Fish Liver Samples Research Articles

Optimization of a Cytochrome-P450-Monooxygenase-1A-Mediated EROD Assay in the Cape Hake Species Merluccius capensis and Merluccius paradoxus (Pisces).

Cytochrome P450 monooxygenase 1A (CYP1A) is induced by several planar toxic compounds, for example, polychlorinated biphenyls (PCBs) and the induction of this protein is often measured in terms of CYP1A-mediated 7-ethoxyresorufin-O-deethylase (EROD) activity. This study was aimed at developing this assay in the Cape hake species Merluccius capensis and Merluccius paradoxus (considered one stock). Microsomal fractions were obtained from frozen fish liver samples by differential centrifugation. Fluorimetric and spectrophotometric analysis of the EROD assay resulted in the spectrophotometric (at 572 nm) detection method being selected, as this method resulted in a lower degree of variability and demonstrated higher reproducibility. The activity in the EROD assay was enhanced in the presence of NADPH, and the addition of dicumarol (phase II enzyme inhibitor) to the reaction mixtures prevented the underestimation of this assay by the inhibition of DT-diaphorase. In summary, an EROD assay was established for use in Cape hake species.

Identification and evaluation of cyp1a transcript expression in fish as molecular biomarker for petroleum contamination in tropical fresh water ecosystems

In order to monitor potential contamination deriving from exploration and transport of oil in the Urucu region (Brazil), there is a need to establish suitable biomarkers for native Amazonian fish. Therefore, the transcript expression of various potentially sensitive genes ( ahr2 1 1 Nomenclature of gene and protein symbols follow those used for zebrafish ( www.zfin.org). Gene name abbreviation are given in lower case letters and as italics. Upper case letters are used for protein name abbreviations. , cyp1a, hmox1, hsp70, maft, mt, nfe212, gstp1 and nqo1) in fish exposed to water soluble fractions of oil (WSF) was compared. The analysis was first performed in an established laboratory model, the zebrafish embryo. The cyp1a gene proved to be the most sensitive and robust marker for oil contamination and, hence, was selected to study the effect of oil-derived contaminants in the Amazonian cichlid Astronotus ocellatus. Induction of cyp1a transcript expression was observed for ≥0.0061% (v/v) WSFs. In liver samples of fish, collected from different lakes in the Urucu oil mining area, no elevated expression of cyp1a transcripts was observed. The data demonstrate the high sensitivity of cyp1a as indicator of oil exposure; further studies should be considered to test its usefulness at known contaminated sites and to evaluate influential factors by, e.g. mesocosm experiments.

Expression of immune-related genes in rohu Labeo rohita (Hamilton) by experimental freshwater lice Argulus siamensis (Wilson) infection

The crustacean ectoparasite, Argulus poses one of the major threats to carp culture due to absence of any suitable control measure. The study was undertaken to determine the expression of immune-related genes in three major immunocompetent organs viz., kidney, skin and liver of rohu (Labeo rohita) during experimental freshwater lice Argulus siamensis infection. Results showed that the expression of TLR 22-like, lysozyme G and β2-microglobulin genes in kidney was significantly (P≤0.05) down-regulated in lice-infected fish. On the other hand, no significant difference (P>0.05) in CXCa, lysozyme C, TNFα and complement component 3 (C3) expression was found between uninfected control and different degrees of lice infected fish. In the skin, the expression of TLR 22-like and TNFα genes were significantly up-regulated whereas that of C3 was significantly (P≤0.05) down-regulated in lice-infected fish with respect to control fish. The expression of CXCa, lysozyme C and transferrin was not detected in the skin samples of fish. In the liver, the expression of CXCa, lysozyme G, β2-microglobulin and transferrin was significantly (P≤0.05) up-regulated in lice-infected fish with respect to control fish whereas expression of C3 was significantly (P≤0.05) down-regulated in lice-infected fish. The expression of TLR 22-like, lysozyme C, TNFα genes was not detected in the liver samples of fish. This study indicates that majority of the genes showed down-regulation in kidney tissue whereas up-regulation in liver and skin tissues except C3 in Argulus-infected fish. We show that infection with this parasite irrespective of intensity can also result in immune gene expression changes in tissues situated away from the site of parasite attachment and feeding. The information obtained here could be useful towards understanding the susceptibility of rohu to argulosis and mechanisms involved in protection of rohu to ectoparasitic infections, which is causing immense economic losses to freshwater aquaculture sector.

