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Biliary 1-hydroxypyrene as a biomarker of exposure to polycyclic aromatic hydrocarbons in fish
Abstract
The aim of this study was the assessment of the Svitava and Svratka rivers contamination by polycyclic aromatic hydrocarbons (PAH) using 1-hydroxypyrene (1-OHP) concentrations in fish bile as a biomarker.
Levels of 1-OHP were determined by reverse phase HPLC with fluorescence detection. For valid assessment of bile accumulation levels, the 1-OHP concentration was normalized to the biliary protein content. The content of 1-OHP was correlated with the PAH content of river bottom sediments.
The highest level of 1-OHP in fish bile (136.1 ng.mg-1 protein) was found at the locality Rajhradice, which is situated downstream of Brno. Also the greatest level of sum of PAHs was found at this locality (17.1 microg.g-1 dry mass). Significant positive correlation (p< or =0.05) between the level of 1-OHP and sum of PAHs in sediment was found only in case of sediments collected in the same month as fish samples (in June).
Our results document that 1-OHP in fish bile is a suitable biochemical marker for the assessment of aquatic ecosystem contamination by PAHs.
... Levels of biliary 1-OHP were determined according to the method described previously by Blahova et al. (2008) [13]. Bile samples were deconjugated with an enzyme mixture of glucuronidase and arylsulphatase (37 ∘ C, 1 hour) and purified on solid phase extraction column (LiChrolut EN, Merck). ...
... Levels of biliary 1-OHP were determined according to the method described previously by Blahova et al. (2008) [13]. Bile samples were deconjugated with an enzyme mixture of glucuronidase and arylsulphatase (37 ∘ C, 1 hour) and purified on solid phase extraction column (LiChrolut EN, Merck). ...
... Based on the previous studies [3,11,19], we chose only one PAH metabolite which we analyzed in the chub bile. In general, many studies have identified 1-OHP as one of the most abundant compounds present in fish bile and this metabolite is regarded as the best general indicator of PAH exposure in fish [7,10,11,13]. Ruddock et al. [9] documented that 1-OHP accounts for up to 76% of all PAH metabolites in fish bile. 1-Hydroxypyrene is the main degradation product of pyrene-a widespread PAH generated by many pyrolytic and petrogenic industrial processes [4,7,10]. ...
This study investigated polycyclic aromatic hydrocarbons (PAH) pollution of selected rivers in the Czech Republic. Integrated evaluation was carried out using combination of chemical and biological monitoring, in which we measured content of 1-hydroxypyrene (1-OHP) in chub bile and priority PAH in water samples obtained by exposing the semipermeable membrane devices at each location. The concentrations of 1-OHP in bile samples and sum of priority PAH in water sampler ranged from 6.8 ng mg protein(-1) to 106.6 ng mg protein(-1) and from 5.2 ng L(-1) to 173.9 ng L(-1), respectively. The highest levels of biliary metabolite and PAH in water were measured at the Odra River (the Bohumín site), which is located in relatively heavily industrialized and polluted region. Statistically significant positive correlation between biliary 1-OHP and sum of PAH in water was also obtained (P < 0.01, r s = 0.806).
... Therefore, hydroxylated metabolites can be analyzed in bile and can be used as biomarkers for exposure to PAH in fish [6,7]. Many laboratory studies have demonstrated that the presence of biliary PAH metabolites is well correlated with the levels of exposure [8,9], and this trend has been corroborated in a number of field studies [10,11]. Analysis of PAH metabolites is effectively and frequently used in the biological monitoring of human exposure to PAH, especially in the working environment [12,13]. ...
... Therefore, sample preparation usually includes an enzymatic treatment for producing free hydroxy PAH. Separation by HPLC [11,161718 or GC [17] prior to fluorescence or mass spectrometry detection are the more frequently used methods for the identification and quantification of individual metabolites [17]. The main metabolite in fish bile is 1-hydroxpyrene (1-OHP), which contributes up to 76% of the sum of PAH metabolites [19]. ...
... Other metabolites, detected in considerably lower levels in fish bile, are 1-hydroxyphenanthrene, 1-hydroxychrysene and three metabolites of benzo(a)pyrene [5,14]. 1-Hydroxypyrene (Figure 1) is the predominant biotransformation product of pyrene, a widespread and common PAH that is generated by many pyrolytic and petrogenic industrial processes [11,18]. Urinary 1-OHP has been used frequently as a biomarker of human exposure to PAH, especially for worker in oil refineries, tar plants, aluminium plants and coal-burning facilities [20].Figure 1. Formula of 1-hydroxypyrene (C 16 H 10 O). ...
