Search

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; P0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P0.001). 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, P0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.

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.

Western analysis of non-peptide bond free carbonyls by derivatisation with 2,4-dinitrophenylhydrazine was applied to hepatic proteins of fish as a potential biomarker of contaminant-mediated oxidative damage. Analysis of 1000 and 9000 g supernatants and sonicated hepatocytes of flounder (Platichthys flesus) liver revealed multiple free carbonyl-containing bands (approximate molecular weight 20–60 kD). RNase and protease treatments and nucleic acid precipitation demonstrated that the assay was specific for protein carbonyls in fish liver. Free carbonyl levels increased with in vitro oxidation by a hydroxyl radical-generating system, indicating that specific target proteins should be identified as possible biomarkers of contaminant-mediated oxidative damage.

The toxic action of the organophosphate pesticide fenitrothion via metabolism to fenitrooxon was investigated in isolated mixed-population cells from the hepatopancreas of the non-target organism Procambarus clarkii. Glutathione-S-transferase (GST; substrate: 1-chloro-2,4-dinitrobenzene), 7-ethoxyresorufin O-deethylase (EROD) andacetylcholin esterase (AChE) specific activities (per mg protein) in 10000 g supernatants were 50–60% lower (GST, AChE), or markedly higher (EROD) in cells compared to whole tissues. However, EROD cell activity was similar to values recorded for whole tissue microsomes. Incubation (12 h) of isolated cells with up to 5 μM fenitrothion resulted in a marked inhibition (84%) of AChE activity, but an increase (50%) in GST activity (p < 0.05). Fenitrothion (5 μM) was readily metabolised to fenitrooxon and 3-methyl-4-nitrophenol. The disappearance of fenitrothion but not the formation of the metabolites was inhibited by 100 μM α-naphthoflavone, indicating the possible involvement of multiple cytochrome P450 forms in the metabolism of fenitrothion.

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.

The use of a suite of different biochemical markers in the deep sea fish Roundnose grenadier and caged common mussels, and analyses of contaminant levels in the mussels, indicate that Skagerrak are more polluted than waters around the Faroe Islands. In Roundnose, induction of ethoxyresorufin-O-deethylase (EROD) is consistent with increased benzo(a)pyrene hydroxylase (BPH) in mussels and elevated levels of polynuclear aromatic hydrocarbons (PAHs) in mussels and sediment from Skagerrak. This evidence of a large-scale environmental impact of pollutants should lead to further efforts to decrease transport into, and deposition of, waste compounds in the Skagerrak area.

Nitroaromatics may be metabolized both to reactive electrophiles and to mediators of reactive oxygen species (ROS) formation. Hepatic effects of nitroaromatic compounds were investigated in three species of flatfish. No changes were seen in 7-ethoxyresorufin O -deethylase (EROD) activity in turbot ( Scophthalmus maximus ) exposed (i.p. 48 h) to either 1-nitropyrene or nitrofurantoin, indicating non-induction of cytochrome P4501A. Similarly, no changes were seen in any antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase, DT-diaphorase, aldehyde dehydrogenase). DNA oxidative damage was indicated to be a widespread endogenous phenomenon by the presence of 8-hydroxydeoxyguanosine (8OHdG) in liver of S. maximus , dab ( Limanda limanda ) and sole ( Solea solea ). No increase in 8OHdG levels were seen in S. maximus, L. limanda or S. solea exposed to nitrofurantoin. The results for 8OHdG were in contrast to those reported for English sole ( Parophrys vetulus ) which showed an increase in hepatic levels following similar nitrofurantoin treatment (Nishimoto et al., 1991). 32 P-post-labelling analysis of liver from exposed S. maximus showed no evidence of DNA adducts with chomatographic conditions able to detect 1-nitro-pyrene-derived DNA adducts in rat. None of the various parameters studied appear to be suitable as biomarkers for the detection of exposure to nitroaromatics in the aquatic environment.

