Effects of waterborne nitrite onphase I–II biotransformation in channel catfish(Ictalurus punctatus)

The effects of waterborne nitrite (3 mg/l NO₂) on channel catfish were studied to evaluate changes in hematological parameters and phase I–II biotransformation in liver slices. Nitrite-exposed fish had significantly higher methemoglobin, blood and liver nitrite, and significantly lower pO₂ than control fish. Total phase I-mediated metabolism of 7-ethoxycoumarin (EC) was not altered in nitrite-exposed fish compared with control fish (291±43 and 312±20 pmol/mg/h, respectively). However, phase II glucuronosyltransferase-mediated metabolism of 7-hydroxycoumarin (HC), both as a phase I metabolite of EC and as a parent substrate, was elevated in nitrite-exposed fish (204±17 and 1007±103 pmol/mg/h, respectively) as compared to control fish (149±14 and 735±87 pmol/mg/h) (P<0.05). Sulfotransferase-mediated metabolism of HC (as a metabolite of EC and as a parent substrate) was not notably altered in nitrite-exposed fish (95±16 and 617±33 pmol/mg protein/h, respectively) as compared with control fish (118±24 and 575±55 pmol/mg/h, respectively). These studies indicate that in vivo nitrite exposure and associated changes in hematological parameters do not appear to affect hepatic phase I EC biotransformation in channel catfish. However, subtle but significant changes in phase II glucuronidation, but not sulfation activity, were observed. The mechanism of these alterations is unclear. However, the data suggest that environmentally realistic concentrations of nitrite may affect the dynamics of conjugative metabolism in exposed fish.     

Effects of waterborne nitrite on phase I-II biotransformation in channel catfish (Ictalurus punctatus).

The effects of waterborne nitrite (3 mg/l NO2) on channel catfish were studied to evaluate changes in hematological parameters and phase I-II biotransformation in liver slices. Nitrite-exposed fish had significantly higher methemoglobin, blood and liver nitrite, and significantly lower pO2 than control fish. Total phase I-mediated metabolism of 7-ethoxycoumarin (EC) was not altered in nitrite-exposed fish compared with control fish (291 +/- 43 and 312 +/- 20 pmol/mg/h, respectively). However, phase II glucuronosyltransferase-mediated metabolism of 7-hydroxycoumarin (HC), both as a phase I metabolite of EC and as a parent substrate, was elevated in nitrite-exposed fish (204 +/- 17 and 1007 +/- 103 pmol/mg/h, respectively) as compared to control fish (149 +/- 14 and 735 +/- 87 pmol/mg/h) (P < 0.05). Sulfotransferase-mediated metabolism of HC (as a metabolite of EC and as a parent substrate) was not notably altered in nitrite-exposed fish (95 +/- 16 and 617 +/- 33 pmol/mg protein/h, respectively) as compared with control fish (118 +/- 24 and 575 +/- 55 pmol/mg/h, respectively). These studies indicate that in vivo nitrite exposure and associated changes in hematological parameters do not appear to affect hepatic phase I EC biotransformation in channel catfish. However, subtle but significant changes in phase II glucuronidation, but not sulfation activity, were observed. The mechanism of these alterations is unclear. However, the data suggest that environmentally realistic concentrations of nitrite may affect the dynamics of conjugative metabolism in exposed fish.     

Induction of Cyp1A in Japanese medaka exposed to carbonaceous pitches via water

The objective of these studies was to characterize hepatic Cyp1A induction by complex carbonaceous pitches both in Japanese medaka exposed via aquarium water and in cultured fish liver cells. Carbonaceous pitches were extracted with DMSO and added to aquaria water of medaka or to cultured medaka or trout liver cells, and CyplA induction was assessed by EROD assay. When medaka were exposed to different carbonaceous pitches, EROD activity was induced to different extents, and increased EROD induction was associated with increasing temperature of hydrogenation. EROD induction in cultured fish liver cells was somewhat less sensitive than that observed in vivo. These studies indicate CyplA induction in medaka can be used to detect compounds, such as polyaromatic hydrocarbons, in complex samples and could be used as a biomarker for the presence of these compounds in the environment.     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17β-estradiol to 19±11%, 27±7%, 63±20% and 61±12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17β-estradiol and 17α-hydroxyprogesterone levels (r=−0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

