L.D Peters

Cytochrome P450 and dependent activities in unexposed and PAH-exposed terrestrial annelids

The cytochrome P450 system of the oligochaetes Eisenia f. fetida (tiger worm) and Enchytraeus crypticus (pot worm) was analysed using ethoxy-, pentoxy- and benzoxyresorufin as substrates for monooxygenase activity. Whole body microsomes of the earthworm E.f. fetida displayed PentROD activity in the range from 0.26 to 1.05 pmol mg protein-1 min-1 and BenzROD activity in the range from 0.14 to 0.30 pmol mg protein-1 min-1. Exposure of the animals for up to four weeks to 100 mg fluoranthene or benzo[a]pyrene kg-1 soil (dry weight) did not induce significant changes in the activity of these monooxygenases. In E. crypticus EROD activity was in the range from 2.10 to 6.18 pmol mg protein-1 min-1 and PentROD activity in the range from 1.75 to 4.78 pmol mg protein-1 min-1. Short-term exposure to BaP by feeding reduced the EROD activity significantly by 45%, but did not effect PentROD activity. After long-term (8 weeks) exposure to BaP in the agar-agar medium EROD activity was not changed but PentROD had decreased to zero. In both species cytochrome P420 and NADPH-cytochrome C reductase activity were present. In E.f. fetida microsomes are associated with the giant haemoglobin. Both can be separated by gel filtration on a Sepharose B2 column or by hydrophobic interaction chromatography after solubilisation with cholate. NADPH-cytochrome C reductase elutes together with haemoglobin. Cytochrome P420 is eluted with Emulgen 911 and can be further purified by ion exchange chromatography using HA-Ultrogel. By SDS-PAGE of the purified microsomal proteins three protein bands are visualised in the range of cytochrome P450 displaying an apparent molecular mass of 54, 56 and 58 kDa. Only the 54-kDa protein interacts weakly with perch (Perca fluviatilis) CYP1A antibodies, while two proteins with an apparent molecular mass of 65 and 71 kDa give a strong antibody signal.

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.

Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay)

Mytilus edulis digestive gland microsomes were prepared from indigenous populations sampled from a clean reference site (Port Quin) and an urban-industrial contaminated site (Blackpool) in the UK. Samples were collected in March/April, May, August and December 1998. Western blot analysis was performed using polyclonal antibodies to fish CYP1A and rat CYP2E using partially purified M. edulis CYP as a positive control, to aid identification. CYP1A- and CYP2E-immunopositive protein levels showed different site-specific seasonal variation with higher levels of CYP2E determined in May (P < 0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P < 0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P < 0.05).

Elevation of cytochrome P450-immunopositive protein and DNA damage in mussels (Mytilus edulis) transplanted to a contaminated site

Mytilus edulis were collected from a reference site (Port Quin) and an urban/industrial contaminated site (New Brighton) in the UK during June 1999. Levels of PCBs (sigma7 congeners) and CB-138 were determined to be, respectively, 21 fold and 16 fold higher in the mussel digestive glands from New Brighton. Levels of CYPIA-immunopositive protein were 1.5 fold higher (P < 0.05) at the polluted site but the levels of DNA strand breaks were 1.3 fold higher (P<0.05) at the reference site. Mussels from Port Quin were placed in cages at both sites and both transplanted and indigenous populations sampled in September (13 weeks). Mussels transplanted from the reference site to the industrial site, reported elevated levels of CYP1A-immunopositive protein (1.4 fold; P < 0.05) and higher levels of DNA damage (1.2 fold; P < 0.05) compared to caged populations at the reference site and a PCB loading similar to the populations from the polluted site. Moreover, transplanted mussels had DNA damage 1.8 fold greater (P < 0.05) than indigenous mussels at the transplant site. These changes were small but significant when compared to the observed temporal changes in the indigenous populations.

Variation in levels of cytochrome P4501A, 2B, 2E, 3A and 4A-immunopositive proteins in digestive gland of indigenous and transplanted mussel Mytilus galloprovincialis in Venice Lagoon, Italy

Mytilus galloprovincialis digestive gland microsomes were prepared from (i) indigenous populations sampled from a clean reference (Lido) and an urban-contaminated site (Salute) and (ii) mussels transplanted for up to 3 weeks from Lido to an industrial-contaminated site (CVE) in Venice Lagoon, Italy. Western blot analysis was performed using antibodies to five mammalian or fish CYP forms (1A, 2B, 2E, 3A, 4A). Simultaneously run M. edulis digestive gland partially purified CYP aided identification of immunopositive bands. Levels of CYP1A, CYP2E, and CYP4A-immunopositive proteins were 50 to 300% higher in indigenous M. galloprovincialis from Salute compared to Lido (p < 0.05). Three weeks after transplantation to CVE, levels of only the CYP1A-immunopositive protein were determined to be higher (63%) than levels for Lido (p < 0.05), indicating that anti-CYP1A shows greater specificity for a contaminant-inducible CYP form than the other antibodies.

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.

