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Components of the cytochrome P450-dependent monooxygenase system and 'NADPH-independent benzo[a]pyrene hydroxylase' activity in a wide range of marine invertebrate species
Abstract
Levels of components of the cytochrome P450 (CYP)-dependent monooxygenase system were characterised in microsomes of major biotransformation tissues, or whole bodies, of 33 species from six phyla of aquatic invertebrates. The phylogenetic distribution of benzo[a]pyrene hydroxylase (BPH) activity in the absence of added NADPH (so-called 'NADPH-independent BPH activity') and presence of NADPH was also examined. Microsomal protein yield was higher in individual tissues than whole tissues. The main components (total CYP and cytochrome b5; NADPH-dependent cytochrome c (CYP) reductase, NADH-dependent cytochrome c reductase and NADH-dependent ferricyanide (b5) reductase activities) were found in most species of the Porifera, Cnidaria, Mollusca, Polychaeta, Crustacea and Echinodermata examined. The so-called '418-peak' of the carbon-monoxide difference spectrum of reduced microsomes was found in all species, indicating the wide distribution of this protein. Total CYP levels (pmol mg(-1) protein; mean+/-SEM) were similar in molluscs (50+/-7), crustaceans (61+/-11) and echinoderms (56+/-9), with the exception of high levels (223-266) in two crustacean species. NADPH-dependent BPH activity (pmol min(-1) mg(-1) protein) was found in 32 species, being lowest in Porifera and Cnidaria (3-4), intermediate in Mollusca (7.8+/-1.3), and highest in Crustacea (25+/-4) and Echinodermata (15+/-4). NADPH-independent BPH activity was evident in 13 out of 15 molluscan species examined, with the addition of NADPH either stimulating (8 species) or inhibiting (5 species) the activity. NADPH-independent BPH activity was also seen in two poriferan species and indicated in three crustacean species, suggesting that the phenomenon is not solely restricted to the Mollusca.
... The S9 fraction was obtained by homogenising the samples in a phosphate buffer (100 mM, pH 7.4) containing 150 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM dithiothreitol (DTT) at a 1:5 (w:v) ratio and centrifuging at 10,000×g for 30 min. Resulting homogenate supernatants were further centrifuged at 100,000×g for 1 h to yield the cytosolic (supernatant) and microsomal (pellet) fractions as described in more detail by Sole and Livingstone (2005). Analyses were carried out (when possible) in each of the three fractions (cytosol, S9 and microsomes) in search of the most appropriate fraction for each assay. ...
... Microsomal CYP-related determinations consisted in the following essays: (i) reductase activities, measured spectrophotometrically at 550 nm using NAD(P)H cytochrome c reductase and NADH-ferricyanide reductase as described by Sole and Livingstone (2005); (ii) catalytic O-deethylase activities of digestive gland CYPs, determined using fluorescent CYP-mediated substrates (ER 7-ethoxyresorufin, PR 7-pentoxyresorufin, BR 7-benzyloxyresorufin, MR methoxyresorufin; CEC 3-cyano-7-ethoxycoumarin); (iii) Odebenzyloxylase activity (BFCOD), using BFC (7benzyloxy-4-trifluoromethylcoumarin) and (iv) Odebenzylase activity using DBF (dibenzylfluorescein). All assay conditions were adapted from fish studies and used 50 μL of mussel microsomes. ...
... Thus, given that we aim to simplify monitoring protocols (by using, for example, a single subcellular fraction), these measurements were excluded from site comparisons. We could, however, observe that the activities (in nmol/min/mg prot) in mussels collected from the Ebre Delta followed the order NADPH cit c red. <NADH cit c red. <NADH-ferricyanide reductase as it was seen in other studies using invertebrates, including mussels (Sole and Livingstone 2005). CYP-related activities were only attempted in the microsomal fraction where these membrane-bound proteins are located. ...
Mussels are worldwide bioindicators in pollution monitoring since they fulfil the requirements for being good sentinels. However, some methodological concerns arise in the use of particular biomarkers, particularly those displaying low enzymatic rates and/or limited responsiveness to chemicals and biological-related variability. In the present study, the suitability of oxidative stress and detoxification parameters when using mussels as sentinels of polycyclic aromatic hydrocarbon (PAH) pollution is addressed. Present results show that the S9 subcellular fraction of the digestive gland in mussels is an adequate and convenient matrix where to measure most pollution-related biomarkers. Furthermore, this work constitutes the first evidence of the potential suitability of using particular carboxylesterase (CE) activities in determining PAHs exposure in mussels. This fact could imply the replacement of more controversial cytochrome P450 components (phase I oxidation), which are only measurable in microsomal fractions, by CEs (measured in S9 fractions) as good alternatives for phase I reactions in PAH-exposed mussels. Some methodological considerations, such as the need of including commercial purified proteins in biomarker determinations for quality assurance, are evaluated.
... The cytochrome P450-dependent monooxygenase system has also been shown to be involved in the detoxification of PAH benzo-a-pyrene, in two marine sponge species (Sol e and Livingstone, 2005). Lower yields of cytochrome P450 protein were detected in sponges compared with other Phyla (Cnidaria, Mollusca, Annelida, Arthropoda, Echinodermata and Chordata), but this could result from overall lower metabolic rates (Sol e and Livingstone, 2005). ...
... The cytochrome P450-dependent monooxygenase system has also been shown to be involved in the detoxification of PAH benzo-a-pyrene, in two marine sponge species (Sol e and Livingstone, 2005). Lower yields of cytochrome P450 protein were detected in sponges compared with other Phyla (Cnidaria, Mollusca, Annelida, Arthropoda, Echinodermata and Chordata), but this could result from overall lower metabolic rates (Sol e and Livingstone, 2005). Under PAH-contaminated conditions produced in the laboratory, PAH molecules interact with the aryl hydrocarbon receptor and induce the cytochrome P450 pathway (Regoli and Giuliani, 2014). ...
... Under PAH-contaminated conditions produced in the laboratory, PAH molecules interact with the aryl hydrocarbon receptor and induce the cytochrome P450 pathway (Regoli and Giuliani, 2014). The cytochrome P450 pathway is known to play an important role in oxidative stress responses (Sol e and Livingstone, 2005), which are induced in many organisms after exposure to PAHs (Nebert et al., 2000;Puga et al., 2002;Regoli and Giuliani, 2014). Oxidative stress is a consequence of an imbalance in the antioxidant system in an organism. ...
Sponges form an important component of benthic ecosystems from shallow littoral to hadal depths. In the deep ocean, beyond the continental shelf, sponges can form high-density fields, constituting important habitats supporting rich benthic communities. Yet these habitats remain relatively unexplored. The oil and gas industry has played an important role in advancing our knowledge of deep-sea environments. Since its inception in the 1960s, offshore oil and gas industry has moved into deeper waters. However, the impacts of these activities on deep-sea sponges and other ecosystems are only starting to become the subject of active research. Throughout the development, operation and closure of an oil or gas field many activities take place, ranging from the seismic exploration of subseafloor geological features to the installation of infrastructure at the seabed to the drilling process itself. These routine activities and accidental releases of hydrocarbons during spills can significantly impact the local marine environment. Each phase of a field development or an accidental oil spill will therefore have different impacts on sponges at community, individual and cellular levels. Legacy issues regarding the future decommissioning of infrastructure and the abandonment of wells are also important environmental management considerations. This chapter reviews our understanding of impacts from hydrocarbon exploration and exploitation activities on deep-sea sponges and the habitats they form. These impacts include those (1) at community level, decreasing the diversity and density of benthic communities associated with deep-sea sponges owing to physical disturbance of the seabed; (2) at individual level, interrupting filtration owing to exposure to increased sedimentation; and (3) at cellular level, decreasing cellular membrane stability owing to exposure to drill muds. However, many potential effects not yet tested in deep-sea sponges but observed in shallow-water sponges or other model organisms should also be taken into account. Furthermore, to the best of our knowledge, no studies have shown impact of oil or dispersed oil on deep-sea sponges. To highlight these significant knowledge gaps, a summary table of potential and known impacts of hydrocarbon extraction and production activities combined with a simple “traffic light” scheme is also provided.