Glutathione-mediated chlorothalonil detoxification in channel catfish gills

Maintenance of the glutathione (GSH) pathway is critical in guarding against the toxic effects of a variety of pollutants. However, despite their importance as an initial line of defense against waterborne toxicants, little is known regarding the role of fish gills in GSH-mediated xenobiotic metabolism. In these studies, the role of channel catfish gills in the detoxification of 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil), an electrophilic fungicide, was examined. Chlorothalonil was metabolized in vitro by gill cytosolic and microsomal glutathione S-transferases (GSTs). Chlorothalonil metabolism by cytosolic fructions was reduced markedly when GSH was omitted from reaction mixtures. Microsomal metabolism of chlorothalonil did not occur in the absence of GSH and in the presence of an NADPH-regenerating system lacking GSH. Channel catfish exposed in vivo to chlorothalonil had increased gill GSH and cysteine concentrations after 72 h exposure, and increased gill gamma-glutamylcysteine synthetase (GCS) activity at 144 h of exposure. Our results show that the gills play an important role in the metabolism and detoxification of chlorothalonil in the channel catfish. Catfish can maintain elevated gill GSH concentrations in the presence of chlorothalonil by increasing gill cysteine concentrations.     

Effects of naphthalene and phenanthrene on visual and prey capture endpoints during early stages of the dourado Salminus brasiliensis

Naphthalene (NAP) and phenanthrene (PHE) effects on Salminus brasiliensis, a carnivorous freshwater fish, were investigated using behavioral tests. Larval stages of S. brasiliensis were exposed to water concentrations of 0, 0.04 mg/l, 0.20mg/l and 0.50mg/l for naphthalene and 0, 0.01 mg/l, 0.05 mg/l and 0.1mg/l for phenanthrene during two developmental phases. The prey fish Prochilodus lineatus were not exposed. Visual acuity was measured at the end of phase 2 in individual S. brasiliensis, which were also tested at the end of each phase for number of attacks on prey, number of prey captured, prey capture efficiency, and distance swam. Vision was impaired by PHE exposure, as acuity angles increased in exposed fish. At Stage I 2.3+/-1.2 prey were captured with 46% efficiency in controls compared to 0.4+/-0.3 prey captured with 13.4% efficiency in fish exposed to 0.05 mg PHEl(-1), the LOEL for both endpoints. At Stage II 4.0+/-1.1 preys were captured in controls compared to 2.5+/-0.9 preys captured in fish exposed to 0.01 mg PHEl(-1), the LOEL. Stage II control fish captured prey with 70% efficiency compared to 30% efficiency at Stage II fish exposed to 0.05 mg PHEl(-1), the LOEL. Distance swam was not affected by either NAP or PHE exposure. The exposure of larval stages of S. brasiliensis to realistic water concentrations of PHE impairs foraging skills and could affect recruitment of the species.     

Cytochromes P-450 in cod (Gadus morhua): Effects of induces on regiospecificity of phenanthrene metabolism and immunochemical properties

The metabolism of some xenobiotics can lead to the formation of reactive intermediates with mutagenic/carcinogenic properties. With the carcinogenic PAH these have been identified as bay-region diol-epoxides.¹ Phenanthrene, a non-carcinogenic, bay-region containing model PAH, is metabolised in vivo by bony fish at the proximate bay-region position, whereas mammals and other marine organisms mainly form the K-region metabolite 9,10-dihydro-9,10-dihydroxy-phenanthrene.² We wanted to investigate this difference more closely by studying the regiospecificity of phenanthrene metabolism in vitro both with microsomes from differently pretreated cod and with isolated cytochrome P-450 isozymes from BNF-induced cod.³ Secondly, by preparing antibodies to the major isozyme isolated (called cod P-450c), we investigated the immunochemical properties of the variously treated cod liver microsomes.     

