Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity,inner filter effect and signal interpretation

Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.     

The detection of biomarkers of genotoxin exposure in the European flounder (Platichthys flesus) collected from the River Tyne Estuary

The Tyne Estuary (North East England) is known to contain elevated levels of polycyclic aromatic hydrocarbons (PAH), compared with other less industrialised English waterways. Previous studies suggest that such contamination is responsible for the toxicity detected in invertebrate bioassays conducted on water and sediment samples collected from the Tyne. Here we present data from a biomonitoring study using hepatic DNA adducts (32P-postlabelling assay) and bile metabolites (synchronous fluorescence spectrometry) to investigate genotoxic exposure in flounder (Platichthys flesus) collected from three sites (Scotswood, Newcastle and Redheugh) along the Tyne Estuary. Flounder were also collected from a clean reference site, the Alde Estuary. Levels of bile metabolites (microgram kg-1 wet weight 1-OH pyrene equivalents) were elevated in flounder caught from the Tyne (Scotswood = 22,247 +/- 3408; Newcastle = 14,572 +/- 1888; Redheugh = 21,872 +/- 2935) compared with those collected from the Alde (632 +/- 56). The levels of DNA adducts (adducted nucleotides per 10(8) normal nucleotides) were also elevated in Tyne flounder (Scotswood = 24.6 +/- 3.2; Newcastle = 34.4 +/- 3.7; Redheugh = 27.6 +/- 6.3) compared with fish collected from the Alde (10.1 +/- 4.8), suggesting that a proportion of the bioavailable PAH was being converted into genotoxic metabolites. All DNA adduct profiles in flounder collected from the Tyne consisted of diagonal radioactive zones of radiolabelled adducts, which were not present in fish sampled from the Alde. The in vivo dosing of flounder with benzo[a]pyrene (BaP) to produced DNA adducts in similar chromatographic positions to the diagonal radioactive zones in the Tyne caught flounder are also described.     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

The effect of UV-B and UV-C radiation on Hibiscus leaves determined by ultraweak luminescence and fluorescence induction

The effects of UV-C (254 nm) and UV-B (280-320 nm) on chlorophyll fluorescence induction and ultraweak luminescence (UL) in detached leaves of Hibiscus rosa-sinensis L. were investigated. UL from leaves exposed to UV-B and UV-C radiation reached a maximum 72 h after irradiation. In both cases most of the light was of a wavelength over 600 nm. An increase in the percentage of long wavelength light with time was detected. UV radiation increased peroxidase activity, which also reached a maximum 72 h after irradiation. UV-B and UV-C both reduced variable chlorophyll fluorescence. No effect on the amount of chlorophyll or UV screening pigments was observed with the short-term irradiation used in this investigation.     

A special pattern of haem catabolism in a marine fish, Clinocottus analis, with green blood plasma

The mechanism responsible for the green colouration of blood plasma in the marine fish Clinocottus analis was studied. Biliverdin in the blood of this fish is unconjugated and is tightly bound to albumin-like proteins. Hydrophobic interactions and hydrogen bonds contributed more than electrostatic interactions to the binding between the bile pigment and the carrier protein. This special biliverdin protein complex was not metabolized by biliverdin reductase, and prevented the further metabolism or excretion of the bile pigment. This accounts for the normal green colouration of the blood plasma in this fish.     

Green-blood pigmentation in lizards

The green pigment in the plasma of the scincid lizard genus Prasinohaema is identified as the bile pigment biliverdin. Concentrations of biliverdin in the plasma of P. flavipes, P. prehensicauda and P. virens are 714 ± 79 μmol/1 (mean ± one standard deviation), 1020 ± 624 μmol/1 and 819 ± 89 μmol/1, respectively. These values represent the highest concentration of plasma biliverdin measured for any organism and are the first report of non-pathological biliverdin accumulation in amniotes. We review the literature for fish species with high concentrations of plasma biliverdin and pathological biliverdin accumulation in humans; we find that Prasinohaema species have plasma biliverdin concentrations approximately 1.5–30 times greater than fish species with green blood plasma and 40 times greater than humans with green jaundice.     

