D.R. Livingstone’s research while affiliated with Plymouth Marine Laboratory and other places

Levels of components of the cytochrome P450 (CYP)-dependent monooxygenase system were characterised in microsomes of major biotransformation tissues, or whole bodies, of 33 species from six phyla of aquatic invertebrates. The phylogenetic distribution of benzo[a]pyrene hydroxylase (BPH) activity in the absence of added NADPH (so-called 'NADPH-independent BPH activity') and presence of NADPH was also examined. Microsomal protein yield was higher in individual tissues than whole tissues. The main components (total CYP and cytochrome b5; NADPH-dependent cytochrome c (CYP) reductase, NADH-dependent cytochrome c reductase and NADH-dependent ferricyanide (b5) reductase activities) were found in most species of the Porifera, Cnidaria, Mollusca, Polychaeta, Crustacea and Echinodermata examined. The so-called '418-peak' of the carbon-monoxide difference spectrum of reduced microsomes was found in all species, indicating the wide distribution of this protein. Total CYP levels (pmol mg(-1) protein; mean+/-SEM) were similar in molluscs (50+/-7), crustaceans (61+/-11) and echinoderms (56+/-9), with the exception of high levels (223-266) in two crustacean species. NADPH-dependent BPH activity (pmol min(-1) mg(-1) protein) was found in 32 species, being lowest in Porifera and Cnidaria (3-4), intermediate in Mollusca (7.8+/-1.3), and highest in Crustacea (25+/-4) and Echinodermata (15+/-4). NADPH-independent BPH activity was evident in 13 out of 15 molluscan species examined, with the addition of NADPH either stimulating (8 species) or inhibiting (5 species) the activity. NADPH-independent BPH activity was also seen in two poriferan species and indicated in three crustacean species, suggesting that the phenomenon is not solely restricted to the Mollusca.

The antioxidant enzymes superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), selenium-dependent glutathione peroxidase (SeGPX; EC 1.11.1.9), glutathione reductase (EC 1.6.4.2) and DT-diaphorase (EC 1.6.99.2), plus total GPX activity (sum of SeGPX and Se-independent GPX activities), were studied in 13 500 g supernatants of embryos and 3-day and 11-day post-hatch larvae of turbot Scophthalmus maximus L. SOD activity decreased progressively during development from embryos to 11-day-old larvae, indicative of a decreased need to detoxify superoxide anion radical (O2−). In contrast, catalase, SeGPX and glutathione reductase activities increased progressively from embryos to 11-day-old larvae, indicative of an increased need to metabolize hydrogen peroxide (H2O2) and organic peroxides. Consistent with the latter changes, levels of lipid peroxides (i.e. thiobarbituric acid reactive substances) increased 13-fold from embryos to 3-day-old larvae, whilst total peroxidizable lipid was indicated to decrease. Increases were seen for NADPH-dependent DT-diaphorase (after hatching) and total GPX (between 3 and 11 days post-hatch) activities, whilst no change was found in NADH-dependent DT-diaphorase activity. Overall, the results demonstrate a capacity for early life-stages of S. maximus to detoxify reactive oxygen species (O2− and H2O2) and other pro-oxidant compounds (organic peroxides, redox cycling chemicals). Furthermore, qualitative and quantitative antioxidant changes occur during hatching and development, possibly linked to such events as altered respiration rates (SOD changes) and tissue reorganization and development (catalase, SeGPX, lipid peroxidation).

The relationship between cytochrome P450 1A- and 2E-immunopositive proteins, lipid peroxidation and DNA strand breaks (SBs) was studied in Mytilus edulis digestive gland at different seasons and at different sites around the UK coast. Cytochrome P4501A (CYP1A)-immunopositive protein and DNA strand breaks were generally lowest in December but there was no correlation between PAH exposure (indicated by chemical measurement and CYP1A-immunopositive protein expression) and DNA strand breaks which was highest at the relatively non-polluted site (Port Quin). As with CYP1A, CYP2E1-immunopositive protein was maximal at most sites in May. Lipid peroxidation, in contrast, did not alter markedly throughout the year. In conclusion, DNA strand breakage was not correlated with any of the above parameters although it did correlate with "scope for growth" as did the inverse of PAH levels. The study highlights the need to establish the relative contribution of DNA damage and DNA repair processes to the production of DNA strand breaks and emphasises the need to consider seasonal variation in interpretation of biomarkers.

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.

