Philippe Lemaire

Assessment of the impact of organic pollutants on goby (Zosterisessor ophiocephalus) and mussel (Mytilus galloprovincialis) from the Venice Lagoon, Italy: Biochemical studies

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.

Pro-oxidant, antioxidant and 7-ethoxyresorufin O-deethylase (EROD) activity responses in liver of Dab (Limanda limanda) exposed to sediment contaminated with hydrocarbons and other chemicals☆

Abstract Dab were exposed to reference sediment (79 μg PAHs g −1 dry wt) or contaminated sediment (309 μg PAHs g −1 dry wt plus low PCBs) for up to 140 days, and the effects on liver cytochrome P4501A (EROD activity), antioxidant enzymes and oxidative damage (lipid peroxidation) examined. EROD activity in the contaminated condition increased ×80 from 25 to 80 days, decreasing to 22% of this activity after 140 days. EROD activity also increased in the reference condition, but only after 140 days. Superoxide dismutase (SOD) and catalase activities and lipid peroxidation (malonaldehyde equivalents) were higher in the contaminated than the reference condition after 80 days but not 140 days. No differences were seen in glutathione peroxidase activity. It is concluded that the changes in SOD, catalase and lipid peroxidation are indicative of contaminant-mediated oxidative stress.

Responses of hepatic biotransformation and antioxidant enzymes to CYP1A-inducers (3-methylcholanthrene, β-naphthoflavone) in sea bass (Dicentrarchus labrax), dab (Limanda limanda) and rainbow trout (Oncorhynchus mykiss)

Abstract The time-course responses of hepatic biotransformation and antioxidant enzymes to cytochrome P4501A-inducers in fish were examined using marine and freshwater species, viz. respectively, sea bass (Dicentrarchus labrax) and dab (Limanda limanda) exposed up to 5 to 11 days to 3-methylcholanthrene (3MC; intraperitoneal (i.p.) 20 mg kg−1), and rainbow trout (Oncorhynchus mykiss) exposed up to 15 days to β-naphthoflavone (BNF; i.p. 5 mg kg−1). The enzymes included those which are part of the mammalian [Ah]-gene locus, viz. cytochrome P4501A (CYP1A), DT-diaphorase (DTD; EC 1.6.99.2), aldehyde dehydrogenase (ADH; EC 1.2.1.3), glutathione S-transferase (GST; EC 2.5.1.18) and UDP-glucuronyltransferase (UDPGT). DTD was assayed as dicoumarol-inhibitable menadione reductase, dicoumarol-inhibitable dichlorophenolindophenol (DCPIP) reductase and resorufin reductase activities; and ADH as benzaldehyde dehydrogenase (BADH) and propionaldehyde dehydrogenase (PADH) activities. The responses varied considerably with enzyme, species and treatment. CYP1A-dependent 7-ethoxyresorufin O-deethylase (EROD) activity was markedly inducible, more so in BNF-treated O. mykiss (maximal 284-fold increase after 2 days) than the 3MC-treated species (maximal 24–30-fold). Modest increases (up to 2-fold) and/or inhibition were seen in UDPGT and GST activities. DTD activities increased about 2-fold after 8 to 15 days in BNF-treated O. mykiss, but were not elevated in the other species. Changes in dicoumarol-inhibitable DCPIP and menadione reductase activities were more similar than resorufin reductase activity. No marked increases were evident in BADH, PADH, superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione peroxidase (GPX; EC 1.11.1.9) activities in any species. Overall, some co-induction of CYP1A and DTD, over different time-courses, was indicated for BNF-treated O. mykiss.