Correction of salinity with flavin-containing monooxygenase activity but not cytochrome P450 activity in the euryhaline fish (Platichthys flesus)

To test the association between flavin-containing monooxygenases (FMOs) and osmoregulation, saltwater-adapted euryhaline flounder (Platichthys flesus) were statically exposed to 34 (ambient), 25, and 15 parts per thousand (/1000) salinity for 1 or 2 weeks. FMO activity (thiourea S-oxidase) was assayed in gill and liver microsomes in P. flesus. Branchial FMO activity was reduced dramatically (98%) in fish exposed to a salinity of 15/1000 as compared with control, while hepatic FMO activity was reduced by 60%. Reduction of FMO activity in response to reduced salinity (15/1000) appeared to occur within 1 week or less in both liver and gill of the flounder. Although hepatic FMO activity continued to fall and was not detected after 2 weeks at 15/1000, branchial FMO activity was still present. A dose-response relationship in FMO reduction was present in liver, but there was no difference observed between 25 and 15/1000 salinity in FMO activity of flounder gill. Serum osmolality and hepatic cytochrome P450 content were unchanged by salinity. In an attempt to determine whether trimethylamine (TMA) plays a role in piscine FMO, the effect of TMA on hepatic and branchial FMO activity was examined. Intraperitoneal injections of TMA failed to induce activity. Thus, an association between osmoregulatory function and FMO expression was observed in a species of euryhaline fish, indicating that alterations by salinity may affect xenobiotic biotransformation in euryhaline animals.

Down-regulation of piscine flavin-containing mono-oxygenase activity by decreased salinity in euryhaline flounder (Platichthys flesus)

Abstract Flavin-containing mono-oxygenases (FMOs) play a significant role in xenobiotic biotransformation in fish. However, endogenous functions of the enzyme have not been documented. Earlier studies in our laboratory indicated a relationship between FMO expression and osmoregulation in euryhaline fish. Consequently, in the present study, euryhaline flounder ( Platichthys flesus ) were exposed to three separate salinity regimes for two weeks. Hepatic and branchial FMO activity was examined in individual animals from each exposure and was found to be directly related to the salinity in which the fish were kept. Thus, an association between osmoregulatory function and FMO expression was observed in euryhaline fish indicating possible divergent or simultaneous evolution of the enzyme system to a xenobiotic metabolizing function.

Differential expression and activity of flavin-containing monooxygenases in euryhaline and stenohaline flatfishes indicates potential osmoregulatory role

N,N-Dimethylaniline (DMA) N-oxidase activity indicative of flavin-containing monooxygenase (FMO) was examined in four tissues (liver, gill, muscle, and kidney) of the flounder (Platichthys flesus). Gill microsomes had the highest levels of activity (456 +/- 343 pmol/min/mg), while kidney (121 +/- 109) and liver (67 +/- 26) had levels just above detection. A single faint band of a 56 kD protein was observed in liver and gill microsomes following Western blot analyses with polyclonal antibodies to FMO 1. DMA N-oxidase activity in gill and liver directly correlated with the expression of the 56 kD protein recognized by polyclonal antibodies against FMO form 1. Likewise a mRNA band of approximately 2.5 kilobases was higher in gill than a 3.0 kb band in liver following hydridization with an FMO 1 cDNA probe. Gill and liver microsomal DMA N-oxidase from the euryhaline P. flesus was compared with that of the stenohaline turbot (Scophthalmus maximus). DMA oxidase activity, FMO protein and mRNA were significantly greater in the gill of P. flesus, while S. maximus had higher levels of enzyme activity in the liver, but also significant levels in gill. Comparison of the enzymatic properties of the P. flesus gill and S. maximus liver enzymes indicated dramatic differences in Km between gill and liver, but were both inhibited by equimolar concentrations of trimethylamine (TMA). Gill microsomal activity in each species was unaffected by the mammalian FMO 2 substrate (competitive inhibitor) n-octylamine. Differential expression of FMO in tissues from stenohaline and euryhaline fish suggests a functional relationship between FMO and osmoregulation.

Random expression of branchial flavin-containing monooxygenase (FMO) activity in the Atlantic flounder (Platichthys flesus)

Abstract In an attempt to better understand the regulatory basis explaining relationships between FMO and osmoregulation, sexually immature Atlantic flounder were treated for 18 days with abdominal implants containing the osmoregulatory modulators cortisol (CO) and 17 beta-estradiol (E2). NaKATPase mRNA expression was significantly repressed in E2 animals, and tended to be elevated in CO-treated animals. FMO mRNA expression was extremely variable with a slight reduction in E2-treated animals, but no significant differences were observed between any treatment groups. TMA oxygenase activity was unchanged by CO or E2 treatment, and detected in only 40% of the animals (8 out of 20). To assess individual variability, 11 male and nine female flounder were examined for branchial FMO expression and activity. Although FMO mRNA was observed in all animals, FMO activity was only observed in 63% (7 out of 11) of males and 33% of females (3 out of 9). No significant correlations were observed between FMO mRNA expression, FMO activity, serum CO, or E2. Results of this study indicate variable expression of FMO mRNA and sporadic deletion of catalytic activity in Atlantic flounder.