Assessment of environmental quality and inland water pollution using biomarker responses in caged carp (Cyprinus carpio): Use of a bioactivation:detoxication ratio as a Biotransformation Index (BTI)

In the present study the bioaccumulation of poly chlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) as well as the responses of a suite of biochemical parameters were investigated in a standardized carp (Cyprinus carpio) fish line. Carps were caged for 2 to 8 weeks at two Amsterdam freshwater sites: the relatively unpolluted Outer IJ and the heavily polluted Volgermeerpolder. The primary objective of the experiments was to develop a reliable and reproducible method to monitor the inland water pollution and to classify the environmental quality of freshwater sites. The highest tissue levels of organic trace pollutants were generally found in carp that was caged for at least four weeks at the polluted site. Hepatic phase I enzymes showed the highest pollution-induced responses, while phase II enzymes and serum transaminases were less sensitive. The antioxidant enzymes showed virtually no response to pollutants. In order to assess effects on the balance between bioactivation and detoxication the ratio between phase I (EROD) and phase II (GST or UDPGT) activities was introduced as a biotransformation index (BTI). Highest BTI values were observed in carp that was caged for 4 to 6 weeks at the polluted site. The BTI values decreased after eight weeks of exposure due to elevated phase II activities.     

Kinetics of hepatic phase I and II biotransformation reactions in eight finfish species

Hepatic microsomes and cytosols of channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterus salmoides), striped bass (Morone saxatilis), hybrid striped bass (M. saxatilis × M. crysops), and bluegill (Lepomis macrochuris) (n = 8) were used to study the kinetics of phase I (ECOD, EROD, PROD, BROD) and phase II (UDP-glucuronosyltransferase (UDPGT)-, sulfotransferase (ST)- and glutathione-s-transferase (GST)-mediated) reactions. The best catalytic efficiency for ECOD and GST activities was performed by channel catfish, Atlantic salmon, rainbow trout and tilapia. The highest EROD catalytic efficiency was for Atlantic salmon. None of the species had either PROD or BROD activities. Rainbow trout had very similar UDPGT catalytic efficiency to tilapia, channel catfish, Atlantic salmon, largemouth bass and bluegill. Sulfotransferase conjugation had no significant differences among the species. In summary, tilapia, channel catfish, Atlantic salmon and rainbow trout had the best biotransforming capabilities; striped bass, hybrid striped bass and bluegill were low metabolizers and largemouth bass shared some capabilities with both groups.     

Spatial variability of biochemical responses in resident fish after the M/V Hebei Spirit Oil Spill (Taean, Korea)

This study describes the spatial variation and the duration of the impacts from the Hebei Spirit oil spill using specific biochemical indices in resident benthic fish. Enzymatic activities and biliary PAHs metabolites were higher at the site closer to the spill area in four months after spill incident. Regarding our results of detoxification response, markers of Phase I followed a similar trend in accordance with levels of biliary metabolites, while markers of phase II and GST appeared relatively unchanged.     

The effect of long-term depuration on phase I and phase II biotransformation in mullets (Mugil cephalus) chronically exposed to pollutants in River Douro Estuary, Portugal

Pollutants such polycyclic aromatic hydrocarbons (PAHs) are released into the environment by urban communities and industries and the enzymes that catalyse the biotransformation of pollutants play a key role regarding the accumulation of these compounds in fish species inhabiting these areas. In this study the relationship between phase I (EROD activity) and phase II (GST activity) and PAH metabolites was measured in grey mullet (Mugil cephalus) after capture in the Douro estuary, and after long-term depuration in an unpolluted laboratory environment. The results showed a significant decrease in EROD activity after 1 month and in bile metabolites after 4 months in captivity, with both maintaining reduced levels at 4 and 8 months depuration. Liver GST activity did not showed significant changes. This study provides evidence that Douro estuary waters contain bioavailable PAHs that can be associated with the induction of cytochrome P450, and that mullets have the ability to metabolise and eliminate PAHs.     

Alternatives to whole animal testing: Use of cDNA probes for studies of phase I and II enzyme induction in isolated plaice hepatocytes

Studies of xenobiotic metabolism and the regulation of enzyme systems for their metabolism (Phase I and II enzyme systems) require large numbers of animals, intensive use of experimental aquarium systems and in some instances can pose major problems when the compounds are scarce, expensive or too toxic to be disposed of easily. Previous studies have demonstrated the usefulness of isolated primary hepatocytes of fish for metabolic and enzyme induction studies. Functionally and structurally competent hepatocytes were isolated from juvenile plaice and after overnight acclimation to culture conditions, were exposed to a variety of PAHs for 24h. The levels of CYP1A1, phenol UDPGT and GST-A mRNAs were then estimated by slot blotting and hybridisation to their cDNA probes. The method enabled rapid and easy determination of the structure/activity relationships of these compounds as inducers of these key Phase I and II enzymes, and is potentially useful for screening large numbers of compounds both as an invitro toxicity test and for mechanistic studies.     