The presence and putative catalytic properties of a CYP1A-like enzyme in the digestive gland of Mytilus edulis L. were investigated by molecular biological and seasonal studies. Reverse-transcriptase PCR using oligonucleotide primers to amplify a sequence around the conserved haem binding cysteine region of hepatic CYP1A1 of trout (Oncorhynchus mykiss) produced several cDNA bands resolved by electrophoresis, including major bands of about 220 and 280 bp compared to the predicted size for O. mykiss of 208 bp. Following Southern blotting and probing with a cDNA probe to O. mykiss CYP1A1 (pfP1450-3′ probe modified by removal of 3′-non-coding region by digestion by Cla1), a single band (280 bp) only was detected using moderate stringency conditions of sequence recognition (i.e. hybridization at 42 °C followed by washing at 55 °C in 1 × SSC containing 0.1% SDS), providing evidence for the presence of a CYP1A orthologous gene sequence. The seasonal variation in levels of putative CYP1A mRNA (Northern analysis using the modified cDNA probe for O. mykiss CYP1A1) over 1 year showed some similarity to seasonal patterns of change in microsomal metabolism of 3H-benzo[a]pyrene (BaP) to polar metabolites (dials, diones and phenols resolved by HPLC). Maxima for putative CYP1A mRNA and BaP metabolism levels were in late spring-early summer. However, differences were also apparent, possibly indicative of other P450s contributing to BaP metabolism. Overall the results indicate the existence of a CYP1A-like enzyme which is, at least, partly responsible for the mono-oxygenase activity of BaP metabolism.

Studies were carried out to examine the role of aromatic hydrocarbon (AH)-quinone metabolism and DT-diaphorase (quinone oxidoreductase; EC 1.6.99.2; DTD) function in pollution-caused oxidative damage in fish. Redox cycling of quinones to produce reactive oxygen species (ROS) was studied by oxygen consumption and oxidation of the hydroxyl radical scavenger 2-keto-4-methiolbutyric acid. NADH-dependent redox cycling by hepatic microsomes of flounder (Platichthys flesus) was seen for 1,4-benzoquinone, 1,2- and 1,4-naphthoquinones, phenanthroquinone and anthraquinone. Combined with previous similar results for duroquinone, menadione and benzo[a]pyrene (BaP) quinones (Lemaire et al., 1994), this demonstrates a general potential for AH-quinone-mediated ROS production. NADPH-dependent 3H-BaP metabolism by hepatic microsomes of P. flesus produced 54% dials, 45% phenols and trace levels of quinones, whereas cumene hydroperoxide-dependent metabolism produced up to 67% quinones (metabolites resolved by HPLC). Exposure to AH-contaminated sediment can lead to enhanced hepatic lipid peroxidation (Livingstone et al., 1993) raising the possibility of an AH-mediated toxicity cycle of peroxidation leading to enhanced quinone and ROS production leading to more peroxidation and oxidative damage. Hepatic DTD purified almost to homogeneity from trout (Oncorhynchus mykiss); Sturve et al., in prep.) also catalysed NADH-dependent AH-quinone-mediated ROS production, indicating a possible function for this enzyme in quinone toxicity.

The use of cytochrome P4501A (CYP1A) and other measurements as biomarkers was investigated in liver of goby (Z. ophiocephalus) and digestive gland of mussel (M. galloprovincialis) from several sites in the Venice lagoon as part of the UNESCO-MURST Venice Lagoon Ecosystem Project. Most tissue contaminants (PAHs, PCBs, DDTs) and biochemical measurements varied seasonally. Elevated 7-ethoxyresorufin O-deethylase activity and CYP1A-protein levels in goby were correlated with high tissue contaminant levels at the industrial Porto Marghera site. On occasions, activities of the antioxidant enzymes catalase and putative DT-diaphorase (resorufin reductase activity) in male but not female goby were also higher at Porto Marghera than other sites, but no differences were seen in Superoxide dismutase (SOD) activity. A range of measurements (SOD, catalase, NADPH-cytochrome c reductase and glutathione S-transferase activities, P450 and ‘418-peak’ contents) in mussel showed little difference between sites. However, indications were obtained of elevated levels of CYP1A1-like mRNA, CYP1A-like protein and metabolism of benzo[a]pyrene to free metabolites in mussels from the Venice lagoon compared to a site in the Adriatic Sea. The studies demonstrate the usefulness of CYP1A as a biomarker for organic pollution in fish and indicate some potential for its application in molluscs.