Sensitization by 5-Bromouracil of DNA in Bacillus subtilis Spores to Ionizing Radiation

Quantitative determination by fluorescence spectroscopy is possible because of the linear relationship between the intensity of emitted fluorescence and the fluorophore concentration. However, concentration quenching may cause the relationship to become nonlinear, and thus, the optimal dilution ratio has to be determined. In the case of fluorescence fingerprint (FF) measurement, fluorescence is measured under multiple wavelength conditions and a method of determining the optimal dilution ratio for multivariate data such as FFs has not been reported. In this study, the FFs of mixed solutions of tryptophan and epicatechin of different concentrations and composition ratios were measured. Principal component analysis was applied, and the resulting loading plots were found to contain useful information about each constituent. The optimal concentration ranges could be determined by identifying the linear region of the PC score plotted against total concentration.     

Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase is a major protein of the cerebrospinal fluid and was originally known as beta-trace. We investigated the binding ability of prostaglandin D synthase toward bile pigments, thyroid hormones, steroid hormones, and fatty acids in this present study. We found that the recombinant enzyme binds bile pigments and thyroid hormones, resulting in quenching of the intrinsic tryptophan fluorescence, the appearance of induced circular dichroism of the lipophilic ligands, and a red shift of the absorption spectra of bilirubin and biliverdin. The binding of prostaglandin D synthase to lipophilic ligands was also demonstrated by the resonant mirror technique and surface plasmon resonance detection. The dissociation constants were calculated to be 33 nM, 37 nM, 660 nM, 820 nM, and 2.08 microM for biliverdin, bilirubin, L-thyroxine, 3,3',5'-triiodo-L-thyronine, and 3,3', 5-triiodo-L-thyronine, respectively. Biliverdin and bilirubin underwent a shift in their absorption peaks from 375 to 380 nm and from 439 to 446 nm, respectively, after binding to prostaglandin D synthase. Bilirubin bound to the enzyme showed a bisignate CD spectrum with a (-) Cotton effect at 422 nm and a (+) Cotton effect at 472 nm, indicating a right-handed chirality. The ligands also inhibited prostaglandin D synthase activity noncompetitively in a concentration-dependent manner, with IC50 values between 3.9 and 10. 9 microM. Epididymal retinoic acid-binding protein and beta-lactoglobulin, two other lipocalin proteins that bind retinoids such as prostaglandin D synthase, did not show any significant interaction with bile pigments or thyroid hormones. These results show that prostaglandin D synthase binds small lipophilic ligands with a specificity distinct from that of other lipocalins.     

Analysis of fish bile with HPLC — fluorescence to determine environmental exposure to benzo(a)pyrene

Brown bullhead from the Black River, Ohio, have a high incidence of liver neoplasia which is associated with elevated concentrations of polynuclear aromatic hydrocarbons (PAHs) in the sediment. We evaluated the use of biliary concentrations of benzo(a)pyrene [B(a)P] equivalents as a means for determining PAH exposure. Bile was collected from 16 brown bullheads and 8 common carp taken from each of two Lake Erie tributaries in Ohio, the industrialized Black River and the non-industrialized Old Woman Creek. Hatchery bullhead (n = 8) were used to determine base levels of PAHs. A high performance liquid chromatography (HPLC) — fluorescence technique was used to determine the concentration of B(a)P equivalents in the bile samples. The area of all peaks fluorescing at 380/430 nm was summed to give a single value for B(a)P equivalents in each sample. Concentrations of B(a)P equivalents generally reflected concentrations of PAH in sediment where fish were collected. Bile taken from Black River carp contained the highest concentration of B(a)P equivalents and was significantly different from all other groups. The value obtained for Black River bullhead was also high and was found to be significantly different from hatchery bullhead. B(a)P equivalents varied between carp and bullhead from the same habitat possibly because of differing food habits or metabolic pathways. However, our results indicate that relative levels of B(a)P equivalents in the bile of fish correspond well to B(a)P levels in sediment and may offer a means of determining environmental exposure of fish to the parent compound.     

New RT-qPCR assay for viral nervous necrosis virus detection in sea bass,Dicentrarchus labrax (L.): application and limits for hatcheries sanitary control

A sensitive and quantitative one step RT-qPCR method was developed to study Viral Nervous Necrosis (VNN) virus loads in sea bass Dicentrarchus labrax (L.) in hatcheries. After determining the limits of this new method, fin tissues were identified as an interesting new simple non-invasive sample source, which might be useful for screening D. labrax (L.) in hatcheries. We observed VNN virus strain V26 associated to D. labrax (L.) eggs and it’s release in tank water during spawning suggesting both vertical transmission to the eggs and the possibility of horizontal transmission by contamination of tank water. VNN is widespread in water bodies and has the ability to infect a large number of fish species, with this in mind, this PCR technique may be used for the surveillance of various fish farms.     