Toxicity of sea-surface microlayer: Effects of hexane extract on baltic herring (Clupea harengus) and atlantic cod (Gadus morhua) embryos

This study was designed to determine whether contaminated sea-surface microlayer was toxic to marine fish embryos in its ntaive form and as a hexane extract. Developing embryos of Atlantic cod (Gadus morhua) and Baltic herring (Clupea harengus) were exposed to hexane extracts of sea-surface microlayer collected from five locations in the North Sea and Baltic Sea. Extracts from two of these locations produced significant embryos mortality as well as severe deformities in live hatched larvae. A control sample of bulk water collected from 20 cm under the surface and extracted in the same way produced no significant mortality or deformities. Significaant changes in timing of hatching were also observed in those samples which produced embryo toxic effects. A comparison of these data with those obtained from code embryos exposed to unextracted microlayer showed a similar biological effect with both unextracted samples and hexane extracts.Chemical analyses revealed the greatest biological effect in samples with petroleum hydrocarbon concentrations between 180 and > 200 μg liter⁻¹. The bulk water control had 1 μg liter⁻¹ while the three samples that showed no biological activity had 3 to 8 μg liter⁻¹ petroleum hydrocarbons. Phthalic acid esters were detected in four samples and chlorinated hydrocarbons in one, but could not be positively correlated with any of the toxic responses. No other chemical contaminants were detected in the five samples.The data presented here show that some sites contain sea-surface microlayer which can be toxic to marine fish embryos: that Baltic herring and Atlantic cod embryos respond similarly to the toxic effects of contaminated microlayer: and that unextracted microlayer and hexane extract of microlayer produce essentially the same toxic effect(s) if only organic contaminants are considered.     

Assessment of the genotoxicity of produced water discharges associated with oil and gas production using a fish embryo and larval test

Widespread discharge of produced waters (PW) into brackish and marine waters of the northwest Gulf of Mexico, which serve as spawning grounds for many commercially important species of fish, has raised concern over the potential impact that these complex mixtures may have on aquatic life. In the present study, the results of investigations which assess the clastogenic effects of production water discharged as the result of oil and gas production activity at two coastal sites are reported. In order to assess the impact of the PW discharges on spawning populations of aquatic organisms, chromosome aberration analysis was performed on Cyprinodon variegatus embryos 4–7 days old, which were exposed directly to varying dilutions of PW collected at Pass Fourchon and Timbalier Island for a period of 5 days. Metaphase chromosomes were scored (percentage aberration per 100 metaphases) for aberrations at a magnification of 1000 ×. When compared to the control treatment, higher levels of chromosomal aberrations were observed for treatments (2 and 4% PW) in which embryos were exposed to PW from Pass Fourchon or Timbalier Island. A maximum frequency of 13% was found to occur at the highest test concentration (4% PW), with most of these expressed as breaks (chromatid and chromosome). In contrast, exposure to water from the enclosed canal at Fourchon resulted in the formation of chromosome breaks and changes in ploidy.     

Effect of in vivo cadmium exposure on the respiratory burst of Marine Fish (Limanda limanda L.) phagocytes