... The cytochrome P450-dependent monooxygenase system has also been shown to be involved in the detoxification of PAH benzo-a-pyrene, in two marine sponge species (Sol e and Livingstone, 2005). Lower yields of cytochrome P450 protein were detected in sponges compared with other Phyla (Cnidaria, Mollusca, Annelida, Arthropoda, Echinodermata and Chordata), but this could result from overall lower metabolic rates (Sol e and Livingstone, 2005). ...
... The cytochrome P450-dependent monooxygenase system has also been shown to be involved in the detoxification of PAH benzo-a-pyrene, in two marine sponge species (Sol e and Livingstone, 2005). Lower yields of cytochrome P450 protein were detected in sponges compared with other Phyla (Cnidaria, Mollusca, Annelida, Arthropoda, Echinodermata and Chordata), but this could result from overall lower metabolic rates (Sol e and Livingstone, 2005). Under PAH-contaminated conditions produced in the laboratory, PAH molecules interact with the aryl hydrocarbon receptor and induce the cytochrome P450 pathway (Regoli and Giuliani, 2014). ...
... Under PAH-contaminated conditions produced in the laboratory, PAH molecules interact with the aryl hydrocarbon receptor and induce the cytochrome P450 pathway (Regoli and Giuliani, 2014). The cytochrome P450 pathway is known to play an important role in oxidative stress responses (Sol e and Livingstone, 2005), which are induced in many organisms after exposure to PAHs (Nebert et al., 2000;Puga et al., 2002;Regoli and Giuliani, 2014). Oxidative stress is a consequence of an imbalance in the antioxidant system in an organism. ...
Sponges form an important component of benthic ecosystems from shallow littoral to hadal depths. In the deep ocean, beyond the continental shelf, sponges can form high-density fields, constituting important habitats supporting rich benthic communities. Yet these habitats remain relatively unexplored. The oil and gas industry has played an important role in advancing our knowledge of deep-sea environments. Since its inception in the 1960s, offshore oil and gas industry has moved into deeper waters. However, the impacts of these activities on deep-sea sponges and other ecosystems are only starting to become the subject of active research. Throughout the development, operation and closure of an oil or gas field many activities take place, ranging from the seismic exploration of subseafloor geological features to the installation of infrastructure at the seabed to the drilling process itself. These routine activities and accidental releases of hydrocarbons during spills can significantly impact the local marine environment. Each phase of a field development or an accidental oil spill will therefore have different impacts on sponges at community, individual and cellular levels. Legacy issues regarding the future decommissioning of infrastructure and the abandonment of wells are also important environmental management considerations. This chapter reviews our understanding of impacts from hydrocarbon exploration and exploitation activities on deep-sea sponges and the habitats they form. These impacts include those (1) at community level, decreasing the diversity and density of benthic communities associated with deep-sea sponges owing to physical disturbance of the seabed; (2) at individual level, interrupting filtration owing to exposure to increased sedimentation; and (3) at cellular level, decreasing cellular membrane stability owing to exposure to drill muds. However, many potential effects not yet tested in deep-sea sponges but observed in shallow-water sponges or other model organisms should also be taken into account. Furthermore, to the best of our knowledge, no studies have shown impact of oil or dispersed oil on deep-sea sponges. To highlight these significant knowledge gaps, a summary table of potential and known impacts of hydrocarbon extraction and production activities combined with a simple "traffic light" scheme is also provided.
... The most currently selected biomarkers in pollution monitoring studies are those from the enzymatic pool involved in the metabolism of foreign (and endogenous) chemicals. Among them, the specific activity of CYP isoforms can be screened using a panel of fluorometric substrates and the effects on the overall cytochrome P450-dependent monooxygenase (MO) system, as alterations in NAD(P)H cytochrome c and NADH ferricyanide reductase activities (Solé and Livingstone, 2005). Hepatic carboxylesterases (CbEs) are also phase I reactions involved in endogenous and drug metabolism (Wheelock et al., 2008). ...
... Microsomal reductases, NAD(P)H-dependent cytochrome c and NADH-dependent ferricyanide reductase activities, were measured by the increase in absorbance at 550 nm and the decrease in absorbance at 420 nm, respectively (Solé and Livingstone, 2005). Assay conditions were: 50 mM Tris-HCl pH 7.6, 1 mM KCN, 0.26 mM NAD(P)H, and 30 μM cytochrome c or 0.2 mM potassium ferricyanide. ...
... Regarding the surfactant NP, no clear action over CYPs was revealed although it caused a 2-fold increase in ECOD activity, 1.4-fold increase in UDPGT (p 4 0.05) and a significant effect on the MO system by increasing the activity of NADH ferricyanide reductase while decreasing the associated NADH cyt c reductase form (p o0.05). The latter activity involved in the particular electron transfer to the cytochrome b 5 and therefore more involved in reproduction (Solé and Livingstone, 2005). Little responsiveness of CYPs to NP injection was in line with a study with Salmo salar in which a 5-fold higher dose (125 mg/kg) was needed to significantly affect EROD activity and CYP3A-like levels, and with no effects of the dose of 25 mg/kg on UDPGT and GST activities (Arukwe et al., 1997). ...
In recent years, Solea senegalensis has increasingly been used in pollution monitoring studies. In order to assess its response to some particular widespread pollutants, juveniles of S. senegalensis were administered an intraperitoneal injection of the model aryl hydrocarbon receptor agonist β-naphtoflavone (βNF) and chemicals of environmental concern, such as the fungicide ketoconazole (KETO), the lipid regulator gemfibrozil (GEM), the surfactant nonylphenol (NP) and the synthetic hormone ethinylestradiol (EE2). Two days after injection, the effect of these chemicals was followed up as alterations of hepatic microsomal activities of the cytochrome P450 (CYPs) and associated reductases, carboxylesterases (CbEs) and the conjugation enzyme uridine diphosphate glucuronyltransferase (UDPGT). In the cytosolic fraction of the liver, the effect on CbEs, glutathione S-transferase (GST) and antioxidant activities was also considered. Alterations on the endocrine reproductive system were evaluated by plasma levels of vitellogenin (VTG) and the sex steroids estradiol (E2), testosterone (T), 11-ketotestosterone (11KT) and the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Injection with the model compound βNF induced the hydrolysis rate of the seven CYP substrates assayed. The xenobiotic GEM induced three CYP-related activities (e.g. ECOD) and UDPGT, but depressed antioxidant defenses. EE2 induced four CYPs, more significantly ECOD and BFCOD activities. The xenoestrogens NP and EE2 altered the activities of CbE in microsomes and catalase, and were the only treatments that induced de novo VTG synthesis. In addition, the progestin 17,20β-P, was induced in NP-injected fish. None of the treatments caused statistically significant effects on steroid plasma levels. In conclusion, the CYP substrates assayed responded specifically to treatments and juveniles of S. senegalensis appear good candidates for assessing xenobiotics exposure.
... This assumption might cause uncertainty in our results because cytochrome P450 activity, which is responsible for biotransforming diazinon to diazoxon, has been shown to be higher in digestive organs, such as digestive glands in gastropods or hepatopancreas in crustaceans, than in other tissues. 37 Even though the P450 enzymatic levels in these digestive organs are comparable in gastropods and crustaceans, there are differences in lipid content of the organs among these two species classes (higher in digestive glands of gastropods than in hepatopancreas of crustaceans 38,39 ). This in turn might affect further distribution of biotransformation products from the digestive tissues throughout the organism and cause uncertainty when we compare diazoxon concentrations among our test species in different organs using the constant ratio of diazoxon/diazinon. ...
... This in turn might affect further distribution of biotransformation products from the digestive tissues throughout the organism and cause uncertainty when we compare diazoxon concentrations among our test species in different organs using the constant ratio of diazoxon/diazinon. However, the P450 pathway occurs also in other tissues, 37 and our approach is the best approximation of diazoxon distribution in tissues that we can achieve without direct tissue specific quantification of metabolites. ...
... The gastrointestinal complex and hepatopancreas of L. stagnalis and gut and hepatopancreas of Gammaridae are major digestive organs with high cytochrome P450 activity. 37,45 As we observed high bioaccumulation of propiconazole in these organs (Table S4), the risk of propiconazole in long-term exposure might be considerable. ...
