Laurence D. Peters

Assessment of the impact of organic pollutants on goby (Zosterisessor ophiocephalus) and mussel (Mytilus galloprovincialis) from the Venice Lagoon, Italy: Biochemical studies

The use of cytochrome P4501A (CYP1A) and other measurements as biomarkers was investigated in liver of goby (Z. ophiocephalus) and digestive gland of mussel (M. galloprovincialis) from several sites in the Venice lagoon as part of the UNESCO-MURST Venice Lagoon Ecosystem Project. Most tissue contaminants (PAHs, PCBs, DDTs) and biochemical measurements varied seasonally. Elevated 7-ethoxyresorufin O-deethylase activity and CYP1A-protein levels in goby were correlated with high tissue contaminant levels at the industrial Porto Marghera site. On occasions, activities of the antioxidant enzymes catalase and putative DT-diaphorase (resorufin reductase activity) in male but not female goby were also higher at Porto Marghera than other sites, but no differences were seen in Superoxide dismutase (SOD) activity. A range of measurements (SOD, catalase, NADPH-cytochrome c reductase and glutathione S-transferase activities, P450 and ‘418-peak’ contents) in mussel showed little difference between sites. However, indications were obtained of elevated levels of CYP1A1-like mRNA, CYP1A-like protein and metabolism of benzo[a]pyrene to free metabolites in mussels from the Venice lagoon compared to a site in the Adriatic Sea. The studies demonstrate the usefulness of CYP1A as a biomarker for organic pollution in fish and indicate some potential for its application in molluscs.

Pro-oxidant, antioxidant and 7-ethoxyresorufin O-deethylase (EROD) activity responses in liver of Dab (Limanda limanda) exposed to sediment contaminated with hydrocarbons and other chemicals☆

Abstract Dab were exposed to reference sediment (79 μg PAHs g −1 dry wt) or contaminated sediment (309 μg PAHs g −1 dry wt plus low PCBs) for up to 140 days, and the effects on liver cytochrome P4501A (EROD activity), antioxidant enzymes and oxidative damage (lipid peroxidation) examined. EROD activity in the contaminated condition increased ×80 from 25 to 80 days, decreasing to 22% of this activity after 140 days. EROD activity also increased in the reference condition, but only after 140 days. Superoxide dismutase (SOD) and catalase activities and lipid peroxidation (malonaldehyde equivalents) were higher in the contaminated than the reference condition after 80 days but not 140 days. No differences were seen in glutathione peroxidase activity. It is concluded that the changes in SOD, catalase and lipid peroxidation are indicative of contaminant-mediated oxidative stress.

Apparent induction of a cytochrome P450 with immunochemical similarities to CYP1A in digestive gland of the common mussel (Mytilus galloprovincialis L.) with exposure to 2,2′,3,4,4′,5′-hexachlorobiphenyl and Arochlor 1254

Abstract The induction of a cytochrome P450 with immunochemical similarities to CYP1A, and accompanying changes in microsomal NADPH-dependent benzo[a]pyrene (BaP) metabolism, were examined in digestive gland of the common mussel (Mytilus galloprovincialis L.) with exposure to 20 ppb water-borne polychlorobiphenyl (PCB) mixture (Arochlor 1254) for 4 or 10 days, or 4 days after a single injection into the mantle cavity of the mixed-type inducer PCB congener 2,2′,3,4,4′,5′-hexachlorobiphenyl (CB-138; 2.5 μg g−1 wet weight). Whole animal tissue levels of PCB following water-column exposure or injection were similar to those for mussel species from polluted field sites, viz. 0.8 to 1.9 μg g−1 wet weight. Levels of microsomal CYP1A-immunopositive protein increased 59% (CB-138) and 72% (Arochlor 1254; 10 days exposure) as determined by Western blot analysis using polyclonal antibodies to hepatic CYP1A of perch (Perca fluviatilis). No changes were seen in levels of digestive gland CYP1A-like mRNA 4 days after injection of CB-138 as determined by Northern analysis using cDNA to hepatic CYP1A1 of rainbow trout (Oncorhynchus mykiss). The increases in levels of CYP1A-immunopositive protein were accompanied by a shift in microsomal NADPH-dependent BaP metabolism towards phenol and diol and away from dione production, the former increasing from 32 to 85% of total free metabolites. The marked decrease in dione production (which is the major BaP metabolite formed in control microsomes) resulted in no increase in total microsomal BaP metabolism with exposure to PCBs. The Type I ligand α-naphthoflavone markedly inhibited microsomal phenol but had no affect on dione production, whereas the Type II ligand clotrimazole markedly inhibited dione, but had much less effect on phenol production. The overall results are interpreted in terms of the existence of an inducible CYP1A-like enzyme catalysing predominantly 2-electron monooxygenation leading to epoxide (and hence phenol and diol) formation, and a constitutive non-inducible cytochrome P450 catalysing predominantly 1-electron oxidation leading to dione formation. Both Arochlor 1254 or CB-138 produced cellular damage in the digestive gland in the form of decreased epithelial digestive cell height and decreased lysosomal membrane stability.

Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels Anguilla anguilla from United Kingdom estuaries

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.

Responses of hepatic cytochrome P450 1A and formation of DNA-adducts in juveniles of turbot (Scophthalmus maximus L.) exposed to water-borne benzo[a]pyrene

The time-course of the induction of hepatic cytochrome P450 1A (CYP1A) and the formation of DNA-adducts in liver and surrounding tissues were studied in juvenile turbot (Scophthalmus maximus) (size range 6.0 ± 0.5 cm) exposed to a single water-column dosage of 1 or 25 ppb benzo[a]pyrene (BaP) for up to 48 h. CYP1A induction was measured in terms of 7-ethoxyresorufin O-deethylase (EROD) activity and CYPlA-immunopositive protein (Western blotting using polyclonal antibody to hepatic CYP1A of perch, Perca fluviatilis). The formation of BaP-DNA-adducts was determined by 32P-postlabelling analysis of extracted DNA. Hepatic EROD activity was not elevated by exposure to 1 ppb BaP at any time but increased 2- to 3-fold, 24 and 48 h after exposure to 25 ppb BaP, indicating induction of CYP1A at the higher BaP exposure concentration. Western blot analysis identified major immunopositive bands of apparent molecular weight 58 kDa (consistent with the presence of a CYP1A protein) and 48.5 kDa. Although levels of the 58kDa CYPlA-immunopositive protein were higher at 25 ppb than 1 ppb BaP 48 h after exposure, no overall consistent meaningful correlation between EROD activity and CYPlA-immunopositive protein could be discerned, probably owing to the relatively low levels of CYP1A induction and the sensitivity of the Western blot analysis. Consistent with the results for EROD activity, formation of DNA-adducts was indicated at 1 ppb BaP, but a 6-fold increase after 16 h was seen at 25 ppb. The latter level of DNA-adducts remained high at 24 h but decreased by 50% after 48 h. The marked formation of DNA-adducts at 16 h, before the increase in EROD activity, indicates a constitutive capacity for the metabolism of BaP to DNA-adducts. The maximal levels of DNA-adducts observed in BaP-exposed juveniles were similar to those in adult S. maximum injected intraperitoneally with 20 mg kg−1 BaP, respectively, 583 ± 131 and 650 ± 106 attomoles adducts μg−1 DNA. Co-chromatography of extracted adducted-bases with (+) and (−)-anti-benzo[a]pyrene diol epoxide-N2-guanine and N6-adenine nucleic acid standards failed to identify any specific adducts.

Partial purification and properties of cytochrome P450 from digestive gland microsomes of the common mussel, Mytilus edulis L.

Abstract Cytochrome P450 from the digestive gland of M. edulis was partially purified by sodium cholate solubilization, 4–15% polyethylene glycol fractionation, and octyl-Sepharose affinity, DEAE-Sephacel ion-exchange and hydroxylapatite chromatography (yields of up to 7–10%). Three peaks were resolved by DEAE-Sephacel chromatography (termed peaks 1–3). P450 specific content was increased from 26 to 800 pmol per mg protein, and the ratio of P450 content to NADPH-cytochrome c (P450) reductase activity reduced by a factor of 250. Oxidised spectrum λmax of P450 was 410.5 ± 1.5 nm. Type II difference spectra were seen with both type II (clotrimazole, metyrapone) and type I (α-naphthoflavone, 7-ethoxycoumarin) compounds. Western blotting with polyclonal anti-P4501A from perch (Perca fluviatilis) gave a single band of approximately 54 kDa molecular weight. A reconstituted system containing peak 2 or 3, rat liver P450 reductase, lipid and NADPH metabolised benzo[a]pyrene to diones, diols, phenols and putative protein adducts. Peak 2 plus cumene hydroperoxide was indicated to produce diones and protein adducts only. Peak 2 alone was indicated to produce diones and phenols. The major free metabolites in all cases were diones (53–100%). The results indicate the existence of a P4501A-like enzyme in M. edulis, possibly with unusual properties as indicated by the difference spectra, metabolism in absence of NADPH and added P450 reductase, and predominance of diones.

Immunochemical investigations of cytochrome P450 forms/epitopes (CYP1A, 2B, 2E, 3A and 4A) in digestive gland of Mytilus sp.

Western blot analysis of microsomes and partially purified cytochrome P450 (CYP) from digestive gland of Mytilus edulis was carried out using polyclonal antibodies to hepatic Perca fluviatilis CYP1A, Oncorhynchus mykiss CYP3A and rat CYP2B, CYP2E and CYP4A isoforms. Multiple CYP bands were detected in partially purified CYP compared to single bands for microsomes for anti-CYP1A, anti-CYP2B, anti-CYP2E and anti-CYP3A. In contrast, anti-CYP4A showed two distinct bands for both. The apparent molecular weights in kD (mean +/- range or S.D.; n = 2-4) for partially purified CYP were 42.5 +/- 0.5 and 48.1 +/- 0.3 (2 bands, anti-CYP1A); 67.4 +/- 0.7, 52.8 +/- 0.6, 44.5 +/- 2.5 (3 bands, anti-CYP3A); 52.8 +/- 0.7, 48.1 +/- 1.1 and 43.9 +/- 1.1 (3 bands, anti-CYP2B); 52.7 +/- 0.8 and 47.2 +/- 0.2 (2 bands, anti-CYP2E); 50.9 +/- 0.3 and 44.1 +/- 0.2 kD (2 bands, anti-CYP4A). Digestive gland microsomes of Mytilus galloprovincialis from a polluted compared to a clean field site showed higher levels of bands recognised by anti-CYP1A, anti-CYP2E and anti-CYP4A, but not anti-CYP2B and anti-CYP3A (P < 0.05), indicative of independent regulation of different CYP forms. Overall, the apparent molecular weight and field studies indicate at least five different digestive gland CYP forms.