Comparison of Metallothionein Detection by Using Brdicka Reaction and Enzyme‐Linked Immunosorbent Assay Employing Chicken Yolk Antibodies

AbstractIn this study, two methods for metallothioneins (MT) determination in a biological sample were compared. Particularly, twenty five human and nine pig blood serum samples and liver and kidney samples from thirty five carps (Cyprinus carpio) were analyzed by enzyme‐linked immunosorbent assay and differential pulse voltammetry Brdicka's reaction. The results obtained by these two methods were in good agreement. For commercially available MT standard the correlation coefficient between the single concentrations signal height was 0.99. In biological samples the correlation coefficients were 0.90 for fish liver and kidney samples, 0.91 for pig blood serum and 0.93 for human blood serum.

Vapor Generation Atomic Absorption Spectrometric Determination of Cadmium in Environmental Samples with In-Line Anion Exchange Separation

A novel in-line anion exchange separation method is described for the removal of Mn(2+), Fe(3+), Ni(2+), Co(2+), Cu(2+), Zn(2+), and Pb(2+) from Cd(2+) and for its subsequent determination by vapor generation atomic absorption spectrometry. High Cd(2+) retention efficiency and maximum exclusion of other metal ions were achieved by using Cl(-), Br(-), or I(-) loaded strong base anion exchange resin and fiber in chloride medium. SO(4)(2-) and NO(3)(-) affected the retention of Cd(2+) on Cl(-) or Br(-) loaded exchangers but were beneficial for elution from I(-) loaded exchangers. Fast and efficient elution from the exchangers was achieved by using ethylenediamine solution. The removal of Zn(2+) was unnecessary as it prevents a decline in the sensitivity of cadmium response when ethylenediamine and high acid concentration were used. Under optimum conditions, the achievable detection limits (3sigma) with sample loadings of 0.48 and 9.6 mL were 40 ng L(-1) and 3 ng L(-1), respectively. Interference of residual organic matrix, in sample digests, with the separation and retention of cadmium were eliminated by use of microwave assisted digestion. The method was successfully applied to the determination of Cd in river sediment, fish liver, and water samples.

Bioaccumulation of organochlorine pollutants in the fish community in Lake Årungen, Norway

Organochlorine pollutants in the major fish species (pike Esox lucius, perch Perca fluviatilis, and roach Rutilus rutilus) of Lake Årungen, Norway, were investigated after an extensive removal of large pike in 2004. The organochlorine pollutants detected in fish liver samples in 2005 were dichlorodiphenyltrichloroethane (DDTs), polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), and heptachlor epoxide (HCE). DDTs were the dominant among all analyzed OCs. ΣPCB and HCB, detected in fish from two clearly distinct trophic levels (prey and predators), give an indication of biomagnification. All OC concentrations in female pike were significantly lower compared to males, which might be due to the removal of high concentrations of pollutants in roe during spawning.

Determination of 17-Ethynylestradiol, Carbamazepine, Diazepam, Simvastatin, and Oxybenzone in Fish Livers

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of 17alpha-ethynylestradiol in fish liver; a second method using LC/MS was developed for the determination of carbamazepine, diazepam, simvastatin, and oxybenzone in fish liver. The fish liver samples were extracted and cleaned up by using liquid-liquid extraction and solid-phase extraction before the extracts were analyzed by LC/MS or LC/MS/MS with electrospray negative and positive ionization. Recoveries of the 5 target compounds from spiked catfish liver ranged from 72 +/- 2 to 100 +/- 3%. Limits of quantification for the 5 compounds were between 4.2 and 12.3 ng/g (wet weight). Ten turbot (Pleuronichthys verticalis) liver samples were analyzed; levels of 17alpha-ethynylestradiol, carbamazepine, simvastatin, and oxybenzone were below the detection limits. Diazepam was detected in all 10 fish liver samples at concentrations ranging from 23 to 110 ng/g (wet weight).