Interspecies variation in sensitivity to synthetic chemicals can be orders of magnitude large. Species traits causing the variation can be related to toxicokinetics (uptake, distribution, biotransformation, elimination), or toxicodynamics (interaction with biological target sites). We present an approach to systematically measure and model the contribution of uptake, biotransformation, internal distribution and elimination kinetics towards species sensitivity differences. The aim is to express sensitivity as target tissue specific, internal lethal concentrations. A case study with the pesticides diazinon, imidacloprid and propiconazole and the aquatic invertebrates Gammarus pulex, Gammarus fossarum and Lymnaea stagnalis illustrates the approach. L. stagnalis accumulates more pesticides than Gammaridae when measured in whole organisms but less in target tissues such as the nervous system. Toxicokinetics, i.e. biotransformation and distribution, explain the higher tolerance of L. stagnalis to the insecticide diazinon when compared to Gammaridae. L. stagnalis was again more tolerant to the other neurotoxicant imidacloprid, however, the difference in sensitivity could not be explained by toxicokinetics alone, indicating the importance of toxicodynamic differences. Sensitivity to propiconazole was comparable among all species, and when expressed as internal lethal concentrations, falls in the range of baseline toxicity.
... In line with these studies, the present work integrates the characterisation of the water soluble PAHs derived from the Prestige fuel oil with measuring several MFO components (flavin reductases i.e. NAD(P)H cytochrome c and NADH ferricyanide reductases) as putative markers of PAH exposure in mussels (Livingstone, 1987;Solé and Livingstone, 2005). The conjugation phase II glutathione S-transferase (GST) was also considered since many xenobiotics undergoing phase I (MFO) reactions may be further eliminated in this way (Fitzpatrik et al., 1997). ...
... Final assay conditions in a final volume of 1 mL were: 50 mM Tris-HCl pH 7.6, 1 mM KCN, 0.26 mM NAD(P)H, and 30 μM cytochrome c or 0.2 mM potassium ferricyanide. Sample volumes were: 50 μL of microsomes for NADPH and 10 μL for NADH dependent reductases (Solé and Livingstone, 2005). ...
... This fact could imply a change in the fatty acid profile as already pointed out in Labarta et al., (2005) in relation to the Prestige spillage. The three reductases followed the trend observed in other molluscs including bivalves (Solé and Livingstone, 2005). That is: NADH-ferricyanide>NADHcytochrome c>NADPH-cytochrome c reductase indicative of cytochrome b5 reductase, cytochrome b5 reductase and cytochrome b5 and CYP reductase respectively. ...
The activity of the antioxidant defences catalase (CAT, EC 1.11.1.6), glutathione peroxidase (t-GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), phase II glutathione S-transferase (GST, EC 2.5.1.18) along with the NADPH-dependent cytochrome c (CYP) reductase (EC 1.6.2.4), NADH-dependent cytochrome c reductase (EC 1.6.2.2), and NADH-dependent ferricyanide (b5) reductase (EC 1.18.1.1) was determined in the digestive gland of mussels Mytilus galloprovincialis fed with Tetraselmis sp. pre-exposed to the water accommodated fraction of the Prestige oil. Mussel gills were also used for measuring acetylcholinesterase activity (AChE, EC 3.1.1.7) and lipid peroxidation (LP) as an indication of neurotoxicity and oxidative stress damage respectively. Bioaccumulation of the selected polycyclic aromatic hydrocarbons (2 to 6 rings PAHs) in mussels after 2, 4, 7 and 10 days of exposure did not show any significant trend; the 2-3 ring PAHs were best represented (51%). A significant (p
... Similarly, GPx induction by exposure to marine diesel oil has been observed in cold water red king crab, Paralithodes camtschaticus (Sagerup et al., 2016). It is important to point out that physiological processes associated with PAH metabolism and depuration have been demonstrated to vary multifold among decapod species and have been extensively reviewed (Livingstone, 1998;Meador et al., 1995b;Solé and Livingstone, 2005). This underlines the necessity to study effects of individual chemicals on different species. ...
... In this respect, these findings allow for doubt on whether measured EROD activity represents CYP1A enzyme in C. irroratus and are thus in support of controversy of EROD measurements in crustaceans. This speculation is based on the advances of genetic studies that have yet failed to isolate CYP1A1 gene in marine invertebrates and rather attribute xenobiotic metabolism to other families of P450 enzymes, related to CYP1A1 (Dam et al., 2008;Rewitz et al., 2006;Solé and Livingstone, 2005;Sparagano et al., 1999). However, as the cytochrome P450 enzyme family is known to serve multiple functions besides PAH metabolism in Carcinus maenas (Dam et al., 2008;Rewitz et al., 2006), some of which individually take part in multiple processes, it is thus likely that EROD measurements in the present study represents one or multiple different members of the P450 family and would explain elevated activity in day 3 control groups. ...
The aims of this study were to estimate suitability of the Atlantic rock crab (Cancer irroratus) for environmental monitoring through investigation of 7-ethoxyresorufin Odeethylase (EROD), glutathione peroxidase (GPx) and glutathione (GSH) biomarker responses to Benzo(a)Pyrene (BaP) exposure through ingestion, i.e. as potential monitoring tool for Polycyclic Aromatic Hydrocarbons (PAHs) through a laboratory exposure to contaminated prey items. Biomarker responses were measured in hepatopancreas tissue shortly after starvation period as well as after long-term exposure. Blue mussels (Mytilus edulis) were exposed to 2, 20 and 50 μg BaP/L for 14 days after which they had accumulated 11.5, 192 and 417 μg BaP/g tissue dw., respectively. Subsequently, mussels were fed to crabs for 21 days in daily portions of about 4.7 g soft tissue ww. per crab. Assimilation rate of BaP through ingestion in crabs was estimated at < 1%. Biomarker responses were measured at days 3 and 21 of the experiment. Biomarker values were generally lower than their relative control levels on day 3, likely reflecting degradation of xenobiotic defence systems induced by prolonged starvation. After 21 day of exposure the crabs exhibited significant biomarker response in the highest exposure group, where significantly higher EROD activity and GSH content as well as significantly lower protein content were observed. Biomarker responses proved insufficiently sensitive for implementation in environmental monitoring. This study provides first insight into physiological responses of Cancer irroratus to PAH induced stress post starvation and during long-term exposure.
... Several studies have attempted to detect CYP1-related enzyme activity, protein expression and CYP1-like transcript levels in bivalve mollusks, specially aiming the use of these parameters as biomarkers once these animals are considered excellent for pollution monitoring (Solé and Livingstone, 2005;Zanette et al., 2013;Lüchmann et al., 2015). Despite bivalve mollusks demonstrated total CYP450 level and CYP1-related activity, such as Benzo[A]Pyrene hydroxylase and EROD (Schlenk and Buhler, 1989;Solé and Livingstone, 2005;Zhang et al., 2010), anti-CYP1 vertebrates antibodies have shown to cross-react with non-CYP proteins in these organisms (Grøsvik et al., 2006) and the function of CYP1-like in mollusks are not yet well understood. ...
... Several studies have attempted to detect CYP1-related enzyme activity, protein expression and CYP1-like transcript levels in bivalve mollusks, specially aiming the use of these parameters as biomarkers once these animals are considered excellent for pollution monitoring (Solé and Livingstone, 2005;Zanette et al., 2013;Lüchmann et al., 2015). Despite bivalve mollusks demonstrated total CYP450 level and CYP1-related activity, such as Benzo[A]Pyrene hydroxylase and EROD (Schlenk and Buhler, 1989;Solé and Livingstone, 2005;Zhang et al., 2010), anti-CYP1 vertebrates antibodies have shown to cross-react with non-CYP proteins in these organisms (Grøsvik et al., 2006) and the function of CYP1-like in mollusks are not yet well understood. ...
Cytochrome P450 family 1 (CYP1) is involved in polycyclic aromatic hydrocarbons (PAHs) biotransformation. PAHs can induce CYP1 protein expression and enzyme activity, the latter being usually quantified as 7-ethoxyresorufin O-deethylase activity (EROD). The aim of this study was to characterize EROD activity in the bivalve mollusk Crassostrea brasiliana. EROD activity was evaluated in cytosolic and microsomal fractions of gills, digestive gland and mantle of C. brasiliana. No EROD activity was detected in mantle, but it was present in microsomal fraction of gills and digestive gland with NADPH as coenzyme. Optima temperature and pH for EROD assay were 30 °C and 7.4, respectively. EROD apparent Km (Kmapp) was 4.32 μM for gills and 5.56 μM for digestive gland. EROD Vmax was 337.3 fmol·min− 1·mg of protein− 1 in gills and 297.7 fmol·min− 1·mg of protein− 1 in digestive gland. Compared to other bivalves, a higher Kmapp and a lower Vmax was found in oyster which may suggest that oyster CYP1-like enzyme has lower affinity for substrate 7-ethoxyresorufin (7-ER) than those species. CYP1 inhibitor ellipticine (ELP) inhibited EROD activity in all tested concentrations in both tissues. The higher ELP concentration, 100 μM, inhibited 78% of EROD activity in gills and 47% in digestive gland. The CYP1 inhibitors α-naphthoflavone and furafylline did not inhibited EROD activity in microsomes of both tissues. In conclusion, EROD activity can be used to determine CYP1-like activity in oysters and possibly a CYP1A1/A2-like enzyme is responsible for this catalysis.