Interactive effects of cadmium and benzo(a)pyrene on cellular structure and biotransformation enzymes of the liver of the european eel Anguilla anguilla

Abstract Interactive effects of cadmium and benzo(a)pyrene (BaP) on eel Anguilla anguilla liver were studied at the ultrastructural and biotransformation enzyme levels. Seawater-adapted eels were held for 24 days in either clean or cadmium-containing (5 μg Cd −1 ) seawater. then given a single intraperitoneal injection of BaP (20 mg kg −1 body weight) and finally sacrificed 24 h later. Cadmium exposure alone caused a hepatic perivascular fibrosis. whereas the short-term BaP intoxication alone produced marked lipid droplet accumulation associated with glycogen depletion. When eels were exposed to both toxicants together, the liver showed a complete disorganisation of the hepatic parenchyma including nuclear degeneration. The existence of elements of a biotransformation enzyme system, i.e. cytochrome P-450 monooxygenase system and glutathione-S-transferase (GST). was demonstrated in the liver. BaP injection alone had no detectable effect on microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity but markedly increased 7-ethoxyresorufin O -deethylase (EROD) and benzo(a)pyrene hydroxylase (BaPMO) activities (induction factors of 15- and 35-fold, respectively). In contrast, cytosolic GST activity was slightly decreased by this treatment. In cadmium-exposed eels, BaP treatment elicited a higher increase in EROD and BaPMO activities (induction factors of 19- and 71-fold. respectively) and also causes a 1.8-fold increase in cylochrome P-450 microsomal content. The effects of cadmium on biotransformation enzymes are discussed in relation to the ultrastructural effects of both toxicants.

Effects of cadmium and benzo(a)pyrene on the immune system, gill ATPase and EROD activity of European sea bass Dicentrarchus labrax

Juvenile European sea bass (Dicentrarchus labrax) were exposed to cadmium (40 μg l−1) for 15 days and then injected with benzo(a)pyrene (BaP) (20 mg kg−1) for 14 h. In the immune system, phagocytosis was reduced both in spleen and kidney macrophages after cadmiumexposure and/or BaP injection, with a synergistic effect of the combined pollutants. Spleen and kidney respiratory burst (measured by hydrogen peroxide production) showed different patterns of response to cadmium and BaP. Cadmium exposure had no effect on spleen macrophages, although BaP totally inhibited the respiratory burst. In kidney macrophages, cadmiumexposure and BaP injection resulted in enhanced hydrogen peroxide production. After BaP injection, hepatic micrososomal 7-ethoxyresorufin-O-deethylase (EROD) activity was increased 21-fold compared with the control, but the induction was even greater (34.5 times compared with the control) in cadmium-exposed fish. However, although an induction (11 times compared to the control) occurred after cadmium exposure the effects of both pollutants were not synergistic (not additional). No perturbation of the gill Na+/K+-ATPase activity was observed in cadmium-exposed fish, but the activity was increased in BaP-injected fish. The role of BaP metabolites and cadmium as both direct effectors and indirect stressors, responsible for the changes in the immune system and gill and liver biochemistry of the fish are discussed.

Stimulation of oxyradical production of hepatic microsomes of flounder (Platichthys flesus) and perch (Perca fluviatilis) by model and pollutant xenobiotics

Stimulation of hepatic microsomal NAD(P)H-dependent hydroxyl radical (·OH) production by model compounds, viz. menadione (2-methyl-1,4-naphthoquinone) and nitrofurantoin (N-(5-nitro-2-furfurylidene)-1-aminohydantoin), and pollutant xenobiotics, viz. benzo[a]pyrene (BaP) diones (products of microsomal BaP metabolism), duroquinone (tetramethyl-1,4-benzoquinone—present in pulp mill effluent), and the pesticide lindane (γ-hexachlorocyclohexane), was examined in flounder Platichthys flesus. Duroquinone was also studied in perch Perca fluviatilis, a freshwater species used in studies of pulp mill effluents in the aquatic environment. Microsomal ·OH production was detected by the oxidation of the scavenging agent 2-keto-4-methiolbutyric acid (KMBA), using FeCl3/EDTA as a promotor of the Haber-Weiss reaction (O2−+H2O2=·OH+OH−+O2). All xenobiotics tested, except lindane, showed synergistic interactions with ferric/EDTA indicative of redox cycling of the xenobiotic. Inhibition of menadione- and nitrofurantoin-stimulated ·OH production by superoxide dismutase (50% inhibition) and catalase (80%) indicated respectively the involvement of O2−and H2O2 in ·OH production. Maximal rates of KMBA oxidation (Vmax in nmol ethylene/min/mg protein) were similar for NADH and NADPH for menadione (4.58–4.61) and duroquinone (0.26–0.3 [flounder] and 0.93–0.99 [perch]), higher for NADPH than NADH for nitrofurantoin (1.21 and 0.77), and higher for NADH than NADPH for BaP diones, decreasing in the order 1,6-dione (1.12 and 0.14), 3,6-dione (0.75 and 0.25), and 6,12-dione (0.31 and 0.09). Rates for lindane, lacking a redox cycling structure, were low (0.01–0.05). Apparent Km (app. Km) values for xenobiotic were 1–2 orders of magnitude lower for BaP diones than the other compounds. App. Km was lower for NADH than NADPH for 3,6-dione (1.23 and 1.66 μM) and 6,12-dione (0.85 and 1.81 μM), but the reverse of this was found for the 1,6-dione (1.41 and 0.78 μM). App. Km values were almost identical for menadione and duroquinone and lower for NADPH (32–44 μM) than NADH (346–382 μM). The reverse was seen for nitrofurantoin, viz., 76 μM (NADH) and 269 μM (NADPH). Hepatic 1000 g supernatants of P. flesus metabolized BaP to oxyradical-generating products, moreso for β-naphthoflavone-induced than control fish, and production was reduced by UDP-glucuronic acid for the latter but not the former. The studies indicate a widespread potential for contaminant-stimulated oxyradical generation via redox cycling and other free radical interactions of xenobiotics and their metabolites.