Halogenated persistent organic pollutants in Scottish deep water fish

Halogenated persistent organic pollutants (chlorobiphenyls (CBs), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD) and tetrabromobisphenol-A (TBBP-A)) and total lipid content were measured in the liver and muscle of three species of deep water fish (black scabbard, roundnose grenadier and black dogfish) collected from the Rockall Trough, to the west of Scotland, in 2006. CB concentrations (SigmaICES (International Council for the Exploration of the Seas)7 CBs) >500 microg kg(-1) lipid weight) were found in 9 of the 31 deep water fish liver samples. Non-ortho CBs were measured in samples with the highest ortho CB concentrations. Non-ortho CBs (CB81, 77, 126 and 169) were not detected in any of the fish muscle samples. In liver, CB81 was not detected in any of the samples while CB169 was detected in all but one sample. The total 'dioxin-like' CB concentration was calculated based on the 5 mono-ortho and 4 non-ortho CBs measured. The non-ortho CB concentration made a very small contribution to the total 'dioxin-like' CB concentrations (<1%). Concentrations for the individual ICES7 CBs in fish liver were above OSPAR Background Assessment Concentrations (BACs) in all three species, except for CB28 and 101 in black dogfish. Toxic Equivalent (TEQs) concentrations calculated for the five mono-ortho and four non-ortho CBs measured, and estimated TEQs calculated using published models in the fish muscle indicated that consumption of deep water fish muscle is unlikely to represent a risk to human health. However, dioxins and furans were not measured and the contribution to the calculated TEQs from these compounds was not taken into account. Calculated and estimated TEQs for some roundnose grenadier liver samples exceeded the 25 pg g(-1) wet weight limit for fish liver and, therefore, there may be a health risk if consumed. PBDEs were detected in both the liver and muscle of the deep water fish, whilst HBCD and TBBP-A were not detected in any of the deep water fish.

Lipid extraction has little effect on the δ15N of aquatic consumers

Proper application of stable isotopes (e.g., δ15N and δ13C) to food web analysis requires an understanding of all nondietary factors that contribute to isotopic variability. Lipid extraction is often used during stable isotope analysis (SIA), because synthesized lipids have a low δ13C and can mask the δ13C of a consumer's diet. Recent studies indicate that lipid extraction intended to adjust δ13C may also cause shifts in δ15N, but the magnitude of and reasons for the shift are highly uncertain. We examined a large data set (n = 854) for effects of lipid extraction (using Bligh and Dyer's [1959] chloroform‐methanol solvent mixtures) on the δ15N of aquatic consumers. We found no effect of chemically extracting lipids on the δ15N of whole zooplankton, unionid mussels, and fish liver samples, and found a small increase in fish muscle δ15N of ~0.4‰. We also detected a negative relationship between the shift in δ15N following extraction and the C:N ratio in muscle tissue, suggesting that effects of extraction were greater for tissue with lower lipid content. As long as appropriate techniques such as those from Bligh and Dyer (1959) are used, effects of lipid extraction on δ15N of aquatic consumers need not be a major consideration in the SIA of food webs.

Immune responses of great sturgeon Huso huso to Aeromonas hydrophila bacterin

The immunocompetent cell population, lysozyme activity, chemiluminescence response and antibody titre were assessed in great sturgeon Huso huso after intraperitoneal immunization with formalin‐inactivated whole cells of Aeromonas hydrophila. Generally an increase was seen in indices of immunocomptent cell counts of immunized fish compared to the control group. The neutrophil count was higher in immunized fish from day 29. Lysozyme activity in liver and spleen samples of immunized fish was initially significantly higher than unimmunized fish, but was not different in serum and kidney. Antibody titre and respiratory burst in immunized fish were mainly significantly higher than unimmunized fish.