Seasonal variation of antioxidant and biotransformation enzymes in barnacle, Balanus balanoides, and their relation with polyaromatic hydrocarbons

Seasonal variations in the antioxidant enzymes (catalase, superoxide dismutase [SOD], NADH-DT diaphorase), biotransformation enzyme, glutathione-S-transferase (GST) and microsomal lipid peroxidation in digestive tissue of barnacle, Balanus balanoides, from polluted and non-polluted populations have been evaluated. Relationships with accumulated polyaromatic hydrocarbon (PAH) concentration in barnacle tissues and environmental parameters (water temperature, salinity, dissolved oxygen concentration, water pH) were determined. As a general trend, maximum antioxidant enzyme and GST activities were detected in the pre-monsoon period or summer (March-June) followed by a gradual decrease during the monsoon (July October) with a minimum in the post-monsoon period or winter (November February). This pattern was similar to tissue concentrations of PAHs, resulting in a significant positive correlation with antioxidant enzymes, mainly catalase and SOD. Microsomal lipid peroxidation exhibited an almost reverse trend of seasonal variation to that of antioxidant enzyme activities indicating an enhanced susceptibility of barnacle tissues to oxidative stress. Among the environmental parameters, only water temperature seemed to have a significant effect on observed variations of antioxidant enzymes and GST activities. The barnacles from polluted and non-polluted populations exhibited seasonal differences in the activities of all the enzymes studied, particularly catalase, SOD and GST, suggesting the possibility of some biochemical adaptation in organisms from a chronically polluted environment. The results indicated that antioxidant defense components, catalase and SOD, are sensitive parameters that could be useful biomarkers for the evaluation of contaminated aquatic ecosystems. The results also suggested the potentiality of barnacle, B. balanoides, as a bioindicator organism against organic pollution.     

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated.The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.     

Monitoring a Marine Coastal Area: Use of Mytilus galloprovincialis and Mullus barbatus as Bioindicators

Abstract. Samples of Mytilus galloprovincialis and Mullus barbatus were collected in eight coastal sites along the South Adriatic and Ionic coasts of Italy in spring 2000 for a survey of coastal pollution in the Mediterranean basin. Specimens were analysed using an integrated approach based on residue analysis of common aquatic pollutants like organochlorines such as hexachlorobenzene (HCB), DDTs and polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), heavy metals and nonylphenols (NPnE) and biomarker responses such as acetylcholinesterase (AChE) activity, and the two specific P450 activities benzo(a)pyrene monooxygenase (BPMO) and 7-ethoxyre-sorufin-(9-deethylase (EROD). Biological and morphological parameters like somatic liver index (SLI), gonadosomatic index (GSI) and gonadal and gamete histology (eggs and sperms) were also evaluated in red mullet samples. A contamination gradient in which several hot spots occur were revealed in this study. The hot spots account for high levels of organochlorines in both species near incinerators and of PAHs in harbour areas. Levels of both NPnE and AChE activity were highest in two protected marine areas and were within detectable limits in others. This finding was confirmed by P450 activities, in which maximum levels were detected in harbours and protected marine areas. No morphological alterations of male and female gonads were observed on the histological level.     

Biomarker responses to pollution in two invertebrate species: Scrobicularia plana and Nereis diversicolor from the Cádiz bay (SW Spain)

The clam Scrobicularia plana and the polychaete worm Nereis diversicolor were collected in several sites from a littoral enclosure in SW Spain. The aim of our study was to relate various biomarker responses in these species to a pollution gradient caused by untreated domestic discharges and to verify the adequacy of the selected species as sentinels in this habitat. The biomarkers selected were the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX) and DT-diaphorase (DT-D). In addition, the activities of cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity, the phase II detoxifying enzyme glutathione S-transferase (GST) and the neurotoxicity marker acetylcholinesterase (AChE) were measured. Metallothionein levels were selected as biomarkers of heavy metals exposure in both species. The results suggest a different response in the water filtering organism (clam) and the sediment eater (polychaete), probably as a consequent of different pollution exposure and that samples from the “Caño Sancti-Petri” were exposed to biologically active compounds that altered some of their biochemical responses. AChE was the most sensitive biomarker in both species and N. diversicolor proved to be a more robust sentinel in this ecosystem.     

