S. C. M. O'Hara

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.

The comparative fate of dinosterol and cholesterol in copepod feeding: Implications for a conservative molecular biomarker in the marine water column

The fate of 4α,23,24-trimethylcholest-22-en-3β-ol (dinosterol) during passage through the gut of the copepod Calanus helgolandicus and fecal pellet sedimentation has been investigated. An artificial diet of starch granules containing 14C-labelled dinosterol was fed to copepods and compared to 14C-cholest-5-en-3β-ol as a common sterol present in copepod tissues. After feeding, the distribution of radioactivity and the detailed lipid composition of animals and their fecal pellets was determined. Cholest-5-en-3β-ol was readily assimilated by the copepod, with over 62% of the total radioactivity residing in animal tissues. In contrast, 4α,23,24-trimethylcholest-22-en-3β-ol was not assimilated and could be quantitatively recovered in fecal pellets. Additional experiments using copepod fecal pellets containing 14C-labeled dinosterol found no structural alteration during 19 days of simulated sedimentation, with all lipid radioactivity present as dinosterol. Small amounts (6.3%) of radioactivity were present as non-lipid material remaining within fecal pellets. Results from these experiments suggest that dinosterol (and by analogy other ring saturated sterols and stanols) is resistant to crustacean and microbial alterations over the relatively short (20 day) periods typical of sedimentation times for large particles in open ocean areas.

Characterization of the presence and seasonal variation of a CYP1A-like enzyme in digestive gland of the common mussel, Mytilus edulis

The presence and putative catalytic properties of a CYP1A-like enzyme in the digestive gland of Mytilus edulis L. were investigated by molecular biological and seasonal studies. Reverse-transcriptase PCR using oligonucleotide primers to amplify a sequence around the conserved haem binding cysteine region of hepatic CYP1A1 of trout (Oncorhynchus mykiss) produced several cDNA bands resolved by electrophoresis, including major bands of about 220 and 280 bp compared to the predicted size for O. mykiss of 208 bp. Following Southern blotting and probing with a cDNA probe to O. mykiss CYP1A1 (pfP1450-3′ probe modified by removal of 3′-non-coding region by digestion by Cla1), a single band (280 bp) only was detected using moderate stringency conditions of sequence recognition (i.e. hybridization at 42 °C followed by washing at 55 °C in 1 × SSC containing 0.1% SDS), providing evidence for the presence of a CYP1A orthologous gene sequence. The seasonal variation in levels of putative CYP1A mRNA (Northern analysis using the modified cDNA probe for O. mykiss CYP1A1) over 1 year showed some similarity to seasonal patterns of change in microsomal metabolism of 3H-benzo[a]pyrene (BaP) to polar metabolites (dials, diones and phenols resolved by HPLC). Maxima for putative CYP1A mRNA and BaP metabolism levels were in late spring-early summer. However, differences were also apparent, possibly indicative of other P450s contributing to BaP metabolism. Overall the results indicate the existence of a CYP1A-like enzyme which is, at least, partly responsible for the mono-oxygenase activity of BaP metabolism.

Lipid Changes During a Planktonic Feeding Sequence Involving Unicellular Algae, Elminius Nauplii and Adult Calanus

Numerous studies have shown that only a small percentage of the organic carbon produced by photosynthesis in the upper layers of the oceans reaches the underlying sediments (see review by Angel, 1984). During intense phytoplankton blooms, plant cells could account for most of the organic carbon contributed to sediments in certain shallow inshore areas (Smetacek, 1980). Examination of the sediments from open ocean environments, however, indicates that the main contribution of organic carbon to these is in the form of faecal material released by zooplankton, for example salps (Iseki, 1981) and larger species of copepod (Schrader, 1971; Krause, 1981).

Increased potential for NAD(P)H-dependent reactive oxygen species production of hepatic subcellular fractions of fish species with in vivo exposure to contaminants

The present study investigated the proposed involvement of contaminant-stimulated reactive oxygen species (ROS) production in disease processes in fish. NAD(P)H-dependent ROS production of subcellular fractions was determined by the iron/EDTA-mediated oxidation of 2-keto-4-methiolbutyric acid. Hepatic cytosolic NADPH-dependent and microsomal NAD(P)H-dependent ROS production were increased 51-160% (P < 0.05) in rainbow trout (Oncorhynchus mykiss) 15 weeks after a single i.p. injection of polychlorobiphenyl (PCB) (100 mg Clophen A50 kg-1 wet wt.). Hepatic microsomal NADH-dependent ROS production was 114% higher in perch (Perca fluviatilis) from PCB-contaminated Lake Järnsjön compared to clean Lake Vänern, Sweden. Hepatic mitochondrial NADH-dependent, cytosolic NADH-dependent and microsomal NADPH-dependent ROS production were variously elevated up to 160% in flounder (Platichthys flesus) at various sites along two pollution transects near to the ports of Rotterdam and Amsterdam, Netherlands. Overall the data indicate increased potential for ROS production in liver of fish exposed to field pollution, and support the hypothesis of oxidative stress as a mechanism of contaminant-mediated disease in fish.