An Undergraduate Experiment for the Measurement of Perfluorinated Surfactants in Fish Liver by Liquid Chromatography–Tandem Mass Spectrometry

Perfluorinated surfactants, including perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), are an emerging class of environmental contaminants that have recently received considerable attention. An undergraduate laboratory experiment to measure perfluorinated surfactants in fish liver has been developed. Fish samples, collected by students from local markets and grocery stores, are extracted using an ion-pairing method and analyzed using liquid chromatography�tandem mass spectrometry (LC�MS/MS) employing student optimized instrumental parameters. In nearly all fish liver samples analyzed by students, perfluorinated surfactants were detected. This undergraduate experiment provides a hands-on opportunity for students to learn new sample preparation and extraction procedures and the specific techniques of LC�MS/MS. Students gain an appreciation of both the sensitivity of LC�MS/MS for the analysis of trace environmental contaminants and the importance of quality control when working with trace analytes in complex biological matrices.

Bioaccumulation, depuration and oxidative stress in fish Carassius auratus under phenanthrene exposure

In this study, laboratory experiment was carried out to determine phenanthrene bioaccumulation, depuration in whole fish and oxidative stress in the liver of freshwater fish Carassius auratus. Fish were exposed to 0.05 mg/l phenanthrene for different periods, while one control group was designated for each exposure group. Some fish after 7 days of exposure were transferred to diluted water. The concentrations of phenanthrene in fish were analyzed by HPLC. Twenty-four hours after the exposure, reactive oxygen species (ROS) were trapped by phenyl- tert-butylnitrone and detected by electron paramagnetic resonance (EPR). The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione-s-transferase (GST) were also determined. The concentrations of phenanthrene in fish increased rapidly shortly after the start of the exposure, reached a maximum level at the 2 days, and then it declined quickly to low-level-steady state. The elimination process of phenanthrene could be divided into two periods—a fast elimination period following a slower loss period. The elimination curve could be fitted mathematically as the sum of two exponential functions according to two-compartment model: C( t) = 2.72e −1.065 t + 0.68e −0.0364 t . The PBN-radical adducts were detected in fish liver samples following the exposure 24 h. The hyperfine splitting constants for the PBN-radical adducts were a N = 13.5 G, a H = 1.77 G and g value was 2.0058, which were consistent with those of PBN/ OH. The results indicated that the hydroxyl radical was probably significantly induced during the exposure of phenanthrene, as compared to the control group. The changes of activities of the antioxidant enzymes also were observed. In addition, after fish were removed from phenanthrene exposure, the recovery status of these antioxidant indices was explored. These results clearly indicated phenanthrene could be accumulated in fish and similar redox cyclings were produced, resulting in the changes of the activities of the antioxidant enzymes and the production of ROS with the oxidative stress.

Electron paramagnetic resonance investigation of in vivo free radical formation and oxidative stress induced by 2,4‐dichlorophenol in the freshwater fishCarassius auratus

In the present study, electron paramagnetic resonance coupled with spin-trapping technique was used, with alpha-phenyl-N-tert-butylnitrone (PBN) as a spin-trapping agent, to investigate free radical generation in freshwater fish with acute 2,4-dichlorophenol (2,4-DCP) poisoning. The PBN-radical adducts were detected in fish liver samples following treatments of 2,4-DCP (0.025, 0.05, 0.5, 5, or 25 mg/kg) 24 h after intraperitoneal (i.p.) injection and 2,4-DCP (0.5 mg/kg) at 2, 4, 8, 24, or 72 h after i.p. injection in Carassius auratus. The hyperfine splitting constants for the PBN-radical adducts are aN = 13.7 G, aH = 1.8 G, and g = 2.0058, which is consistent with those of PBN/hydroxyl radical (*OH). The results indicate that the hydroxyl radical is probably produced during acute intoxication of 2,4-DCP. The relative similarity in the kinetics (from 2 to 72 h) of superoxide dismutase activity induction and *OH generation implies that the generation of *OH possibly depends on the superoxide anion (O2*-). Superoxide anion (O2*-) might be the precursor radical undergoing the Haber-Weiss reaction to form *OH. Possible pathways for radical chain reactions in the formation of the hydroxyl radical in vivo after 2,4-DCP administration are proposed. Other parameters with respect to antioxidant defense (e.g., superoxide dismutase and catalase) and oxidative damage (lipid peroxidation level) indicate that the fish were subjected to oxidative stress induced by 2,4-DCP and that the mechanisms of oxidative stress possibly involve the in vivo stimulation of hydroxyl radical formation.