Enhancement of polycyclic aromatic hydrocarbons in estuarine invertebrates by surface runoff at a decommissioned military fuel depot

Accumulation of polycyclic aromatic hydrocarbons (PAHs) was determined in blue mussels (Mytilus spp.) and shore crabs (Hemigrapsus sp.) at a recently closed military fuel depot in central San Francisco Bay, California. In April 1996, during a period of above average precipitation, specimens were collected at the depot, near the depot, and at sites 10 and 20 km south of the depot. Four weeks after the rains ended, blue mussels were again collected at the depot, and at two additional sites in the central Bay region. In April, total PAHs in mussels from the depot were significantly higher only than that in mussels collected 20 km from the depot; however, seven specific, substituted PAHs were higher at the depot than at all other sites. In June, only two of the 38 PAHs common in mussels in April were detected at the depot; these concentrations were comparable to ambient concentrations in mussels at the Bay. It seemed that bioavailability of PAHs at the depot was enhanced by rainfall, probably due to the mobilization of PAHs via groundwater into the Bay. Concentrations in mussels from chronically contaminated sites were about five times higher than mussels collected from the depot. Low PAH concentrations were detected in shore crabs near the depot, and the highest levels were not associated with the depot. Observed PAH concentrations are discussed in relation to upper trophic organisms.     

Xenobiotic metabolizing enzyme activity in sockeye salmon (Oncorhynchus nerka) during spawning migration

Activities of the cytochrome P450 monooxygenase (MO) system and GSH-transferase (GST) were monitored about every 2 weeks in sockeye salmon during their sexual migration and ultimate death. Liver cytosol and microsomes were prepared from the tissue of 50 individuals, five males and five females at each collection. The activities observed were not significantly different between the sexes. Substantial changes occurred in cytochrome P450, cytochrome b₅ and the monitored activities during the migration. The most dramatic changes occurred at the spawning grounds and were evident even before spawning occurred. The cytochrome P450 and cytochrome b₅ contents increased while the MO and GST activities decreased. Pre-spawning MO and GST activities were similar to those reported for other species undergoing sexual maturation. Migrating salmon offer an interesting model for studying the regulation of sex-dependent and terminal forms of cytochromes P450, MO and phase II activities.     

Biotransformation of polycyclic aromatic hydrocarbons in marine polychaetes

Deposit-feeding polychaetes constitute the dominant macrofauna in marine environments that tend to be depositional centers for organic matter and contaminants. Polychaetes are known to accumulate polycyclic aromatic hydrocarbons (PAHs) from both particulate and dissolved phases but less is known about the mechanisms underlying elimination of accumulated PAHs. An important pathway of elimination is through biotransformation which results in increased aqueous solubility of the otherwise hydrophobic PAHs. Biotransformation in marine polychaetes proceeds in a two phased process similar to those well studied in vertebrates, phase I enzymes belonging to the Cytochrome P450 (CYP) enzyme family, along with a few phase II enzymes have been identified in marine polychaetes. In this review we aim at highlighting advances in the mechanistic understanding of PAH biotransformation in marine polychaetes by including data obtained using analytical chemistry and molecular techniques. In marine polychaetes induction of CYP enzyme activity after exposure to PAHs and the mechanism behind this is currently not well established. Conflicting results regarding the inducibility of CYP enzymes from polychaetes have led to the suggestion that induction in polychaetes is mediated through a different mechanistic pathway, which is corroborated by the apparent lack of an AhR homologous in marine polychaetes. Also, none of the currently identified CYP genes from marine polychaetes are isoforms of those regulated by the AhR in vertebrates. Relatively few studies of phase II enzymes in marine polychaetes are currently available and most of these studies have not measured the activity of specific phase II enzymes and identified phase II metabolites but used an extraction technique only allowing determination of the overall amount of phase II metabolites. Studies in insects and various marine invertebrates suggest that in invertebrates, enzymes in the important phase II enzyme family, UDP-glucuronosyl transferases primarily use glucoside as co-substrate as opposed to the vertebrate cosubstrate glucuronic acid. Recent studies in marine polychaetes have however identified glucuronidation of PAHs indicating no mechanistic difference in co-substrate preference among UDP-glucuronosyl transferases between vertebrates and marine polychaetes but it might suggest a mechanistic difference between marine polychaetes and insects.     

