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.     

DNA adducts and polycyclic aromatic hydrocarbon (PAH) tissue levels in blue mussels (Mytilus spp.) from Nordic coastal sites

DNA adducts in gills and digestive gland, as well as polycyclic aromatic hydrocarbon (PAH) tissue levels were analysed in blue mussels (Mytilus spp.) from Nordic coastal areas (Iceland, Norway and Sweden) with diffuse or point sources of PAHs of various origins. Both DNA adduct and PAH tissue levels were generally low, indicating low PAH exposure to the mussels in the areas studied. DNA adducts were found to be higher in gills than in digestive gland of the mussels at all sites studied. Elevated DNA adduct levels in gills were found at 6 sites out of 18 compared to reference sites in respective coastal zones. Adduct levels ranged from 0.5 to 10 nmol adducts/mol normal nucleotides, being highest in mussels from Reykjavík harbour, Iceland (intertidal mussels), and from Fiskaatangen, Norway (subtidal mussels). Total PAH tissue levels in the mussels ranged between 40 and 11,670 ng/g dry wt., and were significantly correlated with DNA adduct levels (r(2)=0.73, p<0.001). PAH ratio values indicated that the PAHs were in most cases of pyrolytic origin, but with petrogenic input near harbours and an oil refinery.     

DNA strand breakage in mussels (Mytilus edulis L.) deployed in intertidal and subtidal zone in Reykjavík harbour

DNA single-strand breaks were analysed in the blue mussel (Mytilus edulis L.) deployed in intertidal and subtidal zones in the PAH contaminated Reykjavík harbour and at a reference site, Hvalfj?rethur, Iceland. DNA strand breaks were analysed by Comet assay in isolated gill and haemocyte cells from six mussels from each site and depth. Increased DNA damage in both gill cells and haemocytes were observed in mussels deployed in Reykjavík harbour compared to the reference site. Intertidal mussels from Reykjavík harbour had higher DNA damage in haemocytes compared to subtidal mussels. The Comet assay seems to be useful for measuring genotoxic exposure in mussels from the field, and that DNA damage might be higher in the intertidal zone either due to higher exposure to contaminants or because of physiological and biochemical responses to variations in oxygen availability.     

DNA damage and apoptosis in the mussel Mytilus galloprovincialis

The effects of known genotoxic substances (4-nitroquinoline-N-oxide, benzo[a]pyrene, teniposide, etoposide, cycloheximide, tributyltin) on human cells (FLC, HL-60) and on mussels were investigated. The correlations between formation of DNA strand breaks and DNA fragmentation characteristic for the process of apoptosis were estimated. Strand breaks induced by 4-nitroquinoline-N-oxide and benzo[a]pyrene did not correlate with DNA fragmentation detected in the process of apoptosis. Induction of internucleosomal DNA fragmentation in HL-60 cells was initiated by teniposide, etoposide and tributyltin, while in the gills of mussels this was detected only with tributyltin. Levels of DNA strand breaks in natural mussel populations, living at locations under the influence of urban and industrial wastes, do not mirror the apoptotic processes.     

Seasonal variation of histopathological and histochemical markers of PAH exposure in blue mussel (Mytilus edulis L.)

The aim of this work was to study seasonal variation of histopathological and histochemical markers in blue mussels (Mytilus edulis L.) exposed to pyrogenic PAH contaminants. Mussels were collected in January, June, September and October from a sampling site in the vicinity of the discharge from an aluminium smelter and from a clean reference site. Histopathological analysis was carried out on the digestive gland (DG) and the gonads, lipofuscin and neutral lipids were analysed in the DG. Clear responses in lipofuscin and neutral lipids were detected in the DG of mussels collected from the polluted site at some sampling times. Moreover, these mussels presented atrophy in digestive tubules and haemocytic aggregates in the gonad and DG. However, in all parameters studied, the magnitude of the response showed clear seasonal variation.     

Measurement of DNA single-strand breaks in gill and hemolymph cells of mussels, Mytilus sp., collected on the French Atlantic Coast

DNA single-strand breaks were measured by the comet assay in both gill and hemolymph cells of mussels collected in 3 sampling areas of the French coast (Pointe du Castelli, Pen Bron and Saint-Nazaire Harbour). Whole mussel tissue samples were also collected for the chemical determination of PAH, PCB and heavy metal concentrations. In mussel, a higher level of DNA strand breaks was measured in gill than in hemolymph cells (p < 0.01). Despite a factor of contamination from 2 to 3 between sites, no difference in the extent of mussel DNA strand breaks was shown between sampling locations (p > 0.05), questioning the sensitivity of the assays used in biomonitoring studies.     

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.     

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).     

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.     

Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis

The effects of water-borne exposure to benzo[a]pyrene (36 h; celite-bound 0.44 mg L(-1) B[a]P) on cytochrome P450 (CYP) and superoxide dismutases (SODs) were examined in digestive gland of the blood clam, Scapharca inaequivalvis. B[a]P accumulation and elimination were rapid, with maximum whole-body concentrations of 1.78 ng g(-1) wet wt after 12 h of treatment, followed by a progressive decline to 0.89 ng g(-1) at 36 h. The presence of B[a]P resulted in an increase in total CYP of digestive gland microsomes from 54+/-14 to 108+/-21 pmol/mg protein (mean+/-SD; p<0.05, 24 h). Increases were also seen in microsomal CYP1A1/1A2-immunopositive protein (50.5 kDa app. mol. wt; p<0.05), but not CYP2E1-immunopositive protein (49 kDa app. mol. wt.), indicating a specific response of the former isoform. Exposure to B[a]P produced a steady increase in Mn-SOD digestive gland activity (p<0.01; p<0.05) but no significant change in Cu/Zn-SOD activity. The respective proteins, measured by western blotting, were not significant induced after B[a]P exposure. Cu/Zn-SOD and Mn-SOD activities were correlated with total CYP levels (r=0.96 and 0.63, respectively), indicating a role for CYP in reactive oxygen species (ROS) production during exposure. Both 'NADPH-independent' and NADPH-dependent metabolism of B[a]P by digestive gland microsomes was seen, producing mainly 1,6-, 3,6- and 6,12-diones, with some phenols and 7,8-dihydrodiol; putative protein adducts were also formed. Redox cycling of the diones may also have contributed to ROS production, leading to the increased SOD activities.     

DNA strand breaks in grass shrimp embryos exposed to highway runoff sediments and sediments with coal fly ash

Embryo production was reduced in female grass shrimp exposed to sediments with added coal fly ash and to sediments collected from an estuarine station containing high PAH concentrations due to its proximity to a highway storm drain. Grass shrimp embryos exposed to pore water from the high PAH and high metal sediments showed both reduced hatching and increases in DNA strand breaks (comet assay). Sediments with added coal fly ash had high concentrations of vanadium and selenium which may have contributed to effects similar to those observed with sediments with high PAH. The embryo pore water bioassay (hatching/DNA strand breaks) gave results comparable to those observed for reproduction effects (reduced embryo production/embryo hatching) with female grass shrimp exposed to whole sediment.     

Alteration of the biochemical properties of female gonads and vitellins in the clam Mya arenaria at contaminated sites in the Saguenay Fjord

Vitellins (Vn) are the major egg yolk proteins that constitute an important energy reserve for mollusc embryos. The purpose of the present study was to examine whether the relative levels of sugars, lipids, phosphates, and labile IIb metals and calcium normally associated with Vn would differ in clam populations living at contaminated sites. Softshell clams (Mya arenaria) were collected at three sites in the area of the Saguenay Fjord: a marina, a municipal sewer outfall zone, and a reference site. The condition factor (weight:length ratio), metallothionein-like proteins, cytochrome P450 1A1 activity and DNA damage were all determined in the clam's digestive gland. Levels of total sugars, lipids, alkali-labile phosphates, proteins, and labile zinc and calcium were determined in female gonad homogenates and in purified Vn. The results show that clam gonads at the contaminated site by a marina displayed a lower index of gonad activity than the reference site. In addition, the condition factor was significantly lower at the marina site as compared to the reference site, with a concomitant increase in DNA damage and metallothionein (MT) induction. In fact, the condition factor was significantly correlated with DNA damage (R = -0.413, P = 0.045) and MT levels (R = -0.622, P = 0.03). Homogenates of female gonads were found to contain higher levels of labile IIb metals and calcium, with lower lipid content at the marina site compared to the reference site, and much higher levels of alkali-labile phosphates (ALP) and calcium at the municipal outfall site. Vn from the marina site were significantly higher in labile IIb metals but lipid content appeared to be somewhat lower than at the reference site. Vn from the municipal site were found to be highly phosphorylated, with higher levels of lipids and calcium. These results suggest that the chemical composition of the gonads and Vn are altered in contaminated sites.     

多环芳烃化合物对栉孔扇贝组织芳烃羟化酶活力的影响

研究了苯并(a)芘(B[a]P)、苯并(k)荧蒽(B[k]F)以及两者的混合物对栉孔扇贝(Chlamys farreri)鳃丝和消化盲囊芳烃羟化酶(AHH)活力的影响。结果表明,多环芳烃化合物(PAHs)对栉孔扇贝鳃丝和消化盲囊AHH活力具有显著的诱导作用(P<0.05),并且呈现了明显的时间、剂量效应特征。B[a]P和B[k]F混合物处理组鳃丝和消化盲囊的AHH活力在实验期间呈现先升高后稳定趋势,B[a]P处理组则表现峰值变化,然后呈下降趋势。消化盲囊各处理组AHH活力显著高于鳃丝。作者认为可以采用消化盲囊的AHH活力来反映周围环境PAHs的污染,并应以最大饱和浓度为标准。3种PAHs中B[a]P的毒性最强,PAHs对栉孔扇贝的毒性大小与PAHs的组成有关,不能以不同种类单一PAHs毒性的总和来代表PAHs混合物的毒性。     

Exposure of grass shrimp to sediments receiving highway runoff: Effects on reproduction and DNA

A grass shrimp bioassay was carried out on sediments from three estuarine stations which were different distances from a highway storm drain. Total polycyclic aromatic hydrocarbon (PAH) concentrations were 29, 1.5 and 0.1 μg/g sediment at stations A (next to drain), B (100 m from drain) and C (500 m from drain), respectively. Lower embryo production and embryo hatching rates and a higher level of DNA strand breaks (comet assay) were observed in grass shrimp exposed to stations A and B sediments. There appeared to be an association between reproduction abnormalities and increased DNA strand breaks as a result of grass shrimp exposure to estuarine sediments receiving highway runoff.     