The potential of benzo[ a]pyrene (BaP) quinones (diones) to stimulate NAD(P)H-dependent hydroxyl radical (·OH) production (2-keto-4-methiolbutyric acid (KMBA) oxidation) by digestive gland microsomes of M. edulis was studied using iron/EDTA as a promotor of the Haber-Weiss reaction (O2− + H2O2 = ·OH + OH− + O2). Stimulation of basal rates of KMBA oxidation was observed for all three diones. Stimulation was similar for the 1,6- and 3,6-diones, but much less for the 6,12-dione, and greater for the NADH than the NADPH-dependent reaction, viz. % increases of 78 to 333 (NADH) compared to 0 to 78 (NADPH). Maximal KMBA oxidation was obtained at dione concentrations of 12.5 to 50 μm. Inhibition of KMBA oxidation by Superoxide dismutase and catalase indicated the involvement of respectively Superoxide anion radical (O2−) and hydrogen peroxide (H2O2) in 1,6-dione stimulated NADPH-dependent · OH formation. The apparent Km values of xenobiotic-stimulated KMBA oxidation were lower for BaP-diones than for the model redox cycling quinone menadione (2-methyl-1,4-naphthoquinone), viz. in μm, 0.6 to 14.6 (NADH) and 4.4 to 28.5 (NADPH) compared to 315 to 457 (NADH) and 72 to 103 (NADPH). The results are consistent with metabolism of BaP to diones and resultant enhanced generation of oxyradicals being a potential mechanism of pollutant-mediated toxicity in molluscs.

The effects of the mammalian inhibitor ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b] carbazole) were examined in a mechanistic study of the cytochrome P450 monooxygenase system of control and β-naphthoflavone (βNF)-induced hepatic microsomes of Platichthys flesus. Ellipticine was indicated to bind to the haem moiety of cytochrome P450s (gave type II binding spectra) and to inhibit the transfer of electrons from both the hydrophobic binding site of cytochrome P450 reductase (P450R) to P450 (inhibited P450R reductase activity) and the hydrophilic binding site of P450R to soluble electron acceptors (inhibited NAD(P)H-cytochrome c reductase activity). No effect was seen on cytochrome b5 reductase activity. Ellipticine inhibition indicated the involvement of (i) P450R (possibly also P450s) in NADPH- but not NADH- dependent hydroxyl radical production, and (ii) electron transfer and P450/P450R interaction in NADPH-dependent cytochrome P450 1A-catalysed monooxygenation (7-ethoxyresorufin O-deethylase activity and benzo(a)pyrene (BaP) metabolism). Differential effects of ellipticine on cumene hydroperoxide (CHP)-dependent BaP metabolism (P450 peroxidase activity) with CHP concentration indicated the existence of at least two forms of P450 with different substrate affinities for CHP, and different mechanisms of formation for protein adducts and free metabolites. Overall, the studies indicate the primary site of action of ellipticine in P. flesus is binding between Fe3+-P450 and P450R.

The presence and expression of four gene families of the cytochrome P450 multi-gene family (CYP450) was investigated in the digestive gland of M. edulis using Southern and Northern blot analysis. The cDNA probes employed were for CYP1A1 (pfP1450-3′, 1.5kb from rainbow trout), CYP3A (phPCN12, human), CYP4A1 (2.1kb, rat) and CYP1A1 (1.9kb, human). Single hybridising mRNAs of approximately 2.1kb were seen for all probes, indicating presence and expression of genes from or similar to the four families. Hybridisation on Southern blots was demonstrated for CYP3A and CYP11A, confirming the presence of related CYP genes. The former gave a strong band at about 4.5kb and a weak one at 7.3kb, and the latter a single band at 13kb. The detection of CYP3A-like sequences is consistent with the reported presence of testosterone 6-β-hydroxylase activity in M. edulis. Marked variation in levels of CYP1A-like mRNA were seen in mussels from several sites in the Venice area (UNESCO-MURST Venice Lagoon Project), indicating modulation of gene expression. Some correlation with contaminant exposure was seen, indicating potential for use of this measurement as a biomarker for organic pollution.

