Development of two novel CYP-antibodies and their use in a PCB exposure experiment with Mytilus edulis

In an attempt to learn more about the cytochrome P450 (CYP) system of mussels, we used protein databases and alignment software to extract highly conserved CYP sequences. From these alignments synthetic peptides were produced and used for rabbit immunisation, which yielded polyclonal antibodies against the CYP families 2 and 4. The antibodies were evaluated with Western Blot and ELISA assays, using digestive gland microsomal samples from the mussel Mytilus edulis. Western Blots revealed immunoreactions for both antibodies. The anti-CYP2 sequence rendered one major immunopositive protein of ≈49 kDa size, and weak signals for proteins of ≈41 and 56 kDa size. The anti-CYP4 sequence rendered two major bands of ≈56 and 59 kDa size, and also a weak immunoreaction with a protein of ≈43 kDa size. ELISA rendered only weak signals even with a 1:50 dilution of IgG-purified serum. A 10-day exposure to Aroclor 1254 did not appear to affect any of the immunopositive proteins, while total PCBs in soft bodies increased from 14–40 ng/g DW in controls to 373–638 ng/g DW in exposed mussels.     

Polycyclic aromatic hydrocarbons in surface seawater and in indigenous mussels (Mytilus galloprovincialis) from coastal areas of the Saronikos Gulf (Greece)

Polycyclic aromatic hydrocarbons (PAHs) were identified and measured in surface seawater and in the tissues (gills and mantle) of indigenous black mussels, Mytilus galloprovincialis, collected from three coastal sites of Saronikos Gulf (Greece), a gulf that exhibits high levels of pollution. The total PAHs measured by spectrofluorometry in the surface seawater were found in the range of 425–459 ng L⁻¹ at the most polluted sites 1 and 2 (Elefsis Bay–Salamis Island) and in the range of 103–124 ng L⁻¹ at site 3 (Aegina Island). PAHs' sources in seawater were identified by application of specific PAH ratios, such as phenanthrene/anthracene and fluoranthene/pyrene. Levels of PAHs in soft tissues (gills and mantle) of indigenous mussels were much higher than those reported for seawater. Total PAH concentrations in mantle tissues were in the range of 1300–1800 ng g⁻¹ dry weight (dw) tissue at sites 1 and 2 and approximately 380 ng g⁻¹ dw at site 3. In gill tissues total PAH concentrations were in the range of 1480–2400 ng g⁻¹ dw at sites 1 and 2 and approximately 430 ng g⁻¹ dw at site 3. PAHs composition was dominated by two-, three- and four-ring compounds in seawater, where 17 different PAH compounds were identified and measured in mussel tissues. Mussels can be used as sentinel organisms to monitoring PAHs' contamination, since they concentrate PAHs from the surrounding water media and therefore making the chemical analysis simpler and less prone to error than that for water. In surface seawater possible weathering and photodegradation due to hot climates contribute to reduced PAHs concentrations.     

Influence of parasitism in controlling the health, reproduction and PAH body burden of petroleum seep mussels