New quantification procedure for the analysis of chlorinated paraffins using electron capture negative ionization mass spectrometry

An improved quantification procedure for the analysis of chlorinated paraffins (CPs) is presented based on electron capture negative ionization mass spectrometry. It compensates differences in response factors between reference CP mixtures and the CP pattern present in environmental samples. The use of a CP standard with a matching degree of chlorination is no longer necessary. It could be shown that the response factors of C 10-, C 11-, C 12- and C 13-CP mixtures of both 50 and 60% chlorine content were only slightly influenced by the carbon chain length. A linear correlation ( R 2 = 0.965) between the total response factor of a CP mixture and its chlorine content was obtained for seven short chain chlorinated paraffin mixtures (SCCP, C 10–C 13) with different composition and chlorine content (51–69%). Maximum single deviations were <7% for this reference set. It allowed to determine the correct total response factor of the CP composition present in a sample. The deviations were not more than 7–33% for five independent SCCP control samples compared to up to 373% for the conventional procedure. The procedure was tested by quantifying the SCCP and MCCP levels in 10 fish liver samples. The proposed method allowed to compensate the influence of the degree of chlorination of the applied reference standard on the total response factor.

Preliminary screening of perfluorooctane sulfonate (PFOS) and other fluorochemicals in fish, birds and marine mammals from Greenland and the Faroe Islands

Extensive screening analyses of perfluorooctane sulfonate (PFOS) and related perfluorinated compounds in biota samples from all over the world have identified PFOS as a global pollutant and have shown its bioaccumulation into higher trophic levels in the food chain. Perfluorinated compounds have been found in remote areas as the Arctic. In this study a preliminary screening of PFOS and related compounds has been performed in liver samples of fish, birds and marine mammals from Greenland and the Faroe Islands. PFOS was the predominant fluorochemical in the biota analyzed, followed by perfluorooctane sulfonamide (PFOSA). PFOS was found at concentrations above LOQ (10 ng/g wet weight) in 13 out of 16 samples from Greenland and in all samples from the Faroe Islands. The results from Greenland showed a biomagnification of PFOS along the marine food chain (shorthorn sculpin<ringed seal<polar bear). The greatest concentration of PFOS was found in liver of polar bear from east Greenland (mean: 1285 ng/g wet weight, n=2). The geographical distribution of perfluorinated compounds in Greenland was similar to that of persistent organohalogenated compounds (OHCs), with the highest concentrations in east Greenland, indicating a similar geographical distribution to that of OHCs, with higher concentrations in east Greenland than in west Greenland.

Development and characterization of a solvent extraction–gas chromatographic/mass spectrometric method for the analysis of perfluorooctanesulfonamide compounds in solid matrices

A method utilizing solvent extraction and analysis by gas chromatography–positive chemical ionization mass spectrometry (SE–GC–PCI-MS) was developed for the analysis of three neutral hydrophobic perfluorooctanesulfonamide compounds [perfluorooctanesulfonamide (PFOSA), N-ethyl perfluorooctanesulfonamide ( N-EtPFOSA), and N, N-diethyl perfluorooctanesulfonamide ( N, N-Et 2PFOSA)]. These compounds are suspected metabolic precursors of perfluorooctane sulfonate. The SE–GC–PCI-MS method was used to analyze all three perfluorooctanesulfonamides in fast food, fish, and Arctic marine mammal liver samples. The SE–GC–PCI-MS method produced relatively higher recoveries of the analytes (averaging 83 ± 6%, 84 ± 9%, and 89 ± 19% for N, N-Et 2PFOSA, N-EtPFOSA, and PFOSA, respectively) with lower coefficients of variation, and less susceptibility to matrix effects, than ion pair extraction–liquid chromatography–tandem mass spectrometric methods. Method detection limits (MDLs) were 100, 120, and 250 pg/g for N, N-Et 2PFOSA, N-EtPFOSA, and PFOSA, respectively. The three compounds were found at concentrations ranging from below the MDL to 22 ng/g wet weight in fast food, fish, and Arctic marine mammal liver samples.