The aim of the present study was to assess aquatic contamination by polycyclic aromatic hydrocarbons (PAH), using the 1-hydroxypyrene (1-OHP) content in fish bile as a biochemical marker. A total of 71 chub (Leuciscus cephalus L.) were collected from seven locations on the Svitava and Svratka rivers in and around the industrial city of Brno, Czech Republic. The levels of 1-OHP were determined by reverse phase HPLC with fluorescence detection after deconjugation. Normalising the molar concentration of the biliary 1-OHP to the biliary protein content reduced sample variation. The content of 1-OHP was correlated with the PAH level in bottom sediment and semi-permeable membrane devices (SPMD), which was analyzed by a combination of HPLC/FLD and GC/MS methods. The highest mean values of 1-OHP were found in fish caught at the Svratka River at locations Modřice (169.2 ± 99.7 ng · mg(-1) protein) and Rajhradice (152.2 ± 79.7 ng · mg(-1) protein), which are located downstream from Brno. These values were significantly (P < 0.05) higher than those obtained from localities Kníničky (98.4 ± 66.1 ng · mg(-1) protein) and Bílovice nad Svitavou (64.1 ± 31.4 ng · mg(-1) protein). The lowest contents of PAH in sediment and SPMD were found at location Kníničky (1.5 mg · kg(-1) dry mass and 19.4 ng · L(-1), respectively). The highest contents of PAH in sediment and SPMD were found in Rajhradice (26.0 mg · kg(-1) dry mass) and Svitava before junction (65.4 ng · L(-1)), respectively. A Spearman correlation test was applied to determine the relationship between biliary 1-OHP and the sum of PAH in sediment and SPMD. A positive, but no statistically significant correlation was found. The main impact sources of elevated level of PAHs in sites located downstream from Brno are most probably intensive industrial and agricultural activities and domestic waste.
... Monitoring of aquatic pollution may also be conducted using liver biotransformation enzymes (phase I and II) (Van der Oost et al. 2003; Randak et al. 2006; Havelkova et al. 2008). The determination of cytochrome P450 (CYP450) content and ...
... This is in agreement with the field study by Rodas-Ortiz et al. (2008), which confirmed that the exposure to HCB and benzo(a)pyrene caused VTG induction in male Nile tilapia (Oreochromis niloticus ). Similarly, Havelkova et al. (2008) demonstrated that the highest VTG concentrations in male chub caught in the Tichá Orlice River (Czech Republic) were found in the Králíky site. Chemical analyses of sediment and muscle tissue samples verified higher levels of PCBs, HCB, DDT and PAHs at this site. ...
... In fish, the activity of these enzymes may be induced or inhibited upon exposure to xenobiotics (Van der Oost et al. 2003). The balance between phase I activation reactions and phase II conjugation pathways can underlie the toxicity of many organic xenobiotics (Havelkova et al. 2008). Although most studies did not demostrate any significant alterations in GST activity, an increase in hepatic GST activity after the exposure of fish to several pollutants has been reported in some studies (Van der Oost et al. 2003). ...
The study was designed to assess the pollution of the Svitava and Svratka rivers in and around the industrial city of Brno (Czech Republic) by persistent organic pollutants using selected biochemical markers in chub.
Levels of selected biochemical markers were measured in liver and plasma samples of chub. The concentrations of persistent organic pollutants (POPs) were determined in bottom sediment, semi-permeable membrane devices (SPMDs) and muscle samples, and consequently used for correlation with biochemical markers.
Significant alterations (p < 0.05) in some biochemical markers were observed and associated with combined exposure to pollutants. The highest levels of pollutants were found at sites situated downstream from Brno. The most widespread changes were identified in the function of phase I detoxifying enzymes. Significant positive correlations were observed in cytochrome P450 content and DDT concentration in the semi-permeable membrane device (p = 0.019, rs = 0.886), and between ethoxyresorufin-O-deethylase activity and content of DDT (p = 0.041, rs = 0.352) and polychlorinated biphenyls (p = 0.034, rs = 0.365) in muscle tissues of indicator fish.
The results presented in our study indicate the highest contamination of sites situated downstream from Brno, where the intensive industrial and agricultural activities as well as domestic waste and sewage most probably comprise the main impact sources of the enhanced level of pollutants and some biochemical markers in fish.
... Biliary compounds have been quantified for monitoring of recent and ongoing exposures to many xenobiotics including PAHs [e.g., [2][3][4][5][6][7]. After biotransformation and conjugation with hydrophilic compounds, PAH metabolites and residual parent compounds accumulate in the gall bladder before being excreted via the intestine. ...
... Bile was drawn by needle through the exposed gall bladder and samples were stored at -85 °C for later analysis. Determination of biliary PAH metabolites (1-OHP, 2-naphtol, 1-hydroxyphenanthrene) was conducted by the reverse phase HPLC/FLD method described by Blahova et al. (2008). For data analyses, the metabolite concentrations were normalized to protein content determined using bicinchoninic acid. ...