Petroleum seep mussels are often exposed to high hydrocarbon concentrations in their natural habitat and, thus, offer the opportunity to examine the relationship between parasitism, disease and contaminant exposure under natural conditions. This is the first report on the histopathology of cold-seep mussels. Seep mussels were collected by submersible from four primary sites in the Gulf of Mexico, lease blocks Green Canyon (GC) 184, GC-234, GC-233, and Garden Banks 425 in 550–650 m water depth. Five types of parasites were identified in section: (1) gill “rosettes” of unknown affinity associated with the gill bacteriocytes, (2) gill “inclusions” similar to chlamydia/rickettsia inclusions, (3) extracellular gill ciliates, (4) body “inclusions” that also resemble chlamydial/rickettsial inclusions, and (5) Bucephalus-like trematodes. Comparison to shallow-water mytilids demonstrates that: (1) both have similar parasite faunas; (2) seep mytilids are relatively heavily parasitized; and (3) infection intensities are extremely high in comparison to shallow-water mytilids for Bucephalus and chlamydia/rickettsia. In this study, the lowest prevalence for chlamydia/rickettsia was 67%. Prevalences of 100% were recorded from three populations. Bucephalus prevalence was 70% in three of 10 populations. The parasite fauna was highly variable between populations. Some important parasites were not observed in some primary sites. Even within primary sites, some important parasites were not observed in some populations. Bucephalus may exert a significant influence on seep mussel population dynamics. Forty percent of the populations in this study are severely reproductively compromised by Bucephalus infection. Only a fraction of petroleum seep mussel populations are maintaining the entire beta-level population structure of this species. Variation in two parasites, gill ciliates and Bucephalus, explained most of the variation in PAH body burden between mussel populations. PAHs are known to be sequestered preferentially in gametic tissue. Bucephalus would be expected to reduce overall body burden, at high infection intensities, by replacing gametic tissue. PAH concentrations exceeded 1 ppm in 4 of 9 populations, a ratio significantly higher than the 8 of 30 mussel locales in the NOAA Mussel Watch Program. Only five Mussel Watch locales exceeded the highest value for a petroleum seep population. Digestive gland and gill tissue atrophy were not significantly correlated with PAH body burden, even though some populations were characterized by body burdens exceeding 1 ppm, suggesting that seep mussels may not be as sensitive to PAH exposure as are some shallow-water mytilid populations.     

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.     

A laboratory and field comparison of sediment polycyclic aromatic hydrocarbon bioaccumulation by the cosmopolitan estuarine polychaete Streblospio benedicti (Webster)

Polycyclic aromatic hydrocarbon (PAH) bioaccumulation by the polychaete worm Streblospio benedicti (Webster) was measured under exposure to PAH-contaminated sediments in the field and for 28 d in the laboratory. Streblospio benedicti collected from field sediments contaminated at 2.94, 1.07, and l.52 μg g⁻¹ fluoranthene (FLU), benz[a]anthracene (BAA), and benzo[a]pyrene (BAP), bioaccumulated those PAHs at 1.53, 0.215, and 0.332 μg g⁻¹, while worms isolated from less contaminated field sediments (0.399, 0.228, 0.288 μg g⁻¹ FLU, BAA, and BAP) had FLU, BAA, and BAP body burdens of 0.543, 0.236, and 0.083 μg g⁻¹. Worms incubated for 28 d in PAH-spiked sediments (1.52, 0.991, 0.504 μg g⁻¹ FLU, BAA, and BAP) bioaccumulated those PAHs at 0.382, 0.966, and 0.602 μg g⁻¹, respectively. Data normalization to organism lipid and sediment organic carbon (biota-sediment accumulation factors [BSAFs] strongly suggest that Streblospio PAH bioaccumulation was directly related to percent sediment organic carbon, but BSAFs were substantially lower than predicted by equilibrium partitioning theory. BSAFs decreased with increasing PAH log K_ow, in worms collected from field sediments, but in spiked sediments BSAFs increased with increasing PAH hydrophobicity. This disparity may have been caused by insufficient spiked-sediment equilibration time (1.5h) in the case of the laboratory test sediments.     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3^′,4,4^′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

Effects of long-term exposure to silver or copper on growth, bioaccumulation and histopathology in the blue mussel Mytilus edulis