High pressure effect on photosynthetic properties of green plant leaves

We investigated the effects of high pressure treatment on green plant leaves of two species, red rose (Rosa rubiginosa L.) and silver birch male (Betula pendula Roth). Both species were treated with pressure up to 100 bar in order to explore stress reactions, including desirable or undesirable metabolites in plant. When increasing the pressure, chlorophyll (Chl) fluorescence maximum shifts to the wavelength of about 680 nm for both red rose and silver birch, with shift rates–0.062 nm/bar and–0.082 nm/bar, respectively. High pressure induces the changes of the position for the second fluorescence maximum at approximately 730 nm in both species with the same shift rate–0.083 nm/bar. When increasing pressure the change of the photosynthetic apparatus efficiency decreases for both plant species slowly and nonlinearly. High-pressure treatments irreversibly damaged the leaf tissue and at this way induced changes of Chl fluorescence and photosynthetic efficiency.     

Bile pigment formation and excretion in the rabbit

Bile and plasma levels of biliverdin and bilirubin, together with the hepatic biliverdin reductase and bilirubin UDP-glucuronosyl transferase activities, were studied in the rabbit. No biliverdin could be detected in the blood plasma. The bilirubin concentration in blood was 7.81 +/- 0.79 mumol/l. Biliverdin was the predominant pigment in bile (63%). Hepatic biliverdin reductase activity was 0.086 +/- 0.016 nmol/mg protein/hr. The synthesis of bilirubin was apparently limited by the enzyme activity. Most of the bilirubin in bile was conjugated (90%) with monoconjugates predominating (75%). Hepatic UDP-glucuronosyl transferase activity was 2.65 +/- 0.18 and 1.14 +/- 0.16 mumol/mg protein/hr with and without activation, respectively.     

Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile

Biliverdin IX was purified from the bile of Atlantic salmon (Salmo salar) using a silica gel (Wakogel C-200) column. The yield was 49.5 mg per 100 ml of fresh bile and purity 95.3%. The biliverdin IX in the bile was quite stable when the bile was frozen at –80°C for a period of 40 days. However, 7.1% of the biliverdin IX was lost when the bile was stored at 4°C for 20 days. The purified biliverdin IX appeared as a single spot with R_f value of 0.25-0.27 on thin layer chromatography (TLC) and one main peak on high performance liquid chromatography (HPLC) at 436 or 650 nm. When the biliverdin IX was subjected to enzymic reduction with highly purified biliverdin reductase, two clear isobestic points were seen, at 384 and 670 nm. When the products of the reaction with biliverdin IX were extracted in butanol after completion of the reaction, one absorbance peak was observed at 468 nm. The time course of the reduction of biliverdin IX to bilirubin IX catalyzed by biliverdin reductase depended on reduced pyridine nucleotide. The time course of the NADPH-dependent reaction is different from that of the reaction with NADH. In the reduction of biliverdin IX , per mole of biliverdin IX reduced or per mole of bilirubin IX formed 1 mole of reduced pyridine nucleotide was consumed in both the NADH and NADPH systems.     

Polyhalogenated biphenyls and phenobarbital: evaluation as inducers of drug metabolizing enzymes in the sheepshead, Archosargus probatocephalus