The aim of the study was to assess polycyclic aromatic hydrocarbon (PAH) contamination on the Bilina River (the Czech Republic) by measurement of three PAH biliary metabolites in fish and 16 PAHs in passive samplers. A total of sixty-one fish were collected; the indicator species were chub (Leuciscus cephalus L.; n = 25), roach (Rutilus rutilus L.; n = 17) and brown trout (Salmo trutta m. fario L.; n = 19). Three biliary PAH metabolites were measured: 1-hydroxypyrene (1-OHP), 2-naphtol, and 1-hydroxyphenanthrene, but only 1-OHP was detected in all fish. The highest median value of 32.3 ng.mg(-1) of 1-OHP was found at Usti nad Labem, while the lowest median value of 27.6 ng.mg(-1) was found in the control site, Brezenec. No significant differences (P < 0.05) among the sites were found. The highest concentration of PAH was detected in Litvinov-Zaluzi (172.0 ng.l(-1)) and the lowest concentration (7.9 ng.l(-1)) was detected in Brezenec. A positive, but non-significant correlation (r(s) = 0.8) was confirmed between biliary 1-OHP and total PAH in passive samplers. These resufts indicate the highest pollution in the middle stretches of the Bilina River, especially at Litvinov-Zaluzi. We confirmed 1-OHP as the most prevalent PAH biliary metabolite in fish that could be used as a biomarker for assessment of PAH pollution of the aquatic ecosystem. The main importance of the present study is in the combination of biochemical and chemical monitoring that provides complex evaluation of aquatic environment contamination. It was the first study on the Bilina River, in which the assessment of PAH contamination in the aquatic ecosystem was realized using combination of biotic and abiotic monitoring.
... OH-phenanthrene) often results from exposure to PAHs from oil spills (Krahn et al., 1986). For the larger PAH metabolites like OH-pyrene and 3-OH-BaP, containing four and five rings respectively, incineration processes are often the main source (Ariese et al., 2004;Blahova et al., 2008). However, due to the rapid metabolism of many PAHs in fish, the concentrations of their metabolites in bile may cause underestimation of the exposure (Whyte et al., 2000). ...
... polycyclic aromatic hydrocarbons (PAH), chlorinated phenols and estrogenic substances (Pettersson et al., 2006). The analysis of bile metabolites is considered a convenient and relatively rapid method for monitoring PAH contamination (Ruddock et al., 2003; Blahova et al., 2008). Bile from rainbow trout caged downstream of sewage treatment plants in Sweden showed to have contaminants (estrogens , bisphenol A and 4-nonylphenol) at levels that could be linked to their water concentrations. ...
... Biomarkers are considered “early warning” tools in environmental assessment (McCarthy and Shugard, 1990) and provide qualitative measure of exposure to toxic chemicals or environmental stresses (Sarkar et al., 2006). Currently, aquatic ecosystems are monitored in long-term studies by measuring concentrations of biochemical markers such as cytochrom P450 (Havelkova et al., 2008), ethoxyresorufin-O-deethylase (Randak et al., 2009), vitellogenin or 1-hydroxypyrene (Blahova et al., 2008) in fish to assess environmental pollution by organic pollutants mainly polycyclic aromatic hydrocarbons (PAHY) and polychlorinated biphenyls (PCB). Several studies have concluded that exposure to metal such as Cd cause increase of thiol compounds in living body (Thomas et al., 1982; Klaverkamp and Duncan, 1987; Kägi and Schäffer, 1988; Olsson et al., 1998; Schlenk and Rice, 1998; Santovito et al., 2000; Kovarova et al., 2009). ...
Can thiol compounds be used as biomarkers of aquatic ecosystem contamination by cadmium?
Due to anthropogenic activities, heavy metals still represent a threat for various trophic levels. If aquatic animals are exposed to heavy metals we can obviously observe considerable toxicity. It is well known that an organism affected by cadmium (Cd) synthesize low molecular mass thiol compounds rich in cysteine (Cys), such as metallothioneins (MT) and glutathione (GSH/GSSG). The aim of this study was to summarize the effect of Cd on level of thiol compounds in aquatic organisms, and evaluate that the concentrations of thiol compounds are effective indicators of Cd water pollution and explain their potential use in biomonitoring applications.
The aim of the present study was to evaluate polycyclic aromatic hydrocarbon (PAH) contamination of the major rivers in the Czech Republic using 1-hydroxypyrene (1-OHP) content in chub bile as a biomarker. The highest concentration of 1-OHP was found in the Otava River at Topělec (80.5 ng mg protein-1); the lowest content of 1-OHP was found in the Vltava at Zelčín (9.6 ng mg protein-1). At all sites, bottom sediment samples were collected and analyzed for PAH content. The PAH content ranged between 1.2 and 15.2 mg kg dry mass-1 at all sites. Statistically significant positive correlations (p < 0.05) between biliary 1-OHP and sediment PAH content were found. Correlation coefficients for total and individual priority PAHs ranged from 0.63 to 0.77.