Evaluation of four mass spectrometric methods for the gas chromatographic analysis of polychlorinated n-alkanes

The suitability of four mass spectrometric methods for the gas chromatographic analysis of polychlorinated n-alkanes (PCAs, also called chlorinated paraffins) was evaluated and compared using spiked and fish liver samples. Electron ionization tandem mass spectrometry (EI–MS/MS) as well as electron capture negative ionization (ECNI) combined with low and high resolution mass spectrometry and CH 4/CH 2Cl 2-negative ion chemical ionization (NICI) low resolution mass spectrometry were investigated. All methods showed an accuracy of <21% for the analysis of spiked fish samples. However, the analysis of real samples showed deviations of up to 46% between the four mass spectrometric methods. The influence of the selected reference standard on quantification was also evaluated. The use of a quantification standard with a degree of chlorination deviating from that of the sample can result in differences of >100% for the ECNI methods. EI–MS/MS and CH 4/CH 2Cl 2-NICI led to errors of maximum 17% and 33%, respectively, independent from the degree of chlorination of the used reference standard.

Determination of total polychlorinated n-alkane concentration in biota by electron ionization-MS/MS.

Electron ionization (EI) tandem mass spectrometry (MS/MS) allowed the fast determination of the total concentration of short- and medium-chained polychlorinated n-alkanes (PCAs) in biota. EI fragment ions common to all PCAs could be identified. Collision-induced dissociations (CIDs) were carried out by ion trap and triple quadrupole EI-MS/MS. CIDs of m/z 91 --> 53 (limit of detection (LOD) 0.15 ng/microL), 102 --> 65 (LOD = 0.2 ng/microL), and 102 --> 67 (LOD = 0.1 ng/microL) were applied for the determination of the total short- and medium-chain PCA concentration in pooled fish liver samples (North Sea dab, cod, flounder) from the North Sea and from the Baltic Sea using both MS technologies. Total PCA concentrations were in the range of 88-607 ng/g. Accuracy was controlled with spiked samples and deviated not more than 15% from expected values.

Potential application of gas chromatography/tandem mass spectrometry in the measurement of coeluting isomers.

Despite the unprecedented popularity of separation chromatography, the measurement of coeluting isomeric chemicals remains an extremely difficult task. We developed an analytical scheme capable of measuring two coeluting isomers using a single chromatographic column and a gas chromatography/tandem mass spectrometry system. The protocol utilized two product ion fragments generated from a common parent ion associated with the isomers for quantitation. The utility of the analytical scheme was demonstrated with the measurements of several pairs of coeluting polychlorinated biphenyl (PCB) isomers in standard solutions and fish liver samples. Best results were given when a set of stringent constraints for the abundance ratio of the two product ion fragments was satisfied. Analyses of seven fish liver samples collected from nearshore San Diego, CA, indicated that the domain that had been previously reported to comprise PCB 153 and PCB 168 actually contained PCB 153 only. Although only a selected number of PCB congeners were examined, the results presented indicate that the analytical scheme has the potential to be used to determine the concentrations of all chromatographically coeluted isomers.

Development of a method for the determination of inorganic cadmium and cadmium metallothioneins in fish liver by continuous preconcentration on fullerene and flame atomic absorption spectrometry

The adsorptive potential of C60 fullerene for the preconcentration of metallothionein-bound cadmium (Cd-MT) traces was assessed for the first time in this work. A simple flow injection system for the on-line preconcentration of Cd-MT was developed. The compounds were directly retained on the sorbent column and subsequently eluted, by hydrolysis of the complexes with diluted HNO3, and the cadmium was transferred to a flame atomic absorption spectrometer. Total cadmium (inorganic and Cd-MT) was also determined by preparing the sample in acid medium and complexing total cadmium with Na-DDC. The neutral chelates of cadmium, retained also on C60, were eluted with IBMK and transferred to the atomic absorption instrument. By the difference between both determinations, inorganic and organometallic forms of cadmium could be distinguished. The analytical figures of merit of the proposed method are: linear range, 0.3–12 ng ml−1 (using 10 ml of sample); limit of detection, 0.1 ng ml−1 and precision (RSD), ca. 4%. No interferences from metals such as Zn, Fe, Pb, Cu and Hg, as inorganic salts, were observed in the determination of total Cd. The method was applied to the determination of inorganic and organometallic cadmium compounds in spiked fish liver samples, with acceptable recoveries.