... For instance, studies have investigated the modulation of major defense systems (e.g., antioxidant and biotransformation) in bivalves exposed to environmental pollutants (Aloıísio Torres et al., 2002;Borković et al., 2005;Dafre et al., 2004;de Almeida et al., 2004;Doyotte et al., 1997;Franco et al., 2006;Nogueira et al., 2015;Osman et al., 2007;Zanette et al., 2011). In this regard, gills and digestive gland are the most commonly studied tissues: gills are considered the main interface between the organism, environment and, consequently, waterborne pollutants (Ravera, 2001), while digestive gland is known as a major detoxification site due to the higher expression of the mixed-function oxidase system (Livingstone and Farrar, 1984;Solé and Livingstone, 2005). Thus, it is essential to characterize how different organs respond mutually to aquatic pollutants. ...
... However, this could be the case for prolonged or dietary exposures to CDNB or other electrophilic substances. It is plausible to assume that the digestive gland would have a much more important role in the presence of electrophilic metabolites derived from oxidative stress and from the phase I of biotransformation, given that this organ has high phase I activity (Livingstone and Farrar, 1984;Solé and Livingstone, 2005) and abundance of MAP enzymes, as suggested by data shown in Section 3. ...
... In recent years, attention has focused on the induction of cytochrome P450-dependent monooxygenases (phase I) as a sensitive biomarker of mollusc exposure to organic contaminants in the marine environment (Stegeman and Hahn, 1994;Snyder, 2000). Activation of the isoenzyme cytochrome P4501A (CYP1A) can be measured by determining the benzo[a]pyrene hydroxylase (BPH) activity which is frequently used as biomarker of exposure to PAH in mussels (Narbonne et al., 1999;Akcha et al., 2000;Sole and Livingstone, 2005). The enzymatic activities of glutathione Stransferases (GST), a family of multi-functional enzymes involved in phase II of biotransformation are related to cellular antioxidant defences due to the conjugation of electrophilic xenobiotics and oxidised components with glutathione (GSH) (Fitzpatrick et al., 1995). ...
... Invertebrates, particularly bivalve molluscs such as mussels are very suitable organisms for studying the biological effects of pollutants . In mussels, a number of studies have described occurrence and B[a]P induction of metabolising enzymes in the digestive gland (Narbonne et al., 1999 , 2000;Sole and Livingstone., 2005). However, the relationship between DNA alteration and enzymatic biomarker activities is less well studied. ...
Résumé
Le présent travail reporte l’effet du cadmium (Cd), du benzo[a]pyrène (B[a]P) ainsi que leur mélange (Cd/B[a]P), à 1 µM, sur les activités d’enzymes impliqués dans la phase I et la phase II de biotransformation chez le polychète Nereis diversicolor en fonction du temps (après 12, 24, 36 et 48 h).
L’effet d’une contamination aiguë par du cadmium à une dose de 1 µM après 12, 24 et 36 h montre une inhibition de l’activité NADPH cytochrome C réductase chez les individus contaminés comparés à leurs témoins relatifs, alors que le seul effet du cadmium sur l’activité glutathion-S-transférase n’est enregistré qu’après 36 h d’exposition. Quant au benzo[a]pyrène, les résultats montrent une augmentation significative de l’activité NADPH cytochrome C réductase après 12, 24 et 36 h d’exposition, alors que pour l’activité glutathion-S‑transférase, la variation significative entre les animaux témoins et traités n’est enregistrée qu’à 36 h d’exposition. Le mélange (Cd/B[a]P) inhibe l’activité NADPH cytochrome C réductase chez les individus traités par comparaison aux témoins relatifs et montre un effet inducteur sur l’activité GST sauf après 36 h d’exposition. Ces résultats montrent ainsi les interactions entre les polluants ainsi que leurs effets sur les organismes.
... Moreover, concerning C. maenas, Solé and Livingstone (2005) found high levels of total CYP enzymes in Hp (223 pmol mg − 1 ). Also, among epidermis, gills, Hp, muscle, and gonads, the Hp was considered by Dam et al. (2008) as the major site of adult male C. maenas's CYPs gene expression. ...
... NADH cytochrome c reductase (NADH-CcR) activity can also be measured as an endpoint since it is catalysed by both cytochrome b5 reductase and cytochrome b5. A high level of NADH-FR activity was found in the Hp of C. maenas (436 nmol min − 1 mg − 1 protein) by Solé and Livingstone (2005). However, low NADPH-CcR and NADH-CcR activities were reported in the same study, with the values of 4.0 and 22.5 nmol min − 1 mg − 1 protein, respectively. ...
Aquatic ecotoxicology broadly focuses on how aquatic organisms interact with pollutants in their environment in order to determine environmental hazard and potential risks to humans. Research has produced increasing evidence on the pivotal role of aquatic invertebrates in the assessment of the impact of pollutants on the environment. Its potential use to replace fish bioassays, which offers ethical advantages, has already been widely studied. Nevertheless, the selection of adequate invertebrate experimental models, appropriate experimental designs and bioassays, as well as the control of potential confounding factors in toxicity testing are of major importance to obtain scientifically valid results. Therefore, the present study reviews more than four decades of published research papers in which the Green crab Carcinus maenas was used as an experimental test organism. In general, the surveyed literature indicates that C.maenas is sensitive to a wide range of aquatic pollutants and that its biological responses are linked to exposure concentrations or doses. Current scientific knowledge regarding the biology and ecology of C. maenas and the extensive studies on toxicology found for the present review recognise the Green crab as a reliable estuarine/marine model for routine testing in ecotoxicology research and environmental quality assessment, especially in what concerns the application of the biomarker approach. Data gathered provide valuable information for the selection of adequate and trustworthy bioassays to be used in C. maenas toxicity testing. Since the final expression of high quality testing is a reliable outcome, the present review recommends gender, size and morphotype separation in C. maenas experimental designs and data evaluation. Moreover, the organisms' nutritional status should be taken into account, especially in long-term studies. Studies should also consider the crabs' resilience when facing historical and concurrent contamination. Finally, experimental temperature and salinity should be harmonised so as to obtain reliable comparisons between different studies. Concerning future reaserch areas, data gathered in the present review reveals that in vitro assays derived from C. maenas are still lacking. Also, a complete C. maenas genome sequencing programme will be essencial for cutting-edge reseach.
... Additionally, an increase in the activity of glutathione S-transferase (GST) may be observed, as it plays a role in the conjugation of lipid peroxides generated during oxidative stress (Singhal et al. 2015). However, if the antioxidant defenses are overwhelmed or insufficient, oxidative stress can result in damage to macromolecules, including genetic material, or even trigger apoptosis (Solé & Livingstone 2005, Burkina et al. 2015, Ribalta et al. 2015. ...
Fish farm effluents are known to affect water quality and freshwater ecosystems, potentially harming non-target organisms and ecosystem processes. We studied the effect of fish farm effluents at different concentrations (3.125-100% v/v) on catalase (CAT) and glutathione S -transferase (GST) activity as well as the DNA integrity of Oncorhynchus mykiss fry over 24-120 h. Biochemical responses and DNA damage analysis were conducted to assess the impact. We found that fish farm effluent had higher conductivity, nitrate, nitrite, and total dissolved solids concentrations downstream compared to upstream of the farm. Interestingly, no antibiotics were detected in the effluent. CAT activity significantly increased in the fish liver at concentrations of 12.5, 50, and 100% of the effluent after 72 h. In the gills, a significant increase was observed at concentrations ranging from 6.25 to 100% of the effluent after both 24 and 72 h. GST activity increased significantly in the liver at a concentration of 100% of the effluent after 72 h and in the gills at concentrations of 25, 50, and 100% after 24 h, with a decrease noted at higher concentrations. DNA damage assessment revealed significant DNA strand breaks in blood cells at concentrations of 12.5, 25, 50, and 100% of the effluent after 120 h of exposure. The results demonstrate that fish farm effluents can induce oxidative stress, causing damage to DNA integrity in blood cells. Our findings emphasize the potential ecological risks posed by fish farm effluents to aquatic organisms.
