6, 24, and 96 h after a benzo[a]pyrene (B[a]P) injection. The induction in cyp1a transcription showed an organ-specific intensity at every tested time time. The EROD (ethoxyresorufin-O-deethylase) activity increased rapidly (6 h) in the liver and the kidney; the gills and the brain showed an increase at 24 h; and the gills demonstrated the highest activity among all the organs tested. There was no increase in glutathione S-transferase (GST) activity or lipoperoxidation. The decreased hepatic glutathione content (GSH) may be due to its role as an antioxidant. B[a]P was detected in the bile, confirming the xenobiotic efflux from the metabolizing organs. The gills, liver, brain, and kidney of P. lineatus presented an integrated mechanism to deal with the xenobiotic biotransformation.

Study on the hepatobiliary behavior of Ermiao wan formula by microdialysis- LC-qTOF-MS.

An improved bile microdialysis sampling technique was established and coupled with liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) analysis. This method was successfully applied to investigate the metabolic profiles of Ermiao wan (EMW) formula in the bile of Sprague-Dawley (SD) rats. Based on accurate mass information and fragment patterns, 23 alkaloids and lactones metabolites were tentatively identified. Their metabolic pathway involved in glucuronidation, sulfation, hydroxylation and hydrolysis. Because of the high time resolution of microdialysis, the metabolic profiles of EMW were also investigated. Jatrorrhizine, columbamine and other components showed a "double-peak" profiles, suggesting the existence of enterohepatic circulation. The developed microdialysis sampling/ LC-qTOF-MS method provides a simple and efficient research tool for understanding and clarifying the mechanism of hepatobiliary excretion of complex components.

The human gallbladder microbiome is related to the physiological state and the biliary metabolic profile.

BACKGROUND: The microbial populations of the human intestinal tract and their relationship to specific diseases have been extensively studied during the last decade. However, the characterization of the human bile microbiota as a whole has been hampered by difficulties in accessing biological samples and the lack of adequate methodologies to assess molecular studies. Although a few reports have described the biliary microbiota in some hepatobiliary diseases, the bile microbiota of healthy individuals has not been described. With this in mind, the goal of the present study was to generate fundamental knowledge on the composition and activity of the human bile microbiota, as well as establishing its potential relationship with human bile-related disorders. RESULTS: Human bile samples from the gallbladder of individuals from a control group, without any record of hepatobiliary disorder, were obtained from liver donors during liver transplantation surgery. A bile DNA extraction method was optimized together with a quantitative PCR (qPCR) assay for determining the bacterial load. This allows the selection of samples to perform functional metagenomic analysis. Bile samples from the gallbladder of individuals suffering from lithiasis were collected during gallbladder resection and the microbial profiles assessed, using a 16S rRNA gene-based sequencing analysis, and compared with those of the control group. Additionally, the metabolic profile of the samples was analyzed by nuclear magnetic resonance (NMR). We detected, for the first time, bacterial communities in gallbladder samples of individuals without any hepatobiliary pathology. In the biliary microecosystem, the main bacterial phyla were represented by Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Significant differences in the relative abundance of different taxa of both groups were found. Sequences belonging to the family Propionibacteriaceae were more abundant in bile samples from control subjects; meanwhile, in patients with cholelithiasis members of the families Bacteroidaceae, Prevotellaceae, Porphyromonadaceae, and Veillonellaceae were more frequently detected. Furthermore, the metabolomics analysis showed that the two study groups have different metabolic profiles. CONCLUSIONS: Our results indicate that the gallbladder of human individuals, without diagnosed hepatobiliary pathology, harbors a microbial ecosystem that is described for the first time in this study. Its bacterial representatives and metabolites are different from those detected in people suffering from cholelithiasis. In this regard, since liver donors have been subjected to the specific conditions of the hospital's intensive care unit, including an antibiotic treatment, we must be cautious in stating that their bile samples contain a physiologically normal biliary microbiome. In any case, our results open up new possibilities to discover bacterial functions in a microbial ecosystem that has not previously been explored.

Biomarker responses and PAH ratios in fish inhabiting an estuarine urban waterway.