Following the in vivo exposure of dab (Limanda limanda L.) to cadmium chloride, kidney phagocytes were collected and their respiratory burst measured in vitro using chemiluminescence. Fish were exposed to mean measured concentrations of 1.3, 2.7 and 5.5 mg Cd litre⁻¹ (as total cadmium ion) for a total of nine weeks, followed by a three week depuration period in clean sea water. Compared with control fish, the respiratory burst of kidney phagocytes from dab sampled after six weeks was significantly reduced in the 2.7 and 5.5 mg Cd litre⁻¹ treatments (Steel's test, p < 0.05). Significant reductions were observed in the respiratory burst of phagocytes from all cadmium exposed fish compared with control fish after nine weeks (Steel's test, p < 0.05). After a further three week depuration period in clean sea water, the respiratory burst of phagocytes from fish previously exposed to 1.3 and 2.7 mg Cd litre⁻¹ were still significantly less than in the control group (Steel's test, p < 0.05). Muscle tissue cadmium concentrations were also analysed, although there was no clear relationship between the muscle total cadmium levels and kidney phagocyte chemiluminescence. The results are discussed with respect to the possible mechanism(s) of cadmium immunotoxicity in dab and recommendations made for future work.     

7-ethylresorufin O-deethylase activity and level of DNA-adducts in trout treated with benzo(a)pyrene

In fish, as well as in mammals, it is well known that the cytochrome P450-dependent oxidative metabolism of xenobiotics can generate DNA-reactive species. Moreover, this metabolism is known to be inducible by several compounds of environmental significance, such as polychlorobiphenyls, polycyclic aromatic hydrocarbons (PAHs) and dioxins. Consequently, we studied the relationship between the degree of induction of the cytochrome P4501A, expressed as that of 7-ethylresorufin O-deethylase (EROD) activity, and the level of DNA-adducts, using the post-labelling assay, in the liver of rainbow trout exposed to benzo(a)pyrene (a representative PAH). The results showed a significant 2- to 4-fold increase in EROD activity 2, 4 and 8 days after treatment, paralleled by an increase in DNA-adduct levels. This work further emphasizes the involvement of cytochrome P4501A in the metabolism of benzo(a)pyrene into genotoxic metabolites in rainbow trout.     

Monitoring of the chemical pollution of the river Rhône through measurement of DNA damage and cytochrome P4501a induction in chub (Leuciscus cephalus)

The in vivo response of freshwater fish exposed to pollutants was assessed using two biomarkers, 7-ethoxyresorufin-O-deethylase (EROD) induction and DNA single strand breaks. Chub (Leuciscus cephalus) were caught in spring and in fall at various locations in the river Rhône watershed. EROD activity was measured in the liver while DNA damage was evaluated in chub erythrocytes using the recently developed Comet assay. Chemical contamination was evaluated both in fish muscle (PCBs) and in sediment (PCBs, PAHs, heavy metals) collected at each sampling station. Sex of individuals was shown to influence the level of EROD activity but not the level of DNA damage. The EROD activity as well as the DNA damage were found to be higher in the mostly contaminated stations compared to the reference one. This study shows that multibiomarker-based approach provides complementary informations about early effects in feral fish exposed to complex chemical pollution and highlights the interest of the Comet assay in genotoxicity assessment.     

Individual variation in patterns of benzo[a]pyrene metabolism in the marine fish scup (Stenotomus chrysops)

The metabolism at specific sites on carcinogenic hydrocarbons such as benzo[a]pyrene (BP) is responsible for activation to the ultimate mutagens and carcinogens, and patterns of metabolism can thus influence the biological effect of such compounds. Marine fish are known to efficiently metabolize BP at the benzo-ring, forming high percentages of the 9,10-dihydrodiol (DHD) and 7,8-DHD, the latter including the penultimate carcinogen.^1,2 Hydrocarbon-induced cytochrome P-450 in fish is responsible for initiating metabolism on the benzo-ring, but epoxide hydrolase (EH) activity is required for DHD formation.^3,4 Both factors could influence formation of the DHD leading to the ultimate carcinogenic diol-epoxide. In the present study, patterns of BP metabolism were evaluated in a number of individual scup Stenotomus chrysops sampled from local Woods Hole waters, and a correlation is described between variation in the DHD formation and EH activity in these feral fish.     