Cytochrome P4501A (CYP1A) has been investigated extensively in the liver of adults of many different fish species. This study reports the presence and elevation of CYP1A measured as 7-ethoxyresorufin O-deethylase (EROD) activity and protein amount (Western blot — semi-quantified by image analysis) in larval, juvenile and adult turbot (Scophthalmus maximus) after exposure to organic contaminant and model inducers. Basal EROD activity was not detectable in embryos, but increased from 3 day larvae (whole body 13 500g supernatants) through 90 day juveniles (hepatic 13 500g supernatants) to adults (hepatic microsomes), respectively in pmol/min/mg protein (± SEM), 0.57 ± 0.06, 10.8 ± 2.4 and 12.3 ± 4.7. Exposure to 5 ppb benzo[a]pyrene (B[a]P), 1 ppb γ-hexachlorocyclohexane (lindane) and 25 ppb B[a]P caused respectively, 3-, 6- and 2-fold elevation of EROD activity in 4 and 9 day larvae and juvenile stages. Image analysis of Western blots of juvenile samples detected a 3-fold difference in CYP1A protein whereas EROD activity of the same sample varied 4-fold. The results confirm the potential of using CYP1A induction as a biomarker for impact by organic pollution on early developmental stages of fish.

Cytochrome P450 from the digestive gland of M. edulis was partially purified by sodium cholate solubilization, 4–15% polyethylene glycol fractionation, and octyl-Sepharose affinity, DEAE-Sephacel ion-exchange and hydroxylapatite chromatography (yields of up to 7–10%). Three peaks were resolved by DEAE-Sephacel chromatography (termed peaks 1–3). P450 specific content was increased from 26 to 800 pmol per mg protein, and the ratio of P450 content to NADPH-cytochrome c (P450) reductase activity reduced by a factor of 250. Oxidised spectrum λmax of P450 was 410.5 ± 1.5 nm. Type II difference spectra were seen with both type II (clotrimazole, metyrapone) and type I (α-naphthoflavone, 7-ethoxycoumarin) compounds. Western blotting with polyclonal anti-P4501A from perch (Perca fluviatilis) gave a single band of approximately 54 kDa molecular weight. A reconstituted system containing peak 2 or 3, rat liver P450 reductase, lipid and NADPH metabolised benzo[a]pyrene to diones, diols, phenols and putative protein adducts. Peak 2 plus cumene hydroperoxide was indicated to produce diones and protein adducts only. Peak 2 alone was indicated to produce diones and phenols. The major free metabolites in all cases were diones (53–100%). The results indicate the existence of a P4501A-like enzyme in M. edulis, possibly with unusual properties as indicated by the difference spectra, metabolism in absence of NADPH and added P450 reductase, and predominance of diones.

The possible utility of glutathione S-transferase isoenzymes in affinity purified extracts of digestive gland from Mytilus galloprovincialis as indicators of exposure to organic pollution has been assessed by fast protein liquid chromatography (FPLC) using a Mono-Q ion exchange column. Four main peaks of protein (isoenzymes 1–4) were detected in these chromatographic studies all of which displayed detectable catalytic activity with 1-chloro-2,4-dinitrobenzene and glutathione as substrates. The bulk of the activity with these substrates was associated with isoenzyme 1. The specific activity of cytosolic extracts from six sites in Venice Lagoon and the Adriatic Sea (UNESCO-MURST Venice Lagoon Ecosystem Project) decreased slightly with increasing tissue levels of chemical contaminants (hydrocarbons, PCBs and organochlorines). FPLC of affinity purified extracts from three of these sites was used to assess the possible variation in the levels of individual isoenzymes relative to pollution status. No difference was observed in the levels of these isoenzymes.