The purpose of this study was to determine the long-term accumulation of either silver or copper from low concentrations in seawater by blue mussels, Mytilus edulis. Mussels raised from eggs in the laboratory to the age of 2·5 months (approximately 4·5 mm in length) were continuously exposed to 0, 1, 5 and 10μg/liter of either silver (nitrate) or copper (chloride) and sampled at 12, 18 and 21 months for growth studies, measurements of metal accumulation and histopathological examination.Whole-body soft tissues were analyzed for the presence of both silver and copper, as background levels of copper in the incoming seawater averaged 2–4 μg/liter. Mussels exposed to silver had accumulated significant amounts of silver only at the highest test concentration (10 μg/liter Ag) after 12 months, but at 18 and 21 months significant levels were accumulated at all three test concentrations. Mussels exposed to copper accumulated significant amounts of copper at 5 and 10 μg/liter Cu after all three sampling periods, but not at 1μg/liter. Silver-exposed animals also accumulated significantly greater amounts of copper than control animals.In a comparative study, field-collected juvenile mussels (approximately 16·1 mm in shell length) and adult mussels (approximately 53·4 mm in shell length) were exposed for 12 months to 0, 5, 25 and 50 μg/liter silver only and subsequently sampled for metal-accumulation analyses and growth measurements. Juvenile mussels accumulated significant amounts of silver at all test concentrations, with the exception of mussels exposed to 5 μg/liter Ag for 6 months. Copper accumulation in the silver-exposed juveniles was significant only at 50 μg/liter Ag after 6 months, but at all test concentrations after 12 months. Adult mussels exposed to silver accumulated significant levels of both silver and copper, but at somewhat lower levels than juveniles.In the growth study, silver had no effect on laboratory reared mussels at the highest concentration of 10 μg/liter tested, whereas copper at 10 μg/liter did appear to affect growth as early as 4 months after the start of experimental exposure. Field-collected juvenile mussels did show inhibition in growth after 6 months' exposure to 25 and 50 μg/liter Ag, with some growth occurring after 12 months. Adults also showed inhibition in growth after 6 months but not at 12 months.Histopathological examination of mussels exposed to either 5 or 10 μg/liter of copper for 18 months showed changes in the digestive diverticula, gastrointestinal tract, reproductive tract and muscle tissues. These changes were more noticeable in mussels exposed to 5 μg/liter Cu than in those exposed to 10 μg/liter. Mussels exposed to silver for 21 months showed yellowish to black particulate deposition in the basement membrane and connective tissue of the various organs and tissues. Silver deposition increased with increasing test concentration.     

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.     

Internal recycling of particle reactive organic chemicals in the Chesapeake Bay water column

Hydrophobic organic contaminants (HOCs) may be used as tracers of particle dynamics in aquatic systems. Internal cycling of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were studied in the mesohaline Chesapeake Bay to assess the role of resuspension in maintaining particle and contaminant inventories in the water column, and to compare settling and suspended particle characteristics. Direct measurements of sediment resuspension and settling conducted in conjunction with one of the sediment trap deployments indicate reasonable agreement between measurements of particle flux using the two different methods. Organic carbon and PCB concentrations in settling solids collected in near-surface sediment traps were remarkably lower than concentrations in suspended particles collected by filtration during the trap deployments, but higher PAH concentrations were found in the settling particles. The different behaviors of PAHs and PCBs in the settling particles are due to their different source types and association to different types of particles. Sediment trap collections in near bottom waters were dominated by resuspension. Resuspension fluxes of HOCs measured 2 m above the bay bottom were as high as 2.5 μg/m² day for total PCBs and 15 μg/m² day for fluoranthene, and were 25 and 10 times higher than their settling fluxes from surface waters, respectively. HOC concentrations in the near bottom traps varied much less between trap deployments than HOC concentrations in the surface traps, indicating that the chemical composition of the resuspended particles collected in the near bottom traps was more time-averaged by repeated resuspension than the surface particles.     

The PAH composition of surface sediments from Stagnone coastal lagoon, Marsala (Italy)

This paper examines the presence, distribution, nature and sources of 22 Aromatic Hydrocarbons (PAHs), which are important environmentally and toxicologically, in sediments from the Stagnone coastal lagoon at Marsala (Italy). Analysis was performed by gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring (SIM). The total concentration of polycyclic aromatic hydrocarbons ranged from 72 to 18381 μg/kg of dry matrix. The relative standard deviation (RSD) of the replicates on the concentrations of individual compounds ranged from 5% to 20%. The accuracy of method was estimated by analyzing “blank” samples added of known quantities of analytes and the recover percentage was 88 ± 9%. The detection limit (LOD) of analytical procedure was less than 0.2 μg/kg d.w. for all analytes. The quantification limit (LOQ) of analytical procedure was less than 0.7 μg/kg d.w.The resulting distributions and weight ratios of specific compounds are discussed in terms of sampling location and origin of organic matter. A comparison with other studies of total PAHs suggests that the levels are within the concentration ranges already reported by other authors. From an eco-toxicological point of view, total PAH concentrations at seven out of the eight sites studied represent a relatively clean environment when compared to other sites.Organic matter content and PAH concentrations were found to be correlated and the compounds present in Stagnone sediments were shown to be mainly of pyrolitic origin, while a negligible quantity of PAHs may derive from biogenic sources since all the sediments contain perylene traces. There is no evidence of coal-tar contamination.Cluster analysis was carried out in order to discriminate between different PAH origins.     

