ComparativeP-postlabeling analysis of DNA Adducts in Perch (Perca fluviatilis) from unpolluted and polluted areas in Swedish coastal waters

Perch (Perca fluviatilis), were sampled from unpolluted and polluted areas in Swedish coastal waters. The level of aromatic/hydrophobic DNA adducts in liver tissue was analyzed using the nucleas P1 version of the³²P-post-labeling assay. The level of total adducts measured in the individual fish from polluted areas was between 6 and 22 nmol of adducts/mol of nucleotides, and in the fish from the reference area between 0.2 and 0.6 nmol of adducts/mol of nucleotides.     

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.     

In vitro mechanistic differences in benzo[a]pyrene-DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by 32P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or beta-naphthoflavone (beta-NF) or digestive glands from mussels. The beta-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10(8) nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the 32P-postlabelled BaP 7,8-diol, 9,10-epoxide-N2-guanine adduct. Fewer adducts (P < 0.05) were generated by BaP-induced microsomes (9.4-30.6 adducts per 108 nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10(8) nucleotides). Co-incubation with 500 microM alpha-naphthoflavone (alpha-NF) resulted in 97-99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

Relationship between heavy metal accumulation and genetic variability decrease in the intertidal crab Pachygrapsus marmoratus (Decapoda; Grapsidae)

The “genetic erosion” hypothesis posits that heavy metal stress is related to a loss of genetic diversity at the population level. The genetic diversity of natural populations can, however, be affected by natural processes as well as by human impact. We studied the relationship between heavy metal bioaccumulation and genetic variability in the intertidal crab Pachygrapsus marmoratus. Tissue samples were collected from 40 individuals inhabiting four polluted and four unpolluted sites along the Tuscan coast (Mediterranean basin), and were examined for four heavy metals (arsenic, As, cadmium, Cd, lead, Pb, and copper, Cu). We also assessed the genetic variability of 235 crabs from the same localities using six microsatellite loci.Our results show that the bioaccumulation levels of these individuals accurately reflect the levels of pollution in their immediate environment, and that heavy metals accumulate more in the hepatopancreas than in the gills. Moreover, populations from polluted sites have significantly less genetic variability, measured as mean standardized d², and a significantly lower percentage of unrelated individuals, than populations from unpolluted sites. This evidence supports the “genetic erosion” hypothesis for metal heavy exposure in natural environments.     

The occurrence of hydrocarbons and histopathological abnormalities in oysters for seven years following the wreck of the Amoco Cadiz in Brittany (France)

A study of the main oyster areas polluted by the Amoco Cadiz oil spill (Abers, Baie de Morlaix) was carried out between 1978 and 1985 by chemical analyses (mainly aromatic hydrocarbon determinations) and histopathological observations of lesions in the digestive tracts and gonads of oysters. The hydrocarbon content in oyster tissues was monitored and analyzed by UV spectrofluorimetry, and gas and liquid chromatographic techniques.Seven years after the Amoco Cadiz wreck, the oysters sampled in the AberBenoit and in the Baie de Morlaix (Carantec) are still polluted with residual aromatic hydrocarbons, corresponding, respectively, to about five and two times the values from an unpolluted site (Baie de Saint-Brieuc). Since petroleum residues are buried in anaerobic sediments, especially in the Abers areas, they are expected to persist and accordingly may still contaminate oysters for several more years.Depuration kinetics of contaminated flat and Pacific oysters from the heavily polluted zones (Abers, Carantec) were studied and compared with that of healthy oysters transplanted in the same areas. The decontamination rates of oysters after transfer to a clean environment were heavily dependent on the duration of oil exposure.Necrosis and inflammation were the most significant and noticeable changes occurring during the first months after the pollution. Atrophy of the gonadal cells was detected in the oysters from the Abers only during the first six months of the study. No neoplastic and parasitic diseases have been related to this oil spill.     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

In vivo formation of (+)-anti-benzo[a]pyrene diol-epoxide-plasma albumin adducts in fish.

