Seasonal variation in cytochrome P450 immunopositive protein levels, lipid peroxidation and genetic toxicity in digestive gland of the mussel Mytilus edulis.

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.

The chaperonins: perspectives from the Archaea.

Heat-shock protein (Hsp) 60 chaperones are almost ubiquitous and almost always essential. They can be divided on the basis of sequence homology into two broad types: group I (found in bacteria, mitochondria and chloroplasts) and group II (found in Archaea and the eukaryotic cytosol). Of the two, the group I chaperones are the better understood. Data on their structure, mechanism of action and cellular role will be briefly presented. The group II chaperones are less well studied. In eukaryotes they form large complexes with 8-fold symmetry containing eight different subunits, all of which are essential. They appear to have a major role in the folding of actin and tubulin, although they may also act on other substrates. No crystal structures are available for these complexes. The situation in the Archaea is simpler, with organisms containing between one and three genes for these chaperones. A 2.6 A structure exists for one archaeal group II chaperone complex. Some progress has been made in defining the reaction cycle of the archaeal group II chaperones and this has shown that they have some properties distinct from the group I chaperones. To date, the in vivo role and importance of the archaeal group II Hsp60 chaperones has not been determined. We have now shown that in the halophilic archaeon Haloferax volcanii not all the genes for these proteins are essential. Further analysis of these proteins in the Archaea should be very productive in yielding more information about these important chaperones and their cellular functions.

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.

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.

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).

Zonal rotor purification and characterization of "mannosomes": a tubular membrane system in gastropod mollusc digestive gland.

Mannitol oxidase (manox) is an H2O2-generating oxidase apparently unique to molluscs and especially abundant in alimentary tissues. In the digestive gland it is localized to an organelle ("mannosome") that forms an unusual tubular membrane system. We have developed a novel centrifugation procedure for >100-fold purification of these membranes in 20% yield from approximately 30 g of digestive gland of the slug Arion ater. Mannosomes from several other gastropod species are also substantially purified by the procedure. Four successive density gradient separations are employed which minimize structural damage by exploiting near isosmotic conditions early on and by completely avoiding traumatic pelleting and resuspension. Plasma membrane contamination is reduced by digitonin-induced density perturbation. The purified preparation is characterized by a predominant 68-kDa integral membrane protein and retains the in situ appearance of hexagonally arranged tubules with an enveloping outer membrane.

Aromatic alcohol oxidase and aldehyde oxidase activities in the digestive gland of three species of terrestrial gastropod (Helix aspersa, Arion ater, Limax flavus).

1. Digestive gland homogenates of three species of terrestrial gastropod mollusc were used to prepare sources of soluble aldehyde oxidase and solubilized aromatic alcohol oxidase activities. 2. The snail (Helix aspersa) had two aldox isoenzymes each able to utilize salicylaldehyde whereas two slug species had a single apparent activity inhibitable by salicylaldehyde. 3. Aromatic alcohol oxidase activities of the three gastropods differed somewhat in their relative abilities to oxidize various methoxy-benzyl and amino-benzyl alcohols but, unlike fungal aromatic alcohol oxidase, were all able to utilize crotonyl alcohol as substrate.