... Further, there appear to be significant differences in biotransformation capacity between closely related species of copepods (e.g., Calanus finmarchicus = 260 L/kg vs Calanus hyperboreus = 2970 L/kg; both 5% lipid-equivalent standardized values) (Jensen et al. 2012;Agersted et al. 2018). Although some knowledge about invertebrate biotransformation enzyme systems is available (e.g., Meador et al. 1995;Snyder 2000;Sole and Livingstone 2005;Rewitz et al. 2006), many data gaps and uncertainties remain. ...
Bioaccumulation (B) assessment is challenging because there are various B‐metrics from laboratory and field studies, various criteria and thresholds for classifying bioaccumulative (“B”), very bioaccumulative (“vB”), and non‐bioaccumulative (“nB”) chemicals, as well as inherent variability and uncertainty in the data. These challenges can be met using a weight of evidence (WoE) approach. The Bioaccumulation Assessment Tool (BAT) provides a transparent WoE assessment framework that follows Organisation for Economic Coordination and Development (OECD) principles for performing a WoE analysis. The BAT guides an evaluator through the process of data collection, generation, evaluation, and integration of various lines of evidence (LoE) (i.e., B‐metrics) to inform decision‐making. Phenanthrene (PHE) is a naturally occurring chemical for which extensive B and toxicokinetics data are available. A B assessment for PHE using the BAT is described that includes a critical evaluation of 74 measured in vivo LoE for fish and invertebrate species from laboratory and field studies. The number of LoE are reasonably well balanced across taxa (i.e., fish and invertebrates) and the different B‐metrics. Additionally, in silico and in vitro biotransformation rate estimates and corresponding model predicted B‐metrics are included as corroborating evidence. Application of the BAT provides a consistent, coherent, and scientifically‐defensible WoE evaluation to conclude that PHE is not bioaccumulative (“nB”) as the overwhelming majority of the bioconcentration, bioaccumulation and biomagnification metrics for both fish and invertebrates are below regulatory thresholds. An analysis of the relevant data using fugacity ratios is also provided showing phenanthrene does not biomagnify in aquatic food webs. The critical review identifies recommendations to increase the consistency of B assessments, such as improved standardization of B testing guidelines, data reporting requirements for invertebrate studies, and consideration of temperature and salinity effects on certain B‐metrics. This article is protected by copyright. All rights reserved.
... In this context, variations in transcript levels, protein expression, biotransformation enzyme activities or histo-cytological alterations are investigated as possible biomarkers of environmental contamination by PAH and other contaminants (Au, 2004;Gagnon and Rawson, 2017;Lu et al., 2009;Mattos et al., 2010;Nahrgang et al., 2010). Although the biotransformation system of xenobiotics in bivalves is not completely elucidated, several studies have shown changes at transcriptional and enzymatic level of biotransformation systems in these organisms (Lüchmann et al., 2015;Rewitz et al., 2006;Rust et al., 2004;Schlenk and Buhler, 1989;Siebert et al., 2017a;Solé and Livingstone, 2005;Zhang et al., 2012). Oysters C. gigas exposed to phenanthrene (PHE) and PYR showed enhanced transcript levels of cytochrome P450 genes (CYP2AU1 and CYP356A1) in gills of oysters Crassostrea gasar (Lüchmann et al., 2014;Zacchi et al., 2019). ...
Pyrene (PYR) and fluorene (FLU) are among the sixteen priority Polycyclic Aromatic Hydrocarbons (PAH) of the United States Environmental Protection Agency and are both frequently detected in contaminated sites. Due to the importance of bivalve mollusks in biomonitoring programs and the scarce information on the biotransformation system in these organisms, the aim of this study was to investigate the effect of PYR and FLU at the transcriptional level and the enzymatic activities of some biotransformation systems in the Pacific oyster Crassostrea gigas, and to evaluate the histological effects in their soft tissues. Oysters C. gigas were exposed for 24 h and 96 h to PYR (0.25 and 0.5 μM) and FLU (0.6 and 1.2 μM). After exposure, transcript levels of cytochrome P450 coding genes (CYP1-like, CYP2-like, CYP2AU2, CYP356A1, CYP17α-like), glutathione S tranferase genes (omega GSTO-like and microsomal, MGST-like) and sulfotransferase gene (SULT-like), and the activity of ethoxyresorufin O-deethylase (EROD), Glutathione S-transferase (GST) and microssomal GST (MGST) were evaluated in gills. Histologic changes were also evaluated after the exposure period. PYR and FLU bioconcentrated in oyster soft tissues. The half-life time of PYR in water was lower than fluorene, which is in accordance to the higher lipophilicity and bioconcentration of the former. EROD activity was below the limit of detection in all oysters exposed for 96 h to PYR and FLU. The reproductive stage of the oysters exposed to PYR was post-spawn. Exposure to PYR caused tubular atrophy in digestive diverticula, but had no effect on transcript levels of biotransformation genes. However, the organisms exposed for 96 h to PYR 0.5 μM showed higher MGST activity, suggesting a protective role against oxidative stress in gills of oysters under higher levels of PYR in the tissues. Increased number of mucous cells in mantle were observed in oysters exposed to the higher FLU concentration, suggesting a defense mechanisms. Oysters exposed for 24 h to FLU 1.2 μM were in the ripe stage of gonadal development and showed higher transcript levels of CYP2AU2, GSTO-like and SULT-like genes, suggesting a role in the FLU biotransformation. In addition, after 96 h of exposure to FLU there was a significant increase of mucous cells in the mantle of oysters but no effect was observed on the EROD, total GST and MGST activities. These results suggest that PAH have different effects on transcript levels of biotransformation genes and enzyme activities, however these differences could also be related to the reproductive stage.
... NADPH-P450 reductase furthermore plays roles in oxidative and ER stress, as well as protein misfolding, also relating to a number of pathologies including ischemia, diabetes, atherosclerosis, neurodegenerative diseases, inflammation and liver and kidney diseases [196]. In crustaceans, P450-dependent xenobiotic monooxygenation is mainly carried out in the hepatopancreas [197] and NADPH-cytochrome P450 reductase protein has been identified in a number of invertebrate marine species, including Porifera, Cnidaria, Mollusca, Polychaeta, Crustacea and Echinodermata [198]. NADPH cytochrome c reductase has amongst other been found to be an important part in detoxifying responses of prawn, following oil pollution [227]. ...
The American lobster (Homarus americanus) is a commercially important crustacean with an unusual long life span up to 100 years and a comparative animal model of longevity. Therefore, research into its immune system and physiology is of considerable importance both for industry and comparative immunology studies. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family that catalyses post-translational protein deimination via the conversion of arginine to citrulline. This can lead to structural and functional protein changes, sometimes contributing to protein moonlighting, in health and disease. PADs also regulate the cellular release of extracellular vesicles (EVs), which is an important part of cellular communication, both in normal physiology and in immune responses. Hitherto, studies on EV in Crustacea are limited and neither PADs nor associated protein deimination have been studied in a Crustacean species. The current study assessed EV and deimination signatures in haemolymph of the American lobster. Lobster EVs were found to be a poly-dispersed population in the 10-500 nm size range, with the majority of smaller EVs, which fell within 22-115 nm. In lobster haemolymph, 9 key immune and metabolic proteins were identified to be post-translationally deiminated, while further 41 deiminated protein hits were identified when searching against a Crustacean database. KEGG (Kyoto encyclopedia of genes and genomes) and GO (gene ontology) enrichment analysis of these deiminated proteins revealed KEGG and GO pathways relating to a number of immune, including anti-pathogenic (viral, bacterial, fungal) and host-pathogen interactions, as well as metabolic pathways, regulation of vesicle and exosome release, mitochondrial function, ATP generation, gene regulation, telomerase homeostasis and developmental processes. The characterisation of EVs, and post-translational deimination signatures, reported in lobster in the current study, and the first time in Crustacea, provides insights into protein moonlighting functions of both species-specific and phylogenetically conserved proteins and EV-mediated communication in this long-lived crustacean. The current study furthermore lays foundation for novel biomarker discovery for lobster aquaculture.