Benzo[a]pyrene (BaP), a procarcinogenic polycyclic aromatic hydrocarbon (PAH), is bioactivated to BaP diol-epoxides (BPDEs) that can form adducts with DNA and blood proteins. We report here for the first time the in vivo formation of adducts between BPDE and plasma albumin (Alb) from two fish species experimentally exposed to BaP. Brook trout (Salvelinus fontinalis) received either a single i.p. dose (10 mg/kg) or two separate i.p. doses (25 mg/kg; 7 days apart) of BaP, and blood was collected 2 (single exposure) or 3 (multiple exposure) days post-treatment. Arctic charr (Salvelinus alpinus) received 10 i.p. doses (3 mg/kg; a single dose every 6 days), and blood was collected 2 days after the second, sixth, and 10th injections. BPDE-Alb adducts were measured by an improved HPLC/fluorescence method developed to detect and quantify BaP-tetrols released after acid hydrolysis of adducted Alb. HPLC/fluorescence chromatograms of Alb from BaP-treated fish revealed only BaP-tetrol I-1, thus indicating the formation of adducts exclusively via the (+)-anti-BPDE metabolite. Levels of (+)-anti-BPDE-Alb adduct ranged from 0.68 to 19.6 ng of tetrol I-1 per gram of Alb. Notably, adduct level was not related to BaP dose and there was no accumulation of adducts with repeated exposure, which may indicate a very short half-life (< 2 days) of plasma Alb in fish. The data suggest that BPDE-Alb adducts in fish could be useful as a non-destructive biomarker of recent exposure to bioactivated BaP.     

DNA adducts in hematopoietic tissues and blood of the mummichog (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River, Virginia.

Hydrophobic DNA adducts were examined in liver, anterior kidney, spleen, and blood of tumor-prone mummichog (Fundulus heterclitus) from the creosote-contaminated Atlantic Wood (AW) site (Elizabeth River, Virginia). DNA adducts eluted in a diagonal radioactive zone, characteristic of polycyclic aromatic hydrocarbon exposure, in all examined tissues of AW fish. Mummichog demonstrated significantly higher levels of DNA adducts in spleen (394 +/- 109 nmol adducts/mol nucleotides) than in liver (201 +/- 77 nmol adducts/mol nucleotides) or anterior kidney (211 +/- 68 nmol adducts/mol nucleotides; P = 0.036). The levels of DNA adducts in the pooled blood (pool of four) were 142 nmol adducts/mol nucleotides. DNA adducts were not detected in the liver, anterior kidney, spleen and blood of fish collected from the reference site (< 2 nmol adducts/mol nucleotides). The high levels of DNA adducts detected in tissues of AW mummichog may be linked to the increased cancer incidence and immunosuppression in this population.     

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.     

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).     

Health status of red mullets from polluted areas of the Spanish Mediterranean coast,with special reference to Portmán (SE Spain)

A suite of general physiological indicators and hepatic biomarker responses were determined in red mullet (Mullus barbatus) from priority pollution areas of W Mediterranean Sea, including the highly metal polluted area of Portmán (Cartagena, SE Spain). Concentrations of metals and persistent organic pollutants in fish muscle tissue and sediment samples were also analysed. Our results showed that fish from Portmán accumulated the highest concentrations of mercury, lead and arsenic and also of some polycyclic aromatic hydrocarbons and polychlorinated biphenyls congeners. In addition, they had significantly lower condition factor, muscle lipid content and gonadosomatic index, as well as the lowest levels of DNA integrity and the highest ethoxyresorufin-O-deethylase (EROD) activity in liver of the areas investigated. Contaminant body burden in fish only partly corresponded to chemical characteristics of the sediments in the areas in which they were collected. Our findings indicate that red mullets from Portmán had suboptimal health status that warrant further study.     

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.     

Correlative changes in metabolism and DNA damage in turbot (Scophthalmus maximus) exposed to benzo[a]pyrene

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P < 0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.     

A seasonal survey of the sterol composition of the cockle cerastoderma edule: A comparison of polluted and non-polluted environments

The sterol composition of a cockle population from an unpolluted environment in South Wales was monitored by monthly sampling over a 12-month period. In spite of the likely variation in dietary sterols during this period, the component sterols of the cockles showed no significant seasonal variation, indicating that the cockle was able to exhibit a high degree of control over the composition of its functional sterols.Dramatic differences were observed in the sterol compositions of a nearby cockle population from a highly polluted environment, near a sewage outfall; only three out of the ten sterols present were common to the unpolluted cockles. Forty-five per cent of the sterols from the ‘polluted’ samples contained fully reduced systems and included three new alkylated C-29, C-30 and C-31 stanols. It is considered possible that these structural changes may affect the stereochemistry of the functional sterols and hence alter the permeability of the animal's membranes. This may reflect a biochemical response to the polluted environment.     