Seven marine and five freshwater fish species were compared in terms of their activities of eight liver xenobiotic and oxyradical metabolizing enzymes namely those of the mammalian [Ah] gene battery, viz. cytochrome P450 1A (7-ethoxyresorufin-O-deethylase-EROD), glutathione S-transferase, UDP glucuronosyl transferase, DT-diaphorase, aldehyde dehydrogenase, and the antioxidant enzymes catalase, glutathione peroxidase and Superoxide dismutase. A number of these enzyme activities are in use or have been proposed for use as biomarkers for aquatic pollution. Inter-species variations were observed in all enzyme activities. EROD exhibited the greatest variation (> 300-fold), followed by putative DT-diaphorase (20–100-fold). In most other enzyme activities the interspecies variations were in the order of 5- to 10-fold. The data from this comparative study of biomarker enzymes in different fish species provide a valuable basis for future enzyme regulation studies and biomonitoring investigations.

Benzo[a]pyrene (BaP) metabolism was studied in pyloric caeca microsomes of the sea star, Asterias rubens. BaP metabolites were identified by HPLC retention and quantified radiometrically. BaP was metabolized into phenols, quinones and dihydrodiols. Monohydroxylated BaP comprised about 40% of the total metabolites produced. Protein adducts of BaP appeared to be formed. Cumene hydroperoxide or NADH also supported BaP metabolism, causing a shift towards dihydrodiol and quinone formation, and at a total rate of metabolism higher than with NADPH.

The modulation of the mutagenicity of 4-nitroquinoline N-oxide (4-NQO) by M. edulis digestive gland cytosolic and microsomal fractions was studied by monitoring the induction of the umu operon by 4-NQO in Salmonella typhimurium 1535/pSK1002. The mutagenicity of 4-NQO was increased by M. edulis cytosol and microsomes in dose-dependent manner with respect to protein and toxicant concentration. The mutagenic enhancement reflected increased NAD(P)H-dependent oxygen consumption by microsomes in the presence of 4-NQO and enhanced nitroreductase activity in cytosol. The former was stimulated by azide, indicating 4-NQO-enhanced oxyradical production by microsomes, and the latter was inhibitable by dicoumarol, indicating involvement of DT-diaphorase in nitroreduction by M. edulis cytosol. Neither dicoumarol nor allopurinol (inhibitors of DT-diaphorase and xanthine oxidase, respectively) affected the enhanced mutagenic expression of 4-NQO by cytosol. The results support the possibility of oxidative stress as a pollution-mediated mechanism of toxicity.

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

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

The present study investigated the proposed involvement of contaminant-stimulated reactive oxygen species (ROS) production in disease processes in fish. NAD(P)H-dependent ROS production of subcellular fractions was determined by the iron/EDTA-mediated oxidation of 2-keto-4-methiolbutyric acid. Hepatic cytosolic NADPH-dependent and microsomal NAD(P)H-dependent ROS production were increased 51--160% (P < 0.05) in rainbow trout (Oncorhynchus mykiss) 15 weeks after a single i.p. injection of polychlorobiphenyl (PCB) (100 mg Clophen A50 kg -1 wet wt.). Hepatic microsomal NADH-dependent ROS production was 114% higher in perch (Perca fluviatilis) from PCB-contaminated Lake Jarnsjon compared to clean Lake Vanern, Sweden. Hepatic mitochondrial NADH-dependent, cytosolic NADH-dependent and microsomal NADPH-dependent ROS production were variously elevated up to 160% in flounder (Platichthys flesus) at various sites along two pollution transects near to the ports of Rotterdam and Amsterdam, Netherlands. Overall the data indicate increased potential for ROS production in liver of fish exposed to field pollution, and support the hypothesis of oxidative stress as a mechanism of contaminant-mediated disease in fish.

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 (P0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P0.05).

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.