Responses of the mussel Mytilus edulis to copper and phenanthrene: Interactive effects

Most investigations of the responses of marine organisms to xenobiotics have concentrated on single contaminants and little is known of possible interactive effects of different classes of xenobiotics. As these latter seldom occur in environmental isolation, it is important to understand any interactions (synergistic or antagonistic) which may occur. This problem has been approached in the mussel Mytilus edulis by exposing estuarine mussels to copper (20 μg litre⁻¹) and phenanthrene (100 μg litre⁻¹) both individually and in combination, and measuring cytochemical subcellular and physiological responses after 3 days exposure and 3 days and 12 days recovery period. Results showed that mussels accumulated both xenobiotics during 3 days exposure. Depuration of copper was complete in 3 days recovery period, while loss of phenanthrene ranged from 30% to 70% of the concentration reached after 3 days exposure. There were no interactive effects on depuration.Both copper and phenanthrene reduced lysosomal hydrolase latency in digestive cells, and copper appeared to have a synergistic effect in preventing recovery of latency of lysosomal N-acetyl-β-hexosaminidase during the recovery period. There was evidence, in the digestive cells, of an antagonistic effect of copper on stimulation of activity of the microsomal respiratory chain (measured as NADPH-neotetrazolium reductase) by phenanthrene. Stimulation of this system by phenanthrene persisted after 12 days recovery period. There was a synergistic interaction of copper and phenanthrene on elevation of oxygen consumption and ammonium excretion. Clearance rates and scope for growth (physiological condition) were depressed by copper but not by phenanthrene after 3 days exposure.These findings are discussed in terms of known effects of copper and phenanthrene and the interactions are considered in terms of environmental effects measurements.     

Polycyclic aromatic hydrocarbons in bivalves from the San Francisco estuary: Spatial distributions, temporal trends, and sources (1993-2001)

Bivalve tissue samples were examined over a range of spatial and temporal scales (1993-2001) to determine PAH distributions, trends, and possible sources. Mussels (Mytilus californianus), oysters (Crassostrea gigas), and clams (Corbicula fluminea) were deployed for three months in the estuary at stations remote from known point source discharges. The range of summation operatorPAH detected in bivalves was oysters 184-6899 microg/kg dry wt (mean 678 microg/kg dry wt), mussels 21-1093 microg/kg dry wt (mean 175 microg/kg dry wt), and clams 78-720 microg/kg dry wt (mean 323 microg/kg dry wt). Linear regression analysis showed no statistically significant (p>0.05) temporal trends in clam and mussel summation operatorPAH at any of the deployment stations or estuary segments. On the other hand, a statistically significant (p<0.05) decreasing trend was found in summation operatorPAH in oysters at the Petaluma River station, and in the North Estuary segment. PAH isomer pair ratios applied as diagnostic indicators suggested that the bioaccumulated PAH were derived primarily from petroleum combustion, with lesser amounts derived from biomass and coal combustion, and unburned petroleum.     

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.     

CYP1A- and CYP3A-immunopositive protein levels in digestive gland of the mussel Mytilus galloprovincialis from the Mediterranean Sea

CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6β-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r²=0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.     