CYP1A and phase II responses of Scophthalmus maximus upon long-term repeated exposure to B(a)P and B(a)P-PCB77 mixtures

In a controlled laboratory experiment, fish in two treatment groups were exposed to B(a)P or B(a)P+PCB77 repeatedly over 1 year. Fish were held in a flow-through system and sampled on day 0, week 3, and on months 3, 6, 9, and 12. Each sampling (except week 3) was followed by exposure in water under static conditions. Exposure was terminated by restoring the flow-through within a maximum period of 24 h. Hepatic CYP1A remained significantly induced in juveniles even 3 months after the first exposure. The induction response in liver was more persistent in comparison to the gill response in juveniles. Sex influenced CYP1A expression in both organs of control fish and in both exposure groups. Suppression of CYP1A occurred in females even under repeated exposure. Liver GST activity showed sex differences, control females having higher activities than control males on the same sampling day. GST activity showed increases and decreases under both exposures possibly indicating the overriding influence of some other factors. Increased levels of fluorescent aromatic compounds (FACs) were detected in bile only in females exposed to B(a)P and PCB77 for 9 months. Liver UDPGT activity, length, weight, and condition showed no differences related to exposure or gender. The study was supported by Norwegian Research Council and the Ministry of Oil and Energy in Norway.     

Oxidative stress responses in two populations of Laeonereis acuta (Polychaeta, Nereididae) after acute and chronic exposure to copper

Acute (4 days) and chronic (14 days) effects of copper were evaluated on the antioxidant defenses of Laeonereis acuta (Polychaeta) collected in unpolluted (UP) and polluted (P) sites. In the acute assay (125 and 250 micro g Cu/l) superoxide dismutase (SOD) and glutathione S-transferase (GST) activities did not change, whereas catalase (CAT) increased in worms from both the sites. Lipid peroxidation was higher in copper exposed worms from the P site. In the chronic assay (62.5 micro g Cu/l) polychaetes from the P site showed enhanced activities of SOD, GST and CAT and higher contents of metallothionein-like proteins and sulfhydrils compared to worms from UP. Differences in responses between polychaetes from UP and P sites suggest that organisms from the polluted site, P, are more susceptible to oxidative stress conditions.     

Applying a fish expert system for ranking the biological effects of polycyclic aromatic hydrocarbons on the rockfish Sebastiscus marmoratus in the Maowei Sea,China

Polycyclic aromatic hydrocarbons (PAHs) pollution, particularly in coastal environments, is a global concern. In this study, the biomonitoring and ranking effects of PAHs in the rockfish Sebastiscus marmoratus were determined in the Maowei Sea, China. The results showed that the concentrations of the 16 priority PAHs detected in the surface seawater were moderate compared with those in other coastal areas worldwide, and the possible sources were rapid industrialization and urbanization combined with atmospheric deposition and runoff. Nested analysis of variance (ANOVA) suggested significant differences in the hepatic ethoxyresorufin-O-deethylase (EROD) activities and phenanthrene-derived metabolites in bile between the port area and the oyster farming area. The fish expert system (FES) was applied to evaluate the biological effects of PAHs on fish. The FES data demonstrated that the biological effect levels of Site S1 (level III, medium stress) were higher than those of the other sampling sites (level II, low stress).     

DNA adducts and polycyclic aromatic hydrocarbon (PAH) tissue levels in blue mussels (Mytilus spp.) from Nordic coastal sites

DNA adducts in gills and digestive gland, as well as polycyclic aromatic hydrocarbon (PAH) tissue levels were analysed in blue mussels (Mytilus spp.) from Nordic coastal areas (Iceland, Norway and Sweden) with diffuse or point sources of PAHs of various origins. Both DNA adduct and PAH tissue levels were generally low, indicating low PAH exposure to the mussels in the areas studied. DNA adducts were found to be higher in gills than in digestive gland of the mussels at all sites studied. Elevated DNA adduct levels in gills were found at 6 sites out of 18 compared to reference sites in respective coastal zones. Adduct levels ranged from 0.5 to 10 nmol adducts/mol normal nucleotides, being highest in mussels from Reykjavík harbour, Iceland (intertidal mussels), and from Fiskaatangen, Norway (subtidal mussels). Total PAH tissue levels in the mussels ranged between 40 and 11,670 ng/g dry wt., and were significantly correlated with DNA adduct levels (r(2)=0.73, p<0.001). PAH ratio values indicated that the PAHs were in most cases of pyrolytic origin, but with petrogenic input near harbours and an oil refinery.     

Mixed-function oxidase activity and conjugating enzymes in two species of antarctic fish

Enzyme activities of phase I and phase II of the hepatic detoxication system were investigated in two species of fish from the Ross Sea in Antarctica. The fish had extremely low phase I enzyme (BPMO and aldrin epoxidase). The reductase regenerating enzyme activities were comparable to those found in fish of temperate seas. Transferases of cytosol showed highest activity in the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and little or no conjugation of other substrates. The GSSG reductase and the glutathione transferase with peroxidase activity (CuOOH substrate) were low; the Sedependent glutathione peroxidase activity was not detectable. Interspecific differences were found for most of the enzymes tested.     

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.