... Stegeman, 1985) and have not been reported to have a large inductive aryl hydrocarbon hydroxylase (Ahr) enzyme or cytochrome P450 (CYP) enzyme induction in response to exposure and uptake of aromatic hydrocarbons (e.g. see Sole and Livingstone, 2005). However, there are reports of correlations between concentrations of the EPA 16 PAH in tissue of Mytilus galloprovincialis and Phase I NADPH-cyt c red and benzo(a) pyrene monooxygenase activity (Trisciani et al., 2012). ...
A small No. 2 fuel oil spill contaminated a Mytilus edulis population in the Cape Cod Canal, Massachusetts, USA during a three day period in April 1983. Retention and release of the fuel oil compounds were assessed over several days and months. Compounds analyzed included n-alkanes, pristane, phytane, C2 -, C3 –naphthalenes, flourene, phenanthrene, C1-, C2-, C3 – phenanthrenes. Biological half-lives were calculated for the release of the compounds up to day 29 and ranged from 1.5 days to 9.9 days. Results compared favorably with similar data from a small No. 2 fuel oil spill contaminating the same population of Mytilus edulis at the same time of year, April 1978. Gas chromatography-mass spectrometer analyses of C2-, and C3- phenanthrenes documented changes in relative abundance within the isomer groupings after day 29. This suggests a within isomer grouping molecular structural control on release or enzymatic catalyzed alteration of these compounds.
... Microsomal reductases, NAD(P)H-cytochrome c reductases and NADH-ferricyanide reductase activities, were measured by the increase in absorbance at λ = 550 nm and the decrease in absorbance at λ = 420 nm, respectively (Solé and Livingstone, 2005). Assay conditions were: 50 mM Tris−HCl buffer pH 7.6, 1 mM KCN, 0.26 mM NAD (P)H, and 60 μM cytochrome c or 0.2 mM potassium ferricyanide. ...
Fipronil is a phenylpyrazole insecticide widely used to control pests in agriculture even though evidence of harmful side effects in non-target species has been reported. A comprehensive study on the effects of dietary administration of Regent®800WG (80 % fipronil) in European sea bass juveniles was carried out under two temperature regimes: a) natural conditions, and b) 3 °C above the natural temperature (an increase predicted for the NW Mediterranean by the end of this century). Fipronil was added to the fish food (10 mg fipronil /Kg feed) and the effects were studied at several time points including right before administration, 7 and 14 days after daily fipronil feed and one-week after the insecticide withdrawal from the diet (depuration period). A wide array of physiological and metabolic biomarkers including feeding rate, general condition indices, plasma and epidermal mucus metabolites, immune response, osmoregulation, detoxification and oxidative-stress markers and digestive enzymes were assessed. General linear models and principal component analyses indicated that regardless of water temperature, fipronil resulted in a significant alteration of several of the above listed biomarkers. Among them, glucose and lactate levels increased in plasma and decreased in epidermal mucus as indicators of a stress response. Similarly, a depletion in catalase activity and higher lipid peroxidation in liver of fipronil-exposed fish were also indicative of an oxidative-stress condition. Fipronil induced a time dependent inhibition of Cytochrome P450-related activities and an increase of phase II glutathione-S-transferase. Moreover, fipronil administration was able to reduce the hypo-osmoregulatory capability as shown by the increase of plasmatic osmolality and altered several digestive enzymes including trypsin, lipase, alpha amylase and maltase. Finally, analyses in bile and muscle confirmed the rapid clearance of fipronil but the persistence of the metabolite fipronil-sulfone in bile even after the 7-day depuration period. Altogether, the results reveal a notable impact of this compound on the physiological condition of the European sea bass. The results should be considered in future environmental risk assessment studies since fipronil could be hazardous to fish species, particularly those inhabiting estuarine ecosystems exposed to the discharge of agriculture runoffs where this pesticide is mainly used.
... Oxidative stress and the production of ROS (reactive oxygen species) which often result from toxicity in the cell, are one of the reasons for the destabilization of lysosomes via the peroxidation of the lysosomal membranes (Kurz et al. 2008). The knowledge on cellular responses of sponges after oil exposure is reduced but it was shown that diesel oil in seawater activated the Mitogen-Activated Protein Kinase (MAPK) in a temperate sponge and that the cytochrome P450dependant monooxygenase pathway of 2 sponges was involved in PAH detoxification (Solé and Livingstone 2005). Both MAPK and the cytochrome P450 pathway are known to play important roles in cellular response to oxidative stress (Vad et al. 2018). ...
Exploration and extraction of petroleum reserves in deep-sea marine ecosystems are ongoing activities world-wide and are continuously expanding with the identification of new reservoirs. Whilst a wealth of knowledge is known on the effects of oil spills on fish and other organisms in shallow water environments, there is a dearth of studies on the effects of subsurface oil spills (blowout events like the Deepwater Horizon) on deep-sea ecosystems. With exploration activities in deep-sea ecosystems predicted to increase, there are concerns regarding the accidental release of hydrocarbons into deep-sea ecosystems and the subsequent cascading effects on associated fauna. Sponges are abundant and ecologically valuable, known to be highly efficient filter feeders, contributing significantly to benthic pelagic coupling and providing habitat for a suite of organisms. However, sponges, which are sessile in nature, accumulate contaminants present in their ambient environment, potentially making them vulnerable to oil spills. Surprisingly, the impacts of oil on deep-sea sponges remain unexplored, despite sponges being particularly dominant around oil and gas exploration locations in the Northern Atlantic. Here are presented the findings from a mesocosm study, where the locally abundant deep-sea sponge Geodia barretti was exposed to three ecologically relevant oil concentrations for a duration of 8 days, followed by a recovery period of 30 days. A holistic approach elucidating the effects of oil on G. barretti focused on measuring changes in physiology (respiration rates), cellular stress (lysosomal membrane stability), and the structure of the sponge-associated microbiome using high-throughput sequencing of 16s rRNA gene amplicons.
G. barretti did not demonstrate strong sub-lethal stress effects in response to an acute crude oil exposure. Respiration showed varying patterns of increased and decreased rates with no significant effect from the treatments while lysosomes were significantly impacted by oil, displaying destabilisation of lysosomal membranes. A 30 day recovery period allowed the sponges to recover to control levels. G. barretti’s microbiome was stable at the phylum and class level across all treatments and days of exposure. Some evidence suggest that this high microbial abundance sponge could naturally host microorganisms which play a role in oil degradation / detoxification. Further research should investigate the effects of a long-term oil exposure on G. barretti’s physiology, cellular stress, metabolism and associated microbial community. Of particular interest is the uncovering of the functions of the sponge microbiome in relation to oil contamination.
Such studies enhance our understanding of the vulnerability and / or resilience of deep-sea sponges to hydrocarbon exposure, providing useful data for managing risks associated with oil and gas exploration in the Northern Atlantic.
... The toxicity of the extracts was determined using two experimental models of crustaceans -Artemia salina and Daphnia magna. These models can be predictive for the toxicity of extracts containing PAs in animals and humans because numerous studies have demonstrated the presence of cytochrome P450 in these organisms, enzymes needed for the biotransformation of PAs to the toxic pyrrole derivatives (42)(43)(44)(45). The assays consisted in the exposure of the invertebrates at increasing concentrations of test substances and determination of the lethal concentrations 50% (LC 50 ) at different time intervals (24-120 h). ...
Pyrrolizidine alkaloids (PAs) are a widespread class of hepatotoxic heterocyclic organic compounds found in approximately 3% of world flora. Some PAs have been shown to have genotoxic and carcinogenic effects. The present study focuses on the toxicity effects of four dry extracts obtained from medicinal plants (Senecio vernalis, Symphytum officinale, Petasites hybridus and Tussilago farfara), on two aquatic organisms, Artemia salina and Daphnia magna, and the correlation with their PAs content. A new GC‑MS method, using a retention time (TR)‑5MS type capillary column was developed. PAs Kovats retention indices, for this type of column were computed for the first time. The lethal dose 50% (LC50) values for the two invertebrate models were correlated (Pearson 's coefficient, >0.9) and the toxicity was PA concentration-dependent, for three of the four extracts. All tested extracts were found to be toxic in both aquatic organism models. The results can be used to develop a GC‑MS validated method for the assay of PAs in medicinal plants with a further potential application in the risk assessment study of PAs toxicity in humans.