Assessment of lysosomal membrane stability and peroxisome proliferation in the head kidney of Atlantic cod (Gadus morhua) following long-term exposure to produced water components

There is a need for sensitive biological effect methods by which to detect impacts of chronic exposure to low concentrations of contaminants. Two methods shown to be potentially useful for monitoring purposes in fish include lysosomal membrane stability and peroxisome proliferation. These biological endpoints were assessed in Atlantic cod (Gadus morhua) head kidney following exposure to a mixture of produced water components including polycyclic aromatic hydrocarbons, phenol, and alkylphenols. Lysosomal damage of head kidney cells occurred within the first two weeks and did not recover during the entire exposure period (32 weeks). Lysosomal membrane stability was not affected by gender and was responsive at low concentrations of contamination, indicating that lysosomal membrane stability measured in the head kidney could be a useful biomarker for effects of offshore pollution. Peroxisome proliferation, measured as acyl-CoA oxidase activity in the head kidney, appeared to be a potential biomarker in male cod exposed less than 16 weeks.     

Candidate biomarker discovery in plasma of juvenile cod (Gadus morhua) exposed to crude North Sea oil, alkyl phenols and polycyclic aromatic hydrocarbons (PAHs)

In this study we have investigated protein changes in plasma of juvenile Atlantic cod (Gadus morhua) induced by crude North Sea oil and North Sea oil spiked with alkyl phenols and polycyclic aromatic hydrocarbons, a surrogate produced water composition. Using a proteomic approach, we identified 137 differentially expressed proteins at different levels of crude oil exposure. Many of the induced protein changes occurred at low levels of exposure. The results obtained with protein expression profiles after exposure to oil and surrogate produced water indicate effects on fibrinolysis and the complement cascade, the immune system, fertility-linked proteins, bone resorption, fatty acid metabolism as well as increased oxidative stress, impaired cell mobility and increased levels of proteins associated with apoptosis. Although the number of individuals and samples in this study is limited within each treatment group, the protein changes observed in this study represent a first screening for potential biomarker candidates in cod plasma reflecting potential effects of crude oil and produced water exposure on fish.     

Biological effects on viviparous blenny exposed to chrysene and held in synthetic as well as in natural brackish water

Viviparous blenny (Zoarces viviparous) were exposed to chrysene and held in natural brackish water as well as in two different synthetic brackish waters, all with a salinity of 6.8%. After 10 weeks of exposure a number of different biomarkers was analysed. The result indicated increased ethoxyresorufin-O-deethylase activity and the formation of two different DNA adduct spots in the liver tissue, as a result of the chrysene exposure. Very similar results of the chrysene exposure were obtained in blenny held in the two synthetic brackish waters. No influence of the synthetically made waters compared to the natural brackish water could be observed on the biomarkers investigated in the study.     

Microsomal estrogen metabolism in channel catfish

Our goal was to study the involvement of cytochrome P450 genes in estrogen metabolism and the extent to which the potentially carcinogenic 4-hydroxyestradiol metabolite is formed by channel catfish (Ictalurus punctatus; CC). Estradiol metabolism and ethoxyresorufin-O-deethylase (EROD) activity were assessed in several tissues from fish collected from three variably contaminated sites in the Mississippi River Delta, from laboratory control fish, and from fish exposed to 20 mg/kg benzo(a)pyrene (BaP) i.p. for 4 days. Liver EROD activity was induced by BaP, but Delta fish EROD activities were not statistically higher than activities in control fish. Gill microsomal EROD activity was also induced by BaP, but activities were 8- to 77-fold lower than those from liver. The predominant estrogen metabolites formed by CC liver, gill and gonad microsomes were 2-hydroxyestradiol and estrone as detected by GC/MS. Liver and gill 2-hydroxyestradiol formation was induced in BaP-dosed fish. The trends in hydroxyestradiol formation in field collected fish were more variable. In all fish liver microsomes there was more 2- compared to 4-hydroxyestradiol formed. However, BaP-treatment increased the 4:2 hydroxyestradiol ratio from 0.04 in control fish to 0.2 in BaP-exposed fish, suggesting that BaP induces the formation of the potentially genotoxic estrogen metabolite. No detectable 4-hydroxyestradiol was produced by gill and gonad microsomes. These results will ultimately help in determining which fish P450 genes are susceptible to environmental contamination and may be involved in estrogen genotoxicity.     