A 15-month study of zooplankton ingestion by farmed mussels (Mytilus edulis) in Bantry Bay, Southwest Ireland

There is a growing body of evidence to suggest that bivalve molluscs routinely ingest zooplankton. To elucidate further these observations, a 15-month study of zooplankton ingestion by farmed mussels was conducted using mussel long-lines in Bantry Bay, Ireland. Stomach content analysis of the mussels showed that there was evidence of zooplankton ingestion throughout the sampling period, but that highest mean numbers of zooplankters were ingested by mussels in the spring and summer months. Various zooplankton species were present in mussel stomachs. Harpacticoid copepods were found more often in stomach contents than calanoid copepods, probably due to their proximity to the bivalves' inhalent siphons. Barnacle cyprids featured in large numbers in stomach contents, but only for a period of 3 months which broadly corresponded with their pelagic phase. Sizes of ingested zooplankton ranged from 126 μm to 6 mm, but more of the smaller zooplankters (e.g. crustacean nauplii) were ingested. When lengths of ingested copepods were compared with those found in plankton net samples, it was found that the net-sampled copepods were significantly larger than those found in mussel stomachs, suggesting that mussels select for smaller categories within the zooplankton available to them. Soft bodied zooplankton was rarely found in mussel stomachs but their absence may be due to rapid digestion or they may have been destroyed in the preservation process. Ingestion of zooplankton by bivalves is discussed in the context of the impacts mussel farms have on resident zooplankton populations.     

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.     

Concentrations of unsubstituted polynuclear aromatic hydrocarbons in bay mussels (Mytilus edulis) from Oregon, USA

Concentrations of fifteen unsubstituted polynuclear aromatic hydrocarbons (PNAH) were measured in Mytilus edulis from two sites in Yaquina Bay, Oregon, USA, during 1979–1980. There were significant differences in PNAH levels between the two populations. The average total concentration in mussels inhabiting the more industrialized bayfront was 986·2 μg/kg compared with 273·9 μg/kg in mussels from a more remote site across the bay. Substantial differences were found in the concentrations of different PNAH in M. edulis examined during this study. The smaller more water soluble, compounds were concentrated to one or two orders of magnitude above the larger, less soluble PNAH.     

Reproductive success, xenobiotic contaminants and hepatic mixed-function oxidase (MFO) activity in Platichthys stellatus populations from San Francisco Bay

To identify some specific effects of organic contaminants on fisheries in an urbanized estuary we compared the reproductive success of starry flounder from San Francisco Bay with concentrations of tissue contaminants and hepatic mixed-function oxidase (MFO) activity. We found significantly lower (P < 0·05) sediment concentrations of total identified polynuclear aromatic hydrocarbons (PAHs) in the less urbanized San Pablo Bay (SP) area (Fig. 1) than in the more urbanized central bay (CB) stations (Table 1). For flounder in early gametogenesis (August and September) the SP fish (n = 20) had significantly lower (P < 0·01) liver concentrations of Aroclor 1260 (0·34 ± 0·14 μg/g) than those at the CB stations: Berkeley (BK, n = 20, 1·6 ± 1·6 μg/g); Oakland (OK, n = 16, 2·3 ± 2·8 μg/g); and Alameda (AL, n = 4, 2·2 ± 1 μg/g). A similar pattern existed for DDT concentrations: SP = 0·2 ± 0·16 μg/g; BK = 0·1 ± 0·34 μg/g; OK = 0·4 ± 0·53 μg/g; and AL = 0·4 ± 0·33 μg/g. Total PAHs in livers were as follows: SP = 0·14 μg/g; BK = 2·6 μg/g; OK = 1·4 μg/g; and AL = 14 μg/g. Although gonad index, liver index, and presence of fin rot are inversely related to aryl hydrocarbon hydroxylase (AHH) activity, healthy fish in a similar reproductive state have lower AHH activities in the SP area. For example, in August and September, 1984, mean AHH activities were as follows: SP = 203 ± 89, and CB = 355 ± 200 pmol 3-OH-B[a]P mg microsomal protein min. We found a log-linear relationship for AHH activity and its percent inhibition by 7,8-benzoflavone (10⁻⁴m) and only a few fish from SP showed enhanced AHH activity after addition of 7,8-benzoflavone. This suggests that most of the starry flounder in San Francisco Bay are induced.     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.