Many cities worldwide are established adjacent to estuaries and their catchments resulting in estuarine contamination due to intense anthropogenic activities. The aim of this study was to evaluate if fish living in an estuarine urban waterway were affected by contamination, via the measurement of a suite of biomarkers of fish health. Black bream (Acanthopagrus butcheri) were sampled in a small urban embayment and a suite of biomarkers of fish health measured. These were condition factor (CF), liver somatic index (LSI), gonadosomatic index (GSI), hepatic EROD activity, polycyclic aromatic hydrocarbon (PAH) biliary metabolites, serum sorbitol dehydrogenase (s-SDH) and branchial enzymes cytochrome C oxidase (CCO), and lactate dehydrogenase (LDH) activities. The biomarkers of exposure EROD activity, and pyrene- and B(a)P-type biliary metabolites confirmed current or recent exposure of the fish and that fish were metabolizing contaminants. Relative to a reference site, LSI was higher in fish collected in the urban inlet as was the metabolic enzyme LDH activity. CF, GSI, s-SDH, CCO, and naphthalene-type metabolites were at similar levels in the urban inlet relative to the reference site. PAH biliary metabolite ratios of high-molecular-weight to low-molecular-weight suggest that fish from the urban inlet were exposed to pyrogenic PAHs, likely from legacy contamination and road runoff entering the embayment. Similarly, the sediment PAH ratios and the freshness indices suggested legacy contamination of a pyrogenic source, likely originating from the adjacent historic gasworks site and a degree of contamination of petrogenic nature entering the inlet via storm water discharge. Biomarkers of exposure and effect confirmed that black bream collected in the Claisebrook Cove inlet, Western Australia, are currently exposed to contamination and are experiencing metabolic perturbations not observed in fish collected at a nearby reference site.

Pristine Arctic: background mapping of PAHs, PAH metabolites and inorganic trace elements in the North-Atlantic Arctic and sub-Arctic coastal environment.

As the ice cap of the Arctic diminishes due to global warming, the polar sailing route will be open larger parts of the year. These changes are likely to increase the pollution load on the pristine Arctic due to large vessel traffic from specific contaminant groups, such as polycyclic aromatic hydrocarbons (PAHs). A well-documented baseline for PAH concentrations in the biota in the remote regions of the Nordic Seas and the sub-Arctic is currently limited, but will be vital in order to assess future changes in PAH contamination in the region. Blue mussels (Mytilus edulis) were collected from remote sites in Greenland, Iceland, the Faroe Islands, Norway and Sweden as well as from urban sites in the same countries for comparison. Cod (Gadus morhua) was caught north of Iceland and along the Norwegian coast. Sixteen priority PAH congeners and the inorganic trace elements arsenic, cadmium, mercury and lead were analysed in the blue mussel samples as well as PAH metabolites in cod bile. Σ16PAHs ranged from 28 ng/g dry weight (d.w.) (Álftafjörður, NW Iceland) to 480 ng/g d.w. (Ísafjörður, NW Iceland). Mussel samples from Mjóifjörður, East Iceland and Maarmorilik, West Greenland, contained elevated levels of Σ16PAHs, 370 and 280 ng/g d.w., respectively. Levels of inorganic trace elements varied with highest levels of arsenic in mussels from Ísafjörður, Iceland (79 ng/g d.w.), cadmium in mussels from Mjóifjörður, Iceland (4.3 ng/g d.w.), mercury in mussels from Sørenfjorden, Norway (0.23 ng/g d.w.) and lead in mussels from Maarmorilik, Greenland (21 ng/g d.w.). 1-OH-pyrene was only found above limits of quantification (0.5 ng/mL) in samples from the Norwegian coast, ranging between 44 and 140 ng/ml bile. Generally, PAH levels were low in mussels from the remote sites investigated in the study, which indicates limited current effect on the environment.

Ultra high resolution SFC-MS as a high throughput platform for metabolic phenotyping: application to metabolic profiling of rat and dog bile.

Ultra high resolution SFC-MS (on sub-2µm particles) coupled to mass spectrometry has been evaluated for the metabolic profiling of rat and dog bile. The selectivity of the SFC separation differed from that seen in previous reversed-phase UPLC-MS studies on bile, with the order of elution for analytes such as e.g., the bile acids showing many differences. The chromatography system showed excellent stability, reproducibility and robustness with relative standard deviation of less than 1% for retention time obtained over the course of the analysis. SFC showed excellent chromatographic performance with chromatographic peak widths in the order of 3s at the base of the peak. The use of supercritical fluid carbon dioxide as a mobile phase solvent also reduced the overall consumption of organic solvent by a factor of 3 and also reduced the overall analysis time by a factor of 30% compared to reversed-phase gradient LC. SFC-MS appear complementary to RPLC for the metabolic profiling of complex samples such as bile.