Anthropogenic pollution stimulates oxidative stress in soft tissues of mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker1853)

The digestive gland and gills of the mussel Crenomytilus grayanus extracted from three locations — (i) sampled from a clean and (ii) polluted site and (iii) transplanted from the nonpolluted to polluted site - were analysed for antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase), total oxyradical scavenging capacity and levels of lipid peroxidation products (malondialdehyde, conjugated dienes and lipofuscin). Perturbation of redox status was found in both digestive gland and gill tissues of mussels living in the polluted area. As the activities of superoxide dismutase and catalase were 1.2–3 times higher, the total oxyradical scavenging capacity was lower by 20–35% and the levels of lipid peroxidation products were 2–7 times higher compared to mussels from the reference site. In transplanted mussels, the lipid peroxidation process in both tissues was significantly stimulated (the level of conjugated dienes was increased 1.7–2.5-fold; malondialdehyde and lipofuscin contents were increased 3.5–5-fold) and the total oxyradical scavenging capacity fell by 50–70%. In addition, the transplantation generally resulted in transient and variable responses of antioxidant enzymes for both tissues. Complex response-behaviour of the antioxidant enzymes strongly points to the necessity of employing a combined approach that takes into account activities of antioxidant enzymes and the total oxyradical scavenging capacity, as well as measurement of oxidative damage (e.g., lipid peroxidation) to evaluate the physiological health of molluscs.     

Assessing DNA damage in cnidarians using the Comet assay

The assessment of DNA damage by the Comet assay has been described as a useful non-specific general biomarker of stress in many marine organisms. In field situations it has successfully been employed to distinguish between reference and polluted sites and in the laboratory it has been widely used as a mechanistic tool to determine pollutant effects and mechanisms of DNA damage. To date a wide range of marine vertebrates and invertebrates have been used, however, the usefulness of this assay as a biomarker in cnidarians has not yet been assessed. The aims of this study were to optimize the Comet assay for cnidarian cells and to assess its utility for detecting genotoxic damage in these cells. Cells were isolated from the North American pacific coast temperate sea anemone Anthopleura elegantissima using a non-enzymatic dissociation procedure and viability was determined to be in excess of 90%. Cells were incubated either with (1 h acute exposures) hydrogen peroxide (H(2)O(2)), ethylmethanesulphonate (EMS) or benzo(a)pyrene (B[a]P). In comparison to other marine species, anemone cells exhibited high control or background levels of DNA strand breaks. Despite this, however, we observed dose responses for each of the study chemicals with no reduction in cell viability. This study demonstrates that anemone cells respond to known DNA damaging agents, including B[a]P which requires metabolism to exert its genotoxic effect, and that the Comet assay may prove to be a useful biomarker of stress in cnidarian species.     

Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels

An aluminium smelter on the west coast of Scotland discharges an aqueous effluent containing polycyclic aromatic hydrocarbons (PAHs) at the head of Loch Leven. The loch also supports two mussel (Mytilus edulis) farms. Data are presented on burdens of PAHs in the soft tissues of mussels and the effect of these contaminants on glutathione S-transferase (GST) activity in mussel hepatopancreas. GST activity is shown to be correlated with total PAH burden and also with the concentrations of certain individual PAHs. These field data show that high molecular weight PAHs are closely correlated to GST activity, whereas low molecular weight PAHs are not. This suggests that 5- and 6-ring PAHs have a more pronounced role than 2- to 4-ring compounds in inducing GST activity in mussels from Loch Leven. It is proposed that it may be more appropriate to link GST activity with 5- and 6-ring compounds only, rather than with the total PAH burden.     

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels

An aluminium smelter on the west coast of Scotland discharges an aqueous effluent containing polycyclic aromatic hydrocarbons (PAHs) at the head of Loch Leven. The loch also supports two mussel (Mytilus edulis) farms. Data are presented on burdens of PAHs in the soft tissues of mussels and the effect of these contaminants on glutathione S-transferase (GST) activity in mussel hepatopancreas. GST activity is shown to be correlated with total PAH burden and also with the concentrations of certain individual PAHs. These field data show that high molecular weight PAHs are closely correlated to GST activity, whereas low molecular weight PAHs are not. This suggests that 5- and 6-ring PAHs have a more pronounced role than 2- to 4-ring compounds in inducing GST activity in mussels from Loch Leven. It is proposed that it may be more appropriate to link GST activity with 5- and 6-ring compounds only, rather than with the total PAH burden.     

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated.The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.