The viviparous blenny (Zoarces viviparus) as a bioindicator of contaminant exposure: application of biomarkers of apoptosis and DNA damage

The viviparous blenny (Zoarces viviparus), also known as eelpout, is considered a suitable biomonitor for use in European estuarine and coastal waters. We have previously shown that Z. viviparus from the industrialised Tyne estuary (UK) display certain histopathologies (e.g., ovotestis and hepatic nuclear and cellular pleomorphism), which are associated with contaminant exposure [Mar. Environ. Res. 55 (2003) 137]. Furthermore, the prevalence of these pathologies was higher than in fish collected from a less contaminated reference site (the Alde estuary, UK). Here, tissue samples were collected from Z. viviparus from the Tyne and Alde estuaries (in the spring and autumn) and analysed for DNA adducts using the (32)P-postlabelling assay and for histopathology. Z. viviparus caught from the Tyne during the spring (56.4+/-18.5 adducts per 10(8) nucleotides) and autumn (63.1+/-7.5 adducts per 10(8) nucleotides) exhibited patterns of DNA adducts which indicated exposure to a complex mixture of genotoxins. In contrast, levels of DNA adducts in Z. viviparus from Alde estuary were significantly lower during both sampling periods. Histopathological changes to the gonads included intersex (ovotestis) and germ cell apoptosis (detected via the TUNEL assay) in male fish captured from the Tyne estuary. Further studies on the use of germ cell apoptotic rate at different estuarine sites are required if it is to be used as a reproductive biomarker of contaminant exposure in male fish.     

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.     

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.     

DNA adducts in mussels and fish exposed to bulky genotoxic compounds

Biological and procedural factors can influence DNA adduct detection in aquatic organisms. Among them, functional structure and metabolic traits represent major biological determinants for adducts formed by lipophilic pro-mutagenic contaminants. In detecting DNA adducts through the 32P-postlabelling assay, efficiency in DNA purification, digestion, labelling, as well as adduct enrichment and quantification may explain differences between independent studies. Reference DNA adducts have been used to verify some 32P-postlabelling aspects. Data obtained for mussels and fish treated with benzo[a]pyrene (B[a]P) and environmentally exposed to genotoxins confirm the above assertions. Although the 32P-postlabelling assay cannot be proposed for routine biomonitoring it appears a reliable and very sensitive index of exposure to genotoxic pollutants in both fish and mollusks.     

Variation in microbial biomass and community structure in sediments of peter the great bay (sea of japan/east sea), as estimated from fatty acid biomarkers

Variation in the microbial biomass and community structure found in sediment of heavily polluted bays and the adjacent unpolluted areas were examined using phospholipid fatty acid analysis. Total microbial biomass and microbial community structure were responding to environmental determinants, sediment grain size, depth of sediment, and pollution due to petroleum hydrocarbons. The marker fatty acids of microeukaryotes and prokaryotes - aerobic, anaerobic, and sulfate-reducing bacteria -were detected in sediments of the areas studied. Analysis of the fatty acid profiles revealed wide variations in the community structure in sediments, depending on the extent of pollution, sediment depth, and sediment grain size. The abundance of specific bacterial fatty acids points to the dominance of prokaryotic organisms, whose composition differed among the stations. Fatty acid distributions in sediments suggest the high contribution of aerobic bacteria. Sediments of polluted sites were significantly enriched with anaerobic bacteria in comparison with clean areas. The contribution of this bacterial group increased with the depth of sediments. Anaerobic bacteria were predominantly present in muddy sediments, as evidenced from the fatty acid profiles. Relatively high concentrations of marker fatty acids of sulfate-reducing bacteria were associated with organic pollution in this site. Specific fatty acids of microeukaryotes were more abundant in surface sediments than in deeper sediment layers. Among the microeukaryotes, diatoms were an important component. Significant amounts of bacterial biomass, the predominance of bacterial biomarker fatty acids with abundance of anaerobic and sulfate-reducing bacteria are indicative of a prokaryotic consortium responsive to organic pollution.     

Development of thyroid abnormalities in mummichogs,Fundulus heteroclitus,from a polluted site

Previous studies showed that adult and 6-month-old young-of-year Fundulus heteroclitus from a polluted site have enlarged thyroid follicles and (in adults) higher thyroxine levels compared to fish from an unpolluted reference site. This study's purpose was to discover when in development these differences begin to appear. For 15-20 mm fish, the average follicle size of the polluted population was significantly larger than that of the reference population, similar to the pattern found in older fish. However, for the smaller size (12-14.5 mm), the average follicle size of fish from the polluted site was significantly smaller. Although not statistically significant, radioimmunoassay measurements of thyroxine levels were consistent with follicle differences: in larger sizes (20-25 and 15-20 mm) fish from the polluted site had higher thyroxine levels but the smaller size (12-14.5 mm) had lower levels. Therefore, the thyroid abnormalities seen in adults begin when the fish are approximately 15-20 mm long. However, the follicle size reversal for the 12-14.5 mm range may also indicate abnormality at that developmental stage.