The aim was to evaluate the extent of pollution of the Svratka River with organochlorinated pollutants using the chub (Leuciscus cephalus) as a suitable bioindicator of the river contamination. The sum of 7 PCB congeners was found in range of 7.6-93.8 ng/g (with median of 31.7 ng/g) wet weight (ww), DDTs of 2.9-93.8 (29.9) ng/g ww, HCHs isomers of 0.1-5.3 (1.0) ng/g ww, HCB of 0.3-8.4 (2.2) ng/g ww and OCS of <0.1-0.5 (0.1) ng/g ww. Our results confirm predominance of metabolite DDE and higher-chlorinated PCB congeners and are comparable with similar studies and findings within the Czech Republic.
Formaldehyde, sodium chloride and potassium permanganate belong to the commonly used substances for fish treatment. The aim is to define and compare their acute toxicity and therapeutic index between two fish species - Danio rerio and Poecilia reticulata.
To determine acute toxicity of these compounds, the semistatic method was implemented in compliance with the OECD No. 203 (Fish acute toxicity test). In each test series, 4 acute toxicity tests were performed. The results were subjected to the probit analysis to determine the 96hLC50 values. Therapeutic index (TI) was calculated with respect to short-term LC50 and effective therapeutic concentrations commonly used for fish treatment.
The mean 96hLC50 values for sodium chloride were 21.69 +/- 0.92 g l-1 (TI = 1.4) for P. reticulata and 10.39 +/- 0.12 g l-1 (TI = 1.0) for D. rerio. The acute toxicity of formaldehyde for D. rerio expressed as 96LC50 was 0.12 +/- 0.003 ml l-1 (TI = 5.24) and for P. reticulata 0.1 +/- 0.003 ml l-1 (TI = 4.9). The acute toxicity of potassium permanganate for D. rerio expressed as 96LC50 was 1.25 +/- 0.15 mg l-1(TI = 1.5) and for P. reticulata 1.43 +/- 0.05 mg l-1 (TI = 2).
P. reticulata showed significantly higher (p < 0.05) tolerance to sodium chloride than D. rerio, whereas D. rerio showed significantly higher (p < 0.05) tolerance to formaldehyde than P. reticulata. The acute toxicity of potassium permanganate was comparable for both fish species. Calculated therapeutic indexes of all tested substances were low; therefore it is important to conduct preliminary tolerance tests before application of treatment baths.
This study assessed the subchronic effects of a triazine compound, simazine, on common carp (Cyprinus carpio L.) though, via by means of biometric, biochemical, hematological, and histological examination.
One-year-old fish were exposed to simazine at four concentrations, 0.06, (reported concentration in Czech rivers), 4, 20, and 50 microg L-1 for 28 days and compared to carp in a non-treated control group.
Exposure of fish to simazine at 0.06 microg L-1 had no effect on measured parameters. However, exposure to simazine at the concentrations of 4, 20, 50 microg L-1 showed significant differences in biochemical, hematological, and histopathological profiles of fish compared to controls.
Our data suggest that simazine in the recorded environmental concentration 0.06 microg L-1 had no effect on common carp. Subchronic exposure to 4, 20, and 50 microg L-1 of simazine was associated with alterations in biochemical and hematological indices and in fish organ tissues.
A controlled laboratory study was carried out to quantify vitellogenin (VTG) concentrations in a common cyprinid freshwater fish, the chub (Leuciscus cephalus L.), exposed to steroid hormones.
The effect of 17betaestradiol, testosterone and testosterone-estradiol mixture was investigated on vitellogenin induction. Gonad status was also determined.
Oral exposure to estradiol and a testosterone-estradiol mixture increased (p < 0.01) blood plasma concentrations of VTG in blood plasma of both sexes. The testosterone-estradiol mixture had a negative effect on the investigated chub gonads. The effects were signified by histological changes when compared to control fish.
Our results showed a significant VTG increase in blood plasma of both sexes, indicating that vitellogenic response in the chub is sensitive to steroid hormones.
The present study was aimed at evaluation of the response of Japanese quails to cyanobacterial biomass administered in feed using biochemical profiles and parameters of reproduction.
Effects of cyanobacterial biomass were studied according to the OECD 206 Guideline on Avian Reproduction Toxicity. A total of 16 control and 16 experimental pairs (32 males and 32 females) were analyzed. The chronic exposure of parent birds lasted eight weeks with the daily sum of 61.62 microg MCs including 26.54 microg MC-RR, 7.62 microg MC-YR and 27.39 microg MC-LR.