Mixed-function oxygenase enzymes as tools for pollution monitoring: field studies on the French coast of the Mediterranean Sea.

1. MFO enzyme activities were measured in microsomes from whole mussels (Mytilus galloprovincialis) comber livers (Serranus cabrilla), or Posidonia oceanica etiolated tissues, and PAH contents were determined in sediments collected in coastal locations of the French Riviera and Corsica during 3 oceanographic cruises in 1987-1988. 2. BaP activities in mussel and EROD activities measured in fish were strongly correlated to the log of PAH content in sediments. The first results for CA4H in Posidonia showed significant differences related to PAH pollution levels. The increase in MFO activities measured in Corsica in summer 1988 indicated a recent petroleum contamination.

Time-course and dose-response of the apparent induction of the cytochrome P450 monooxygenase system of pyloric caeca musomes of the female sea star Asterias rubens L. by benzo[a]pyrene and polychlorinated biphenyls

Abstract A study was made on the time-course of putative monooxygenase (MO) induction and the dose-response relationships for the effects of 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC congener 126), 2,3′,4,4′,5-pentachlorobiphenyl (congener 118), 2,2′,4,4′,5,5′-hexachlorobiphenyl (congener 153) and benzo[a]pyrene (BaP) on MO-system activities in female sea stars Asterias rubens L. Sea stars received a single injection of PCB or BaP at different concentrations. Cytochrome P450 concentration and activities of NADPH-cytochrome c (P450) reductase, BaP hydroxylase (BPH) and prenenolone hydroxylase were determined in musomal fractions of the pyloric caeca. Apparent induction of BPH activity by PCB was observed only with CB-126 (3-methylcholanthrene (3-MC)-type inducer) at 10 μmol/kg. BPH activity was not affected by CB-153 (phenobarbital-type inducer) or CB-118 (mixed-type inducer). BaP (3-MC-type inducer) elicited a clear dose-dependent BPH induction in the range 10–160 μmol/kg. The maximum observed increase in BPH activity was about 350%. In time-course studies with CB-126 and BaP, highest BPH activities were found 3–4 days after injection. HPLC analysis of musomal BaP metabolites revealed that the elevated metabolism was shifted towards phenol production. PCB or BaP had no effect on the level of cytochrome P450 or the NADPH-cytochrome c reductase activity. All three PCBs and BaP inhibited pregnenolone hydroxylase activity, but no dose-response relationships were found. Overall the results suggest that the echinoderm MO system is inducible in response to exposure to cytochrome P450 1A-type inducers. However, the mechanism of induction remains unclear.

Comparative studies of hepatic xenobiotic metabolizing and antioxidant enzymes in different fish species

Abstract 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.

Partial purification and properties of cytochrome P450 from digestive gland microsomes of the common mussel, Mytilus edulis L.

Abstract 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.