Several doses of Aroclor 1254 (polychlorinated biphenyl (PCB) mixture), Firemaster FF1 (polybrominated biphenyl (PBB) mixture), 2,2',4,4',5,5'-hexabromobiphenyl (HBB), 3,3',4,4',5,5'-hexachlorobiphenyl (HCB) and phenobarbital (PB) were administered to the marine fish sheepshead (Archosargus probatocephalus). The PCB and PBB mixtures caused induction of hepatic microsomal benzo[a]pyrene hydroxylase (AHH), 7-ethoxycoumarin O-deethylase (7-EC) and 7-ethoxyresorufin O-deethylase (ERF) activities, but not benzphetamine N-demethylase (BND), epoxide hydrolase (EH) or glutathione S-transferase (GSH-T) activities. This induction pattern is typical of that caused by polycyclic aromatic hydrocarbons (PAH) in fish and mammals or by tetrachlorodibenzo-p-dioxin (TCDD) in mammals. The extent of induction of AHH-activity and cytochrome P-450 content was higher when experiments were carried out in summer (water temperature 25 +/- 4 degrees C) than in winter (water temperature 11 +/- 3 degrees C). Firemaster FF1 (15 mg/kg) induced fish for at least 56 days in both summer and winter at which time the liver concentrations of PBB were in the ppm range. PCB concentrations in the ppm range have been found in fish from polluted lakes and seas, thus we may expect that environmental exposure to PCB is sufficient to induce hepatic mixed function oxidase (MFO) activities. The PCB isomer 3,3'4,4'5,5'-HCB, which induces the same spectrum of hepatic drug-metabolizing activities as TCDD and PAH in rats, had a broadly similar effect in the sheepshead. Another purified isomer, 2,2',4,4',5,5'-HBB, which induces the same enzymes as PB in rats, had no effect on drug-metabolizing activities in sheepshead. PB was also without effect on sheepshead hepatic drug-metabolizing enzymes, although a typical narcotic effect was produced in PB-treated sheepshead. Our studies provide further evidence that drug-metabolizing activities in fish liver are readily induced by chemicals like TCDD or PAH, but we fail to demonstrate induction after treatment of sheepshead with inducers of the PB type.     

Fluorescence modeling in a multicomponent system

An extensive fluorescence database for binary tyrosinetryptophan mixtures utilizing 280 nm excitation was collected. The database spanned three orders of magnitude (10(-6)M-10(-3)M) and covered all compositions within this range. A generalized model for describing the multicomponent fluorescence signals as a function of emission wavelength, excitation wavelength, and sample composition was derived. A geometric integral that contained all the geometric factors affecting fluorescence was introduced; thus the model was applicable to various configurations, including the three used in this study: an NADH probe, a backscatter laser-induced fluorescence setup, and a commercial spectroflurometer. A correction factor was proposed that allowed linearization of the fluorescence signals with respect to fluorophore concentrations. The effect of the water Raman on fluorescence spectra was also modeled. The model contains only two wavelength-dependent parameters for each of the components present in a sample, one specifying absorption of the excitation energy and the other specifying the species' fluorescence tendency. These wavelength-dependent parameters were correlated with polynomials. The average prediction error at each wavelength was 10-20%, a major portion of which was attributed to experimental uncertainties.     

Modelling of fish contamination with 90Sr in relation to the calcium concentrations in water

A set of data on the equilibrium concentration factors of 90Sr in fish in relation to the Ca concentrations in water was collected and analyzed. An empiric relationship was obtained on the basis of statistical processing of this dataset: CF(90Sr, Bq/kg w.w.) = 3940(1770-6110)/[Ca, mg/L]water. The range of observed data on the concentration factors of 90Sr in fish is from 20 to 4000 L/kg (about 200 times). The statistical relationship obtained in the paper allows one to reduce this uncertainty by more than 50 times. The formula adequately describes equilibrium concentration factors of 90Sr both in freshwater and in marine fish. A dynamics model approach is described for the cases of accidental contamination of water bodies, when the equilibrium approaches are not appropriate.     

DNA damage in eelpout (Zoarces viviparus) from Göteborg harbour

The relationship between DNA damage and the exposure of marine organisms to environmental contaminants was examined in the G?teborg harbour area. This research is part of a wider ecotoxicological study planned to evaluate the biological impact of chemical contamination in the River G?ta estuary, following a bunker oil (10-100 tonnes) spill occurred in June 2003. Here we present data on the DNA strand breaks derived using the comet assay and the presence of apoptotic cells using the diffusion assay in nucleated erythrocytes of the eelpout (Zoarces viviparus) from the study area and at a clean reference site. Polycyclic aromatic hydrocarbon metabolites were also analyzed in the bile of exposed fish. The results showed a high level of damaged DNA, paralleled by a peak in bile PAH metabolites, in fish from the most impacted site, 3 weeks after the oil spill. A significant recovery was observed in specimens from the spill site, 5 months later, but not in fish caught in the middle part of G?teborg harbour, which is chronically subjected to heavy chemical pollution. The levels of apoptic cells did not show any marked variations, but a significant recovery was observed in fish from the oil impacted site 5 months after the spill.