Responses of the cytochrome P450 dependent monooxygenase and other protective enzyme systems in digestive gland of transplanted common mussel Mytilus edulis L. to organic contaminants in the Skagerrak and Kattegat (North Sea)

In order to determine the biological impact of contaminants in the Skagerrak and Kattegat, mussels (Mytilus edulis L.) (4 5-6 cm in length) from a clean area (Faroe Islands) were transplanted for 6-8 weeks in 1993 and 1994 to sites in the Faroe Islands (reference control),to the Skagerrak deep-waterregion between Norway and Sweden, and to suspected contaminant-influx sites near the Hvaler Archipelago (Norway) and Goteborg (Sweden). Similar results were obtained in both years. Whole body total polynuclear aromatic hydrocarbons (PAHs) were 57-206 % higher in M. edulis from the Skagerrak, Norway and Sweden sites (up to 62 ng g-1 dry wt) compared with the Faroe Islands reference control, whereas no differences were seen in organochlorines (PCBs, chlordanes, DDTs, hexachlorocyclohexanes, hexachlorobenzene). Digestive gland microsomal benzo\ [a] pyrene hydroxylase (BPH) activity (formation of phenols) was elevated at all the contaminated sites compared with the Faroe Islands reference control (p < 0 05). BPH...In order to determine the biological impact of contaminants in the Skagerrak and Kattegat, mussels (Mytilus edulis L.) (4 5-6 cm in length) from a clean area (Faroe Islands) were transplanted for 6-8 weeks in 1993 and 1994 to sites in the Faroe Islands (reference control),to the Skagerrak deep-waterregion between Norway and Sweden, and to suspected contaminant-influx sites near the Hvaler Archipelago (Norway) and Goteborg (Sweden). Similar results were obtained in both years. Whole body total polynuclear aromatic hydrocarbons (PAHs) were 57-206 % higher in M. edulis from the Skagerrak, Norway and Sweden sites (up to 62 ng g-1 dry wt) compared with the Faroe Islands reference control, whereas no differences were seen in organochlorines (PCBs, chlordanes, DDTs, hexachlorocyclohexanes, hexachlorobenzene). Digestive gland microsomal benzo\ [a] pyrene hydroxylase (BPH) activity (formation of phenols) was elevated at all the contaminated sites compared with the Faroe Islands reference control (p < 0 05). BPH turnover (BPH activity pmol-1 P450) was elevated 132-288 % compared with the the Faroe Islands (p < 0 05) and showed limited correlation with total PAHs (r 2=0 58). Overall, the results are indicative of impact by PAHs and induction of the cytochrome P450 monooxygenase system. In contrast to previous studies on M. edulis exposed to higher tissue levels of PAHs or PCBs, no elevation of cytochrome P4501Aimmunopositive protein (CYP1A) was detected using antibodies to fish hepatic CYP1A. Little or no differences between any sites were seen in digestive gland glutathione S-transferase (EC 2.5.1.18), superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) activities.

Studies on cytochrome P4501A in early and adult life stages of turbot (Scophthalmus maximus L.)

Cytochrome P4501A (CYP1A) has been investigated extensively in the liver of adults of many different fish species. This study reports the presence and elevation of CYP1A measured as 7-ethoxyresorufin O-deethylase (EROD) activity and protein amount (Western blot — semi-quantified by image analysis) in larval, juvenile and adult turbot (Scophthalmus maximus) after exposure to organic contaminant and model inducers. Basal EROD activity was not detectable in embryos, but increased from 3 day larvae (whole body 13 500g supernatants) through 90 day juveniles (hepatic 13 500g supernatants) to adults (hepatic microsomes), respectively in pmol/min/mg protein (± SEM), 0.57 ± 0.06, 10.8 ± 2.4 and 12.3 ± 4.7. Exposure to 5 ppb benzo[a]pyrene (B[a]P), 1 ppb γ-hexachlorocyclohexane (lindane) and 25 ppb B[a]P caused respectively, 3-, 6- and 2-fold elevation of EROD activity in 4 and 9 day larvae and juvenile stages. Image analysis of Western blots of juvenile samples detected a 3-fold difference in CYP1A protein whereas EROD activity of the same sample varied 4-fold. The results confirm the potential of using CYP1A induction as a biomarker for impact by organic pollution on early developmental stages of fish.

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goksøyr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.