... Where biochemical studies have been carried out on other tropical and temperate marine invertebrates (i.e. annelids, crustaceans, echinoderms), direct correlation of responses to hydrocarbons have sometimes been weak or absent (den Besten, 1998;Fossi et al., 2000;Payne and May, 1979;Pérez et al., 2004;Snyder, 2000;Solé and Livingstone, 2005). At present, information is scarce on the optimal conditions for sample collection/processing, due to variations in biotic and abiotic factors (Shaw et al., 2004;Sheehan and Power, 1999;Viarengo et al., 1991), and the identification of suitable biomarkers in cold-water species necessitates a case-by-case approach (Morales-Caselles et al., 2008a). ...
In spite of increasing naval activities and petroleum exploration in cold environments, there is currently a paucity of tools available to monitor oil contamination in boreal marine life, especially in sedentary (non-fish) species that dominate benthic communities. This research aimed to identify biotic sources of variation in biomarkers using subarctic echinoderms, and to identify suitable biomarkers of their exposure to hydrocarbons. The focal species included the sea star Asterias rubens, the brittle star Ophiopholis aculeata, the sea urchin Strongylocentrotus droebachiensis, and the sea cucumber Cucumaria frondosa, which are among the most abundant echinoderms in the North Atlantic and Arctic Oceans. The latter two species are also commercially exploited. A series of 96-h acute exposures of the water-accommodating fraction (WAF) of used lubricating oil (ULO) were performed in different seasons (i.e. distinct reproductive stages). Digestive and reproductive tissues were analyzed for baseline and response levels of glutathione peroxidase (GPx) and ethoxyresorufin-O-deethylase (EROD). GPx activity was detected in the pyloric caecum, stomach, and gonad of sea stars, the intestine and gonad of sea cucumbers, and the gonad of brittle stars and sea urchins. No seasonal variation in baseline GPx activity occurred. Upon exposure to the ULO WAF, sex-based differences were elicited in the GPx activity of sea star stomachs (lower in females than males). EROD activity was present in the pyloric caeca of sea stars, and the gonads of brittle stars and sea urchins. An interaction between season and sex on baseline EROD activity was measured in the gonads of sea urchins. Ovaries exhibited significant seasonal variation in EROD activity and had greater activity than testes during the spawning and post-spawning seasons. Seasonal variation in EROD activity also occurred in sea star pyloric caeca and brittle star gonads. Furthermore, testes of sea urchins exposed to the ULO WAF exhibited suppressed EROD activity compared to baseline levels. The nearly universal presence of GPx activity highlights its potential as a useful biomarker, while EROD activity was much more limited. Findings suggest a complex relationship between temporal and biotic factors on both the baseline and response levels of enzymatic activity, emphasizing the need to consider sex and sampling season in studies of biomarkers of hydrocarbon exposure in boreal indicator species that display annual reproductive cycles.
... However, the single use of sediments, water, or bivalves to assess the environmental impact of the oil and gas production activities may not indicate the whole of ecosystem health, in which the biota comprises different phyla and trophic levels (Fang et al. 2009). On the other hand, contaminants in sediments and water may not be bioavailable for the organisms, and the capability of metabolization in bivalves also might underestimate the human health risks caused by these pollutants when consumed (Sole and Livingstone 2005). Hence, the use of a suite of feral fishes for pollution monitoring is advisable, and there is a need to evaluate some other species, which are representative of the local conditions, to accompany bivalves for assessment of the state of the ecosystem (Fang et al. 2009). ...
The main effluent of oil and gas production is the discharge of produced water (PW). Despite the potential environmental impact, the structure of oil and gas platforms can act as artificial reefs. Two species of fish, Caranx crysos and Tylosurus acus, were captured under two discharging platforms at Campos Basin, P19 and P40. A nondischarging platform, P25, was taken as reference. The highest median concentration of PAH in muscle tissue was observed for C. Crysos at P40 (219.38 ng g−1) followed by P19 (68.26 ng g−1). For T. acus, the highest median concentrations were 40.07 and 42.21 ng g−1 from P19 and P40 respectively. P25 presented the smallest recorded concentrations. The results of PAH in the muscle tissue of C. crysos enabled to distinguish the platforms with PW discharge from the platform without discharge. Trace elements concentrations reflected the background levels and were not influenced by the PW release.
... Previous works have reported capacity of transformation of xenobiotics by AS, modifying disposition, residence time, and degree of toxicity of xenobiotic into the fish (Livingstone, 1994(Livingstone, , 1998Fontagné-Dicharry et al., 2014). A well-documented example is the phase II biotransformation enzyme GST (Solé and Livingstone, 2005;Ricciardi et al., 2006), but also GSH has been involved in detoxification of xenobiotics through their eventually excretion in form of mercapturic acids (Pastore et al., 2003). In general, the AS promotes the reduction or detoxification of xenobiotics in three phases (Park et al., 2005). ...
... The high concentrations of FLX observed in the DG are not surprising, given that this organ is primarily responsible for xenobiotic detoxification (Sol e and Livingstone, 2005) and can make up for up to 6% of the total body weight (Ceccaldi, 1989). The DG also appears to be the primary site of metabolism for FLX based on the higher concentration of nor-FLX observed in this tissue. ...
The disposition and metabolism of fluoxetine in the European shore crab and the Dungeness crab were assessed. Crabs received itracardiac doses of either 0.13 μg/kg or 0.5 mg/kg fluoxetine, respectively. In addition, fluoxetine was administered to Metacarcinus cancer by oral gavage at 7.8 mg/kg. The distribution of fluoxetine was quantified in haemolymph and digestive gland for both crabs, as well as brain, muscle, and testis of Carceinus maenas, over 12 days. The metabolite norfluoxetine, was also measured in C. maenas. Fluoxetine was mainly found in lipid rich tissues. Distribution coefficients increased for digestive gland until three days after fluoxetine administration and then decreased until the end of the observations. The highest distribution coefficients were obtained for brain. Norfluoxetine displayed continuously high levels in digestive gland and brain. The strong decrease in fluoxetine and the concomitant increase in norfluoxetine demonstrates that decapod crustaceans metabolise fluoxetine into the more biologically active norfluoxetine. Fluoxetine levels in the haemolymph of M. cancer declined within 20 hours, but showed a second peak 25 hours later, suggesting remobilisation from tissues sequestering the compound. The steady state volume distribution and the total body clearance of fluoxetine were high, consistent with high diffusion of fluoxetine into the peripheral tissues and biotransformation as an important elimination pathway. Oral administration of fluoxetine prolonged its half-life in M. cancer, but bioavailability was low. These results confirm the high volume of distribution into nervous tissue, extensive biotransformation into the highly active nor-fluoxetine and a half-life similar to that observed in vertebrates.
... Available data seems to indicate that gastropods biotransform TBT by the cytochrome P450 enzymes (Fent 1996). However, P450-mediated detoxification/elimination reactions appear to be limited in mollusks (Livingstone et al. 1989;Solé and Livingstone 2005) resulting in incomplete biotransformation of TBT and increased accumulation (Sternberg et al. 2010). This tendency to accumulate TBT may contribute to the high sensitivity of gastropods to TBT toxicity (Sternberg et al. 2010). ...
... In this species, bioaccumulated PAHs are mainly released by passive elimination (Rantam€ aki, 1997). However, biotransformation of organic contaminants by the cytochrome P450 has been reported in a wide range of invertebrate species including bivalves (Sol e and Livingstone, 2005). Moreover, in the high exposure group, concentrations of S16PAHs were 2-fold higher than the low exposure group in both bivalve species. ...
Shipping activities are expected to increase in the Arctic Seas. Today, the majority of vessels are using marine diesel oil (MDO) as propulsion fuel. However, there is a general lack of knowledge of how cold-water marine species respond to acute exposures to MDO. Arctic red king crabs (Paralithodes camtschaticus) were exposed to mechanically dispersed MDO in a flow-through exposure system for one week followed by three weeks of recovery. Observations of increased movements in exposed crabs were interpreted as avoidance behaviour. Further, glutathione peroxidase activity increased in high exposed crab, the catalase activity showed a insignificant increase with exposure, while no differences between groups were observed for lipid peroxidation and acetylcholinesterase activity. After three weeks of recovery in clean seawater, PAH concentrations in the crabs were significantly reduced, with no specific biomarker responses in exposed groups compared to the control. The results suggest that effects from instantaneous MDO spill only will have short-term effects on the red king crab.