There was no mortality both in control and cyanobacterial-biomass-exposed adults during the present study. Nor did the birds show any clinical signs of intoxication. Lactate dehydrogenase activity was increased about three-fold in exposed birds. No other biochemical parameters were showing significant differences. A total of 824 and 821 eggs were laid by control and exposed birds, respectively, during the eight-week study period. Eggs laid by cyanobacterial-biomass-exposed hens had lower weight than in controls (11.99+/-1.13g and 12.40+/-1.27g, respectively; p<0.01). Egg viability, hatchability, and the effect of hatching in control and experimental birds were 79.6+/-9.3 and 86.8+/-8.2% (p<0.05), 83.2+/-12.6 and 90.1+/-9.3%, and 65.2+/-17.7 and 77.7+/-15.2% (p<0.05), respectively. There was also a statistically significant difference in the number of 14-day old survivors per hen per day in control and experimental birds (0.38+/-0.02 and 0.43+/-0.01 %, respectively).
The lower weight of eggs produced by exposed parental hens was not reflected in their biological quality. On the contrary, reproductive parameters in cyanobacterial-biomass-exposed birds were better than in the control group. It might be hypothesized that compounds of hormonal activity could be present in the complex cyanobacterial biomass. However, further research into this issue is necessary.
The aim of this study was to assess the effect of metals, with special attention of cadmium (Cd), content of the Svitava and Svratka rivers (Czech Republic) on levels of thiol compounds such as metallothioneins (MT), glutathione (GSH/GSSG), and cysteine (Cys) concentrations in chub (Leuciscus cephalus L.) liver and their use as biomarkers.
Levels of thiol compounds in fish liver were determined by the differential pulse voltammetry Brdicka reaction. Cadmium content in L. cephalus liver was measured by atomic absorption spectometry (AAS) with electrothermal atomization. The contents of thiols and Cd were correlated with the Cd and metals content of river bottom sediments.
Results were inconsistent with those of experimental studies. Correlations between liver Cd content and MT, Cys, GSSG were not significant in any locality (p > 0.05). Cd liver content was negatively (p< 0.05) correlated with GSH at one (Zidlochovice) of the seven tested sites. GSH and GSSG correlated negatively in five localities (p > 0.05). Correlations between Cd in sediment and Cys and GSSG (p > 0.05), and MT was negative (p< 0.05). The relationship between sum of metals value and MT was negative (p< 0.05), similarly with GSSG (p > 0.05).
Many factors influence content of MT, GSH, and GSSG, and data for Cd were equivocal. Monitoring the aquatic ecosystem to identify metal content using thiol compounds in fish liver as biomarkers is complex. More relevant data including laboratory experiments will be required to assess most of the factors influencing thiols and their relevance to Cd contamination.
Havelková M., Z. Svobodová, J. Kolářová, J. Krijt, D. Némethová, J. Jarkovský, R. Pospíšil: Organic Pollutant Contamination of the River Tichá Orlice as Assessed by Biochemical Markers. Acta Vet. Brno 2008, 77: 133-141. This study used biochemical markers to assess contamination at two contaminated sites (Králíky and Lichkov) and one control site (Červená Voda) on the River Tichá Orlice, a left-side tributary of the River Elbe. The brown trout (Salmo trutta fario) was selected as an indicator species. Enzymes of the first stage of xenobiotic conversion, namely cytochrome P450 (CYP 450) and ethoxyresorufin- O-deethylase (EROD) in the liver were selected as biochemical markers. Blood plasma vitellogenin concentrations were used to evaluate xenoestrogenic effects of contamination. Results were compared with the most important inductors of these markers, i.e. with organic pollutants (PCB, HCH, HCB, OCS and DDT and their metabolites in fish muscle and with PAH concentrations in bottom sediments). The highest contamination with organic pollutants was at Králíky, and this was reflected in increased cytochrome P450, EROD activity and vitellogenin concentrations. Significant differences were demonstrated in EROD activity and vitellogenin concentrations between Králíky and Červená Voda (P < 0.001). At the most contaminated site (Králíky), a significant negative correlation (rs = -0.964) between EROD activity and vitellogenin concentrations was demonstrated. This relationship was discussed from the point of view of a possible induction or inhibition of the assessed biomarkers at persistently highly contaminated sites. Cytochrome P450, EROD, vitellogenin, Salmo trutta fario, PCB, river contamination