The main aim of this work was to provide baseline data on aspects of pro-oxidant and antioxidant processes in different life-stages of the marine amphipod Gammarus locusta. The activities of antioxidant enzymes and levels of lipid peroxidation were determined in whole body juveniles, subadults, and male and female adults of a laboratory population of G. locusta. Fatty acid composition of individuals at these different stages of development was also characterised in order to examine the contribution that polyunsaturated fatty acids (PUFA) might make to the peroxidation status of animals. The antioxidant enzymes, measured in whole body 100,000 supernatants, comprised catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GPX; EC 1.11.1.9). Fatty acids were analysed as fatty acid methyl esters (FAME). Lipid peroxidation was examined in terms of the levels of lipid peroxides determined as thiobarbituric acid-reactive malondialdehyde equivalents. Age-related changes were seen in antioxidant enzyme status: levels of SOD (p<0.01) and GPX (p<0.001) activities decreased progressively during development from juveniles to adults. Sex-related changes in GPX activity were also seen, the levels being higher in adult males than females (p<0.001). The amount of FAME present in whole body amphipod also changed over the life span. Among PUFA, the eicosapentaenoic (C20:5n-3), arachidonic (C20:4n-6) and docosahexaenoic (C22:6n-3) were the most abundant acids in this species, and both their individual concentrations and total PUFA increased progressively with age (up to 3.3-fold; p<0.001). The latter changes may contribute to the explanation of the observed differences in peroxidation status of the animals with age; thus, levels of lipid peroxides increased up to 40% in adult males compared to other age-classes (p<0.01). Overall, the decline in antioxidant enzyme activities, coupled with increased levels of PUFA, as the individual grows older, may render the older animals more susceptible to lipid peroxidation and oxidative stress.

The aim of the current work was to determine over 10 days the effects of water-borne exposure of the marine amphipod Gammarus locusta to 4 microgCu l(-1) on the metallothionein (MT; measured by differential pulse polarography) protection system and lipid peroxidation (LP: thiobarbituric acid-reactive malondialdehyde equivalents) as a measure of oxidative damage. MT levels in exposed animals increased significantly at day 2 (36% > control; P < 0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P < 0.001). The maximum level of MT at day 6 coincided with the highest Cu body-burden. LP increased within I day of exposure, indicative of Cu as an oxidative stressor. However, in contrast to MT, the highest LP level was seen at day 4 (68% > control, P < 0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.

Blue mussels (Mytilus edulis, L.) were exposed to a single dose of 1 ppb benzo[a]pyrene (BaP) under subtidal (SC) or tidal conditions (TC; 6 h immersion, 6 h emersion) in order to follow its bioaccumulation in whole mussel and mantle tissue, and to compare BaP-mediated toxicity on lipids (malonaldehyde formation, MDA) in the mantle. Rapid uptake of BaP (70-80% of BaP initially introduced in tanks) was observed in both conditions after 12 h, but subsequent depuration in clean water was slower in TC mussels. BaP levels decreased in whole tissue in both conditions between 12 and 24 h, but increased in mantle. The mantle BaP levels were similar during the first 4 days in SC and TC, but whereas they decreased in SC after 7 days. BaP was retained at high levels in mantle in TC until the end of the study (14 days). In both conditions, significant increases (P < 0.05) in lipid peroxidation were observed after 4 days, but MDA levels were approximately 3 times higher in the mantle of TC than SC mussels, although BaP tissue concentrations were similar. These observations suggested that increased BaP-mediated toxicity in mantle lipid was due to the interactive effect of the tidal cycle of immersion/emersion on BaP-mediated oxidative damage.

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.

Sublethal exposures of the marine amphipod Gammarus locusta to a concentration range of copper (Cu) in water (4 days' exposure; 3, 5 and 10 micro g Cu l(-1)) or spiked sediments (28 days' exposure; 1, 3 and 6 mg Cu kg(-1) dry weight) were performed, and the resulting bioaccumulation of Cu and effects on putative metallothionein (MT) and lipid peroxidation (LP) were investigated. A time-course exposure study (over 10 days) to a single water-borne concentration of Cu (4 micro g l(-1)) was also carried out. MT and LP were quantified, respectively, by differential pulse polarography and as thiobarbituric acid-reactive malondialdehyde equivalents. The increasing levels of Cu in water and sediment exposures resulted in enhanced uptake of the metal by G. locusta. Synthesis of putative MT occurred in response to exposure to water-borne Cu, the levels being higher (p < 0.05) over the dose range of Cu compared with controls. A positive correlation was observed between putative MT levels and the Cu body-burden concentration (p < 0.001). However, no increase in LP was observed in these animals. In contrast, in the time-course experiment, LP levels increased within 1 day of exposure, subsequently peaking at 4 days (68% greater than control, p < 0.001), before returning to control values by day 6. Higher levels of MT were also observed in this exposure, but at days 6 and 10 (55% and 38%, respectively), paralleling the decrease in LP. No increase in MT levels was recorded with exposure to Cu-contaminated sediments, whereas higher levels of LP were seen in comparison with controls (p < 0.001). Overall, the inverse relationship between putative MT induction and the occurrence of LP indicates that MT may protect against the prooxidant effects of Cu. It is concluded that MT and LP offer potential for application as biomarkers in G. locusta.