Effects of toxic chemicals on macrophage phagocytosis in two estuarine fishes

The phagocytic efficiency of macrophages isolated from the kidney of spot, Leiostomus xanthurus, and hogchoker, Trinectes maculatus, was determined in fish captured from the Elizabeth River (Virginia) Macrophage phagocytosis was found to be markedly reduced in the Elizabeth River fish as compared to Ware River controls. The macrophage phagocytic activity of Elizabeth River fish returned to normal after the fish were held in clean water for several weeks. This indicates that the decreased phagocytic activity was related to exposure to Elizabeth River pollutants but may be reversible.     

Study of the N, P and Si fluxes between fish farm sediment and seawater. Results of simulation experiments employing a benthic chamber under various redox conditions

The rapidly expanding industry of marine cage fish farming of sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) in the Mediterranean Sea over the last decade has often had damaging effects on the benthic aquatic environment near the fish farm installations. It has been observed that the food-remains, together with the pellets and metabolic products from fish, frequently form a “nepheloid” sediment layer covering large areas of the seabed. Under these conditions anoxia and/or hypoxia develop and affect benthic communities while the quality of the marine environment deteriorates for long periods, extending even beyond the life span of the fish farm itself. In most cases the affected areas act initially as sediment traps and in a second phase as secondary sources of organic carbon, nutrients and other substances.The aim of this paper is to explore the behaviour of such an affected area under different redox regimes by measuring the fluxes of ammonium, nitrite, nitrate, TDN, phosphate, TDP and silicate between the surface sediment and the overlying waters. To achieve this goal we constructed a prototype benthic chamber capable of sampling considerable quantities of affected undisturbed sediment along with its overlying water. The chamber was transported to the laboratory where simulation experiments reproducing the conditions occurring in nature, including the extreme ones, were carried out and studied carefully. The chamber allows the full and fine control of the dissolved oxygen concentration – and thus of the redox potential – as well as water temperature while subsamples of both water and sediment could be obtained and analysed for a series of chemical substances. The controlled laboratory chamber experiments reproduced four successional phases: 1) deoxygenation, 2) hypoxia, 3) reoxygenation and 4) anoxia.The results showed that even minor changes in the redox conditions at a relatively narrow zone near the water sediment interface have significant impacts on the concentrations of dissolved nitrogen, phosphorus and silicate compounds.With decreasing oxygen supply (phases 1, 2 and 4), the concentrations of ammonium, nitrite, TDN, phosphate, TDP and silicate rapidly increase, those of nitrate decrease. DON and DOP exhibit remarkable fluctuations.During reoxygenation (phase 3) the concentrations of ammonium stabilise, the nitrate concentration decreases while nitrite shows an increasing trend. Decreases in phosphate and silicate concentrations were also observed paralleled by TDP stabilization and DOP increasing trend. TDN shows a relatively small increase while the DON concentration fluctuates significantly.     

Unbleached pulp mill effluents affect cytochrome P450 monooxygenase enzyme activities

The polysubstrate monooxygenase system has been shown to be highly responsive to chemical pollution. The present study summarizes the enzyme based biomonitoring of the waste waters released by a pulp mill producing unbleached pulp and paperboard. Cytochrome P4501A enzyme activities of feral and caged fish, as well as cultures of fish hepatocytes, were tested. The 7-ethoxyresorufin-O-deethylase (EROD) activity was clearly induced in fish hepatocytes exposed to biotreated unbleached pulp mill effluent fractions in vitro. The effluent increased EROD activities also in feral perch, compared with controls. Caging experiments showed similar effects to those seen in feral fish: however, the maximal induction coefficients observed were higher. Unbleached effluents contain compounds that are able to affect the P4501A activities in fish.