Benzo[a]pyrene metabolism and xenobiotic-stimulated reactive oxygen species generation by subcellular fraction of larvae of turbot (Scophthalmus maximus L.)☆

Abstract NADPH-dependent 3 H-benzo[a]pyrene (BaP) metabolism and basal and xenobiotic-stimulated NAD(P)H-dependent reactive oxygen species (ROS) production were investigated in 11,600 g supernatants of 4-day-old (yolk sac) larvae of turbot ( Scophthalmus maximus L.). BaP metabolites were resolved by HPLC and detected radiometrically. ROS were quantified by the iron-EDTA mediated production of hydroxyl radical (·OH) that was detected by its oxidation of 2-keto-4-methiolbutyric acid (KMBA) to yield ethylene. BaP metabolism produced phenols, dihydrodiols and diones (quinones) (respectively, 54, 32, and 14% of free metabolites) and putative protein adducts. Metabolites identified by retention time included the 7,8-dihydrodiol, the 1,6-, 3,6- and 6,12-diones and the 3- and 9-phenols. Pre-exposure of turbot larvae to 5 ppb BaP for 24 hr caused an approximate 2-fold increase in both BaP metabolism and 7-ethoxyresorufin O-deethylase activity, indicative of the induction of cytochrome P4501A and its involvement in BaP metabolism. Basal KMBA oxidation rates were similar for NADH and NADPH. Inhibition studies indicate that ·OH was formed via the production of Superoxide anion radical and hydrogen peroxide. Basal ROS production was stimulated up to 3-fold by a range of redox cycling aromatic hydrocarbon quinones and indicated to be stimulated by other xenobiotics, including nitroaromatics. The results indicate biotransformation and ROS production as potential mechanisms of toxicity in larval fish.

Assimilation of Dietary Sterols and Faecal Contribution of Lipids by the Marine Invertebrates Neomysis Integer, Scrobicularia Plana and Nereis Diversicolor

Feeding experiments were conducted to characterise the contribution of fatty acids, sterols and fatty alcohols to the faeces produced during feeding on starch by the marine invertebrates Neomysis integer Scrobicularia plana and Hediste diversicolor

Interaction of tributyltin with hepatic cytochrome P450 and uridine diphosphate-glucuronosyl transferase systems of fish: In vitro studies

Hepatic microsomes of red mullet (Mullus barbatus) and flounder (Platichthys flesus) were preincubated in the presence of a concentration range of the antifouling agent tributyltin (TBT) chloride, and the interactions of TBT with cytochrome P450 and uridine diphosphate-glucuronyl transferase systems were investigated. The enzyme systems were examined in terms of cytochrome P4501A (CYP1A)-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity and benzo[a]pyrene (BaP) metabolism and in terms of glucuronidation of testosterone and 17beta-estradiol, respectively. Ethoxyresorufin O-deethylase and BaP hydroxylase (BPH) activities of both fish species were progressively inhibited by increasing concentrations of TBT, and the effects were more pronounced for EROD than for BPH (maximal inhibition at 100 microM TBT for EROD and 250-500 microM TBT for BPH). Hydroxylated metabolites of BaP (3-hydroxy-, 7,8-dihydrodiol, and 9,10-dihydrodiol), representing 95% of the total metabolites formed, were reduced up to 75% in the presence of 100 to 500 microM TBT, whereas the formation of other metabolites was less affected. This may alter BaP toxicity and carcinogenicity. Overall, the results were consistent with a specific inhibitory effect of TBT on CYP1A in the two fish species. Additionally, the conjugation of testosterone was significantly inhibited (20%) at low TBT doses (5 microM), with no effect on the glucuronidation of estradiol.

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.

Oxyradical generation and redox cycling mechanisms in digestive gland microsomes of the common mussel, Mytilus edulis L.

Abstract Mechanisms of xenobiotic-stimulated oxyradical generation were investigated in digestive gland microsomes using menadione (MD) and nitrofurantoin (NF) as model redox cycling compounds, and iron/EDTA as a promoter of the Haber-Weiss reaction. Hydroxyl radical, ·OH (or species of corresponding oxidizing power), was detected by oxidation of the scavenging agents α-keto-γ-methiolbutyric acid (KMBA) and sodium benzoate. MD and NF both stimulated ·OH production from NADH or NADPH. NADPH dependent ·OH production became limited at lower concentrations of xenobiotic (MD), or stimulation was less marked (NF), than for NADH. In the case of MD, the existence of an NADPH-dependent microsomal antioxidant enzyme, DT-diaphorase, limiting the formation of the MD semiquinone is indicated. Inhibitor studies indicated the involvement of superoxide anion radical, O2− (and redox cycling) and hydrogen peroxide, H2O2, in the formation of ·OH. The studies support the possibility of enhanced oxyradical generation as a mechanism of pollution-mediated toxicity in molluscs.