Abstract ·iroká Z., J. Krijt, T. Randák, Z. Svobodová, G. Pe‰ková, J. Fuksa, J. Haj‰lová, J. Jarkovsk˘, M. Jánská: Organic Pollutant Contamination of the River Elbe as Assessed by Biochemical Markers. Acta Vet Brno 2005, 74: 293-303. The aim of the ,study was to assess contamination ,of the ,River Elbe basin using selected biochemical markers. Biochemical markers selected were enzymes of the first stage of xenobiotic transformation, namely cytochrome P450 (CYP 450) and ethoxyresorufin-O-deethylase (EROD). The results were correlated with the most important inducers of the enzymes, i.e. polychlorinated biphenyls (PCB) concentrations in muscle tissue of fish, polyaromatic hydrocarbons (PAH) values in bottom sediments and 1-hydroxypyrene (1-OHPY) values in fish bile (terminal metabolite of PAH, or, rather, of one of them, i.e. pyrene), which were determined during the chemical monitoring of the River Elbe basin. The indicator species selected was chub (Leuciscus cephalus L.), which was captured at ten locations in the River Elbe basin. A comparison between the EROD activity and the CYP 450 content along the longitudinal profile of the Elbe showed,a significant correlation at the level of significance of p ,). In Blanice (control location), EROD activity was significantly lower than in any ,of the ,other locations studied (p < 0.05). The study failed to produce ,an unambiguous,proof of any,correlations between detoxification enzyme,activity (CYP 450 and EROD) in the liver and their two important inducers (PCB and PAHs). The possibility that other
Samples of sediment and eel taken from six sites in Amsterdam with different levels of water pollution were analyzed for 16 parental PAHs In addition, biliary PAH metabolites and hepatic PAH-DNA adducts were determined in the eel to evaluate biomomtoring techniques for PAH exposure There was a clear difference between PAH profiles in sediments and eel Mainly two- and three-ring PAHs were detected in eel, whereas four-ring PAHs predominated in the sediments Because PAH bioaccumulation was highest in eel from the reference sites, tissue levels of the parental PAH are probably not the most accurate monitor of PAH exposure in fish An elevated excretion of 1 OH pyrene (determined by synchronous scan fluores cence) was observed in the bile of fish from three of the four polluted sites, indicating that this parameter may be used as a biomarker for PAH exposure A significant increase in PAH-DNA adduct levels (determined by 32P postlabeling) was observed in the liver of eel from all polluted sites Therefore, this parameter seems to be a sensitive biomarker for exposure to mutagenic and carcinogenic PAHs
During field campaigns of the BEEP project (Biological Effects of Environmental Pollution in Marine Coastal Ecosystems) in 2001-2002, metabolites of polycyclic aromatic hydrocarbons (PAHs) were determined in bile samples from three fish species, flounder (Platichthys flesus), perch (Perca fluviatilis) and eelpout (Zoarces viviparus), from four separate areas in the Baltic Sea. Two determination methods were applied: fixed wavelength fluorescence (FF) for pyrene-type metabolites and high-performance liquid chromatography with fluorescence detection (HPLC). There was a good correlation between the FF method and 1-OH pyrene determined by HPLC. Normalisation of the FF data for absorbance at 380 nm or bile protein concentrations greatly increased variance in one third and decreased it in two thirds of the cases and resulted in a loss of significant differences (protein normalisation) between the sampling stations, but normalisation of the HPLC data had little effect on the results. The biliary PAH metabolite content was usually higher in males than in females. In perch and eelpout the biliary PAH contents were at similar levels, whereas in flounder the levels were lower. The sampling areas arranged in decreasing order of biliary PAH contents were: Wismar Bay > Gulf of Gdansk > Lithuanian coast > Kvadofjärden (reference area). It is concluded that FF with un-normalised data is a reliable and simple method for monitoring purposes and only one sex of a selected species should be used.
To study whether the urinary 1-hydroxypyrene (1-OHP) could be the biomarker of atmospheric PAHs, a small-scale pilot study was carried out on the relation of 1-OHP vs PAHs with the traffic policemen in Beijing of smokers and nonsmokers to be subgroups in both the exposure and control groups. Both the PAHs and 1-OHP were analyzed with high performance liquid chromatography (HPLC). The ambient concentrations of PAHs were different at the different sites (the average sum of PAHs (TPAH) were 12.36, 16.27, 18.37 ng/m(3) at the suburban residential, police station and high traffic area, respectively.), but considerably lower than the personal-exposure concentrations (the average TPAH were 65.84 and 47.28 ng/m(3) for patrol cars and inspection station, respectively). Pyrene was correlated well with BaP and the summed PAHs (TPAH), with the correlation coefficients (R) of 0.79, 0.87 for ambient level and 0.92, 0.96 for personal exposure, respectively. The average of 1-hydroxypyrene of smokers and nonsmokers were 0.39, 0.15 mumol/mol creatinine in control group and 0.57, 0.33 mumol/mol creatinine in exposure group, respectively. The better correlation of pyrene to BaP and TPAH especially for personal exposure samples indicated that the probability of urinary 1-hydroxypyrene, the metabolite of pyrene, to be the biomarker of total PAH. Nonsmokers in the exposure and control groups had indistinguishable levels of 1-OHP, presumably because the ambient levels of pyrene were so similar (the average were 3.25, 3.20 ng/m(3) at the police station and high traffic area, respectively.). Smokers in the control group had significantly higher 1-OHP than that of the nonsmokers, but showed indistinguishable differences in the exposure group. These results suggested that urinary 1-OHP could be a biomarker of PAHs only when the level of PAHs was at a relatively higher level. Smoking as an important influencing factor need to be controlled carefully.
Oocyte maturation and yolk incorporation (vitellogenesis) is a hormonally controlled and regulated process. In fish, the primarily exogenous synthesis of vitellogenin is initiated by gonadotropins and regulated by estrogens. Vitellogenin is a species-specific protein synthesized by hepatocytes, released into the bloodstream and actively sequestered by maturing oocytes. A large amount of research has been directed at assessing the effects of polycyclic aromatic hydrocarbons (PAHs) on different phases of vitellogenesis in fish and identifying their mechanisms of action. PAHs have been found to have a deleterious effect on the vitellogenesis of fish from feral populations as well as in laboratory experiments. This is of particular concern as any of the stages of the vitellogenic cycle could be affected which would result in harmful sub-lethal effects with trans-generational consequences. The reported effects include reduction in circulating hormones and plasma vitellogenin, estrogenic and antiestrogenic effects, retardation of oocyte maturation and reduction of reproductive success. However, the lack of agreement between some of the results indicates that some of the mechanisms at play are not yet completely understood and require further investigation. This review attempts to summarize the present understanding of the effects of PAHs on vitellogenesis in fish.
Polycyclic Aromatic Hydrocarbons (PAH) are common environmental pollutants that have been linked to cancerous lesions in bottom fish. In this research, the feasibility of using nitrate as an alternative electron acceptor to stimulate PAH-degradation in anoxic marine sediment was investigated. PAH could be degraded under denitrifying conditions as long as other nutrients were not limiting. The half lives of low molecular weight PAH ranged from approximately 33–88 days. Degradation of high molecular weight PAH was slower, or not observed. Half lives ranged from 143-812 days. Nitrate may be applied to sediments in situ or used in bioreactors to reduce the cost of bioremediation operations ex situ.
Since little is known about the effects of contaminants on Antarctic organisms, the effects of polynuclear aromatic hydrocarbons (PAH) on Antarctic fish were evaluated. Fish captured near Palmer Station on the Antarctic Peninsula exhibited induced ethoxyresorufin O-deethylase (EROD) activities and elevated concentrations of biliary PAH metabolites compared to fish from control sites. Naphthalene and phenanthrene PAH metabolite levels were significantly higher in the bile of fish captured near McMurdo Station than in fish from remote sites in McMurdo Sound. Laboratory experiments were conducted in which Notothenia gibberifrons were treated with benzo[a]pyrene (BaP) and diesel fuel Arctic (DFA). Although DFA is composed primarily of 2- and 3-ring PAH which are not known to be potent CYPIA inducers, the maximal hepatic EROD activity of DFA-treated fish was approximately 80% of that observed in BaP-treated fish. Additionally, 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were determined for hepatic extracts of laboratory-dosed and field-captured fish using rat hepatoma H4IIE cell bioassays. The TEQ values of H4IIE cells dosed with hepatic extracts of DFA-treated fish correlated more closely with hepatic concentrations of 3-ring and >3-ring PAH than with 2-ring and PAH concentrations. However, bioassay-derived TEQs were higher than expected based on the measured levels of 3-ring PAH in the hepatic extracts of DFA-treated fish. The TEQs for hepatic tissue extracts of BaP-treated fish paralleled tissue concentrations of PAH. The TEQs for field captured fish were significantly lower than those derived from dosed fish extracts.
The uptake of polycyclic aromatic hydrocarbons (PAHs) by fish can be determined by screening the gall bladder bile for PAH metabolites. Conjugated 1-hydroxy pyrene is a major metabolite in bile of fish exposed to PAH polluted sediment. Synchronous fluorescence spectrometry is presented as a rapid screening technique for the determination of this compound; the complete analysis takes only 2–3 min. HPLC with fluorescence detection was used to validate the assay. Calibration methods, using either free 1-hydroxy pyrene or the pyrene-1-glucuronide conjugate as a reference standard, were investigated. The technique was applied to a mesocosm study in which the uptake of PAHs by flounder (Platichthys flesus) from polluted sediment was studied. Direct contact with the sediment proved the most important factor; the uptake through water phase or diet was less significant. The usefulness of 1-hydroxy pyrene analysis in bile as a monitoring tool for PAH exposure is discussed.
Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
Polycyclic aromatic hydrocarbons (PAHs) in both sediment and brown bullhead catfish tissues from the Black River in Lorain County, Ohio, declined by 65% and 93%, respectively, between 1980 and 1982. Sediment PAHs declined an additional 99% by 1987, coincident with the closure of a coking facility in 1983. Contemporaneously, liver cancer in 3- to 4-year-old brown bullheads declined to about one-quarter the 1982 frequency (10% versus 39%) by 1987, while the percentage of livers without any proliferative lesions doubled (42% versus 20%). These changes were significant within age group. Our data affirm a cause-and-effect relationship between PAH exposure and liver cancer in wild fish. The data also support the efficacy of natural, unassisted remediation once the source of the pollution is eliminated.
A total of 94 European eels (Anguilla anguilla) were collected from five estuaries in the UK. The deconjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) in the bile of the eels were separated using HPLC. Six PAH metabolites were identified: 1-hydroxy (1-OH) metabolites of phenanthrene, pyrene and chrysene; and the 1-OH, 3-OH and 7,8 dihydrodiol metabolites of benzo[a]pyrene (BaP). The mean concentration of the six metabolites was greatest in eels from the Tyne (49 microM) followed by the Wear (33 microM), Tees (19 microM), Thames (4 microM) and Severn (2 microM) estuaries. Although 1-OH pyrene was always the dominant compound, there were significant differences (P<0.05) between sites and between estuaries for some metabolites. Normalising the molar concentration of the bile metabolites to the bile biliverdin absorbance reduced sample variation. When the metabolites identified were each expressed as a percentage of the total detected, the metabolite profile was characteristic for each estuary.
Xenoestrogenic potential of propylparaben (PP), one of the most commonly used preservatives in drugs, cosmetics and food, was investigated in vivo using zebrafish (Danio rerio).
Juvenile zebrafish (20 days post hatching) were exposed to three different concentrations of propylparaben (PP) dissolved in ethanol and added into the water. After 20 days of exposure the fish were euthanized and vitellogenin concentrations in their whole body homogenates were measured by the method of direct sandwich ELISA. Simultaneously, vitellogenin concentrations in either fish from the control group (exposed to solvent without the substance tested) and in fish from the positive control group (exposed to natural estrogen 17beta-estradiol) were measured.
Vitellogenin concentration in whole body homogenates of control fish was 400 (396-540) ng/ml(-1) (geometric mean (95% CI)). Zebrafish exposure to propylparaben at the concentrations of 0.1; 0.4 and 0.9 mg/l(-1) elicited statistically significant decline (P<0.001) of vitellogenin production, i.e. geometric means of vitellogenin concentrations in whole body homogenates were 240 (186-311); 218 (175-270) and 270 (234-311) ng/ml(-1), respectively. Conversely, the geometric mean of vitellogenin concentration in whole body homogenates of zebrafish exposed to 100 ng/ml(-1) of 17beta-estradiol (positive control) was significantly higher (P<0.001) than values in all other groups, i.e. 35,553 (16,860-74,968) ng/ml(-1).
Our results suggest an antiestrogenic potential of propylparaben tested in vivo in juvenile zebrafish (Danio rerio). The estrogenic effect of 17beta-estradiol was confirmed.
The detoxifying metabolism of a potent rodent carcinogen, 2-nitroanisole (2-NA) by human, rabbit and rat cytochromes P450 (P450) was investigated. Comparison between P450s of experimental animals and humans is essential for the extrapolation of animal carcinogenicity data to the human situation and to assess health risk.
HPLC with UV detection was employed for the separation and characterization of 2-NA metabolites formed by hepatic microsomes, human recombinant P450s and purified rat and rabbit P450s.
An O-demethylated metabolite of 2-NA, 2-nitrophenol (2-NP), and two oxidation products of this metabolite [2,5-dihydroxynitrobenzene (2,5-DNB) and 2,6-dihydroxynitrobenzene (2,6-DNB)] were generated by microsomes and P450s from the species investigated, but at different levels. All the metabolites are detoxication products. 2-NP is the major metabolite generated by rabbit and rat microsomes, but 2,5-DNB is the predominant product in human microsomes. Using human recombinant P450s and purified rodent P450s, we found that human P450 2E1, 1A1 and 2B6 as well as orthologous animal P450s were the most efficient enzymes oxidizing 2-NA to 2-NP, while P450 2E1 and 1A1 were the most effective in the formation of 2,5-DNB and 2,6-DNB. In human hepatic microsomes, 2-NA was oxidized mainly by P4502E1. 2-NA and its reductive metabolite o-anisidine induced rat hepatic and renal P450 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1), thus modifying their own detoxication and/or activation pathways.
The data demonstrated the participation of orthologous P450s in 2-NA oxidation by all species and indicated that the rat and rabbit might serve as suitable models to mimic 2-NA oxidation in man.