... 30,31,42,69,70 Predicting BTPs based on common drug biotransformation reactions such as oxidations by CYP enzymes is feasible and was successfully applied in this study, assuming CYPs to be present among different organismal levels and species. 42,61,71,72 However, toxicokinetic processes that determine the internal concentration quantitatively and thus the effect of a chemical are extremely difficult to predict. Uptake rate constants for small aquatic invertebrates seem to be mainly diffusion driven and dependent on the physicochemical properties as well as on the individual test species, whereas no simple relationship exists between elimination rate constants and hydrophobicity. ...
... In December 2011, 9 months after the beginning of this study, the cargo ship TK Bremen ran aground on the Kerminihy beach (Erdeven, Brittany, France) spilling tens of thousands litres of its fuel oil (Intermediate Fuel Oil 120) at the mouth of the Etel River. Not surprisingly, PAHs amounts decreased with the time suggesting this species has the capacities to metabolize PAHs probably through the components of its cytochrome P450-dependent monooxygenase system (Solé & Livingstone, 2005). The role of pollutants is not clearly incriminated in the invasion process of C. ciocalyptoides. ...
The distribution and abundance of the marine invasive sponge Celtodoryx ciocalyptoides is described for the first time along the Etel River, a Natura 2000 site of the French Atlantic coast. This invasive species is now well established along both sides of the Etel River and has colonized about 29.3% of the surface of the rocky reef at a depth of 10-18 m. In the Etel marina, C. ciocalyptoides covers all the pilings with a covering rate estimated to 17.4% between 5 and 9 m deep. The bioaccumulation capacities of this species exposed to metallic and organic pollutants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organotin derivatives) was also measured during a three-year study in the Etel marina. Preliminary results suggest this marine invasive species has the capacities to adapt in few months to its changing environment by sequestering and/or metabolizing those pollutants.
... The low concentrations of PPCP residues measured in the G. pulex and unspiked AFW post-exposure highlights the ability for G. pulex to readily metabolise and eliminate xenobiotics, as previously shown by Nyman et al. (2014) and Ashauer et al. (2012). This evidence suggests there is conservation of cytochrome P450 enzymes, similarly observed in other aquatic invertebrate species (Solé & Livingstone, 2005). ...
This study considers whether the current standard toxicokinetic methods are an accurate and applicable assessment of xenobiotic exposure in an aquatic freshwater invertebrate. An in vivo exposure examined the uptake and elimination kinetics for eight pharmaceutical compounds in the amphipod crustacean, Gammarus pulex by measuring their concentrations in both biological material and in the exposure medium over a 96 h period. Selected pharmaceuticals included two anti-inflammatories (diclofenac and ibuprofen), two beta-blockers (propranolol and metoprolol), an anti-depressant (imipramine), an anti-histamine (ranitidine) and two beta-agonists (formoterol and terbutaline). Kinetic bioconcentration factors (BCFs) for the selected pharmaceuticals were derived from a first-order one-compartment model using either the simultaneous or sequential modelling methods. Using the simultaneous method for parameter estimation, BCF values ranged from 12 to 212. In contrast, the sequential method for parameter estimation resulted in bioconcentration factors ranging from 19 to 4533. Observed toxicokinetic plots showed statistically significant lack-of-fits and further interrogation of the models revealed a decreasing trend in the uptake rate constant over time for rantidine, diclofenac, imipramine, metoprolol, formoterol and terbutaline. Previous published toxicokinetic data for 14 organic micro-pollutants were also assessed and similar trends were identified to those observed in this study. The decreasing trend of the uptake rate constant over time highlights the need to interpret modelled data more comprehensively to ensure uncertainties associated with uptake and elimination parameters for determining bioconcentration factors are minimised.
... A welldocumented example is the phase II bio transformation enzyme glutathione transferase (GST; EC 2.5.1.18) (Sole´and Livingstone, 2005;Ricciardi et al., 2006). In addition, oxidative stress enzymes such as catalase (CAT; EC 1.11.1.6) ...
The mRNA expression profile of some antioxidant genes in skin, gills, livers, and muscles of Siganus canaliculatus and Epinephelus morio was used as an indicator of petroleum hydrocarbons pollution in six areas at Jeddah and Yanbu coasts in KSA. Total petroleum hydrocarbons (TPHs) were determined in both sea water and sediments collected from the studied areas. The mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were determined. The highest level of total petroleum hydrocarbons was observed in front of the petromine refinery at Jeddah and in S. canaliculatus when compared to E. morio. There was a significant high expression level of studied antioxidant enzymes genes in the polluted areas and the level of the expression profile tended to correlate with the degree of pollution and fish species feed habit. This was confirmed by the level of MDA which in the same way increased with an increase in the level of total hydrocarbons. In conclusion; the expression profile of antioxidant enzymes of S. canaliculatus and E. morio tissues can be used as a strong bio-indicator of total hydrocarbons pollution especially in S. canaliculatus.
... Further, tissue measurements are more challenging than aqueous exposure analyses and often impractical to routinely perform, particularly for small aquatic test organisms. Lastly, many aquatic species can effectively metabolize petroleum hydrocarbons (Sole and Livingstone, 2005; Arnot et al., 2008). Thus, measurement of parent hydrocarbons in tissues omits the potential action of the metabolites, which may contribute to observed toxicity. ...
The complex nature and limited aqueous solubility of petroleum substances pose challenges for consistently characterizing exposures in aquatic life hazard assessments. This paper reviews important considerations for the design, conduct and interpretation of laboratory toxicity tests with physically and chemically dispersed oils based on an understanding of the behavior and toxicity of the hydrocarbons that comprise these substances. Guiding principles are provided that emphasize the critical need to understand and, when possible, characterize dissolved hydrocarbon exposures that dictate observed toxicity in these tests. These principles provide a consistent framework for interpreting toxicity studies performed using different substances and test methods by allowing varying dissolved exposures to be expressed in terms of a common metric based on toxic units (TUs). The use of passive sampling methods is also advocated since such analyses provide an analytical surrogate for TUs. The proposed guidance is translated into a series of questions that can be used in evaluating existing data and in guiding design of future studies. Application of these questions to a number of recent publications indicates such considerations are often ignored, thus perpetuating the difficulty of interpreting and comparing results between studies and limiting data use in objective hazard assessment. Greater attention to these principles will increase the comparability and utility of oil toxicity data in decision-making.
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... The CYP genes of marine invertebrates, including mollusks, polychaetes and crustaceans, have been observed in a wide range of tissues and are generally enriched in organs associated with food processing [36,41]. CYPs show tissue specificity with respect to their functions; for instance, CYPs in hepatic tissues are generally involved in the detoxification of a wide range of xenobiotics [36]. ...
Background:
The tropical green-lipped mussel Perna viridis is a common biomonitor throughout the Indo-Pacific region that is used for environmental monitoring and ecotoxicological investigations. However, there is limited molecular data available regarding this species. We sought to establish a global transcriptome database from the tissues of adductor muscle, gills and the hepatopancreas of P. viridis in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses in this sentinel species.
Results:
Illumina sequencing results yielded 544,272,542 high-quality filtered reads. After de novo assembly using Trinity, 233,257 contigs were generated with an average length of 1,264 bp and an N50 length of 2,868 bp; 192,879 assembled transcripts and 150,111 assembled unigenes were obtained after clustering. A total of 93,668 assembled transcripts (66,692 assembled genes) with putative functions for protein domains were predicted based on InterProScan analysis. Based on similarity searches, 44,713 assembled transcripts and 25,319 assembled unigenes were annotated with at least one BLAST hit. A total of 21,262 assembled transcripts (11,947 assembled genes) were annotated with at least one well-defined Gene Ontology (GO) and 5,131 assembled transcripts (3,181 assembled unigenes) were assigned to 329 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The quantity of assembled unigenes and transcripts obtained from male and female mussels were similar but varied among the three studied tissues, with the highest numbers recorded in the gills, followed by the hepatopancreas, and then the adductor muscle. Multivariate analyses revealed strong tissue-specific patterns among the three different tissues, but not between sexes in terms of expression profiles for annotated genes in various GO terms, and genes associated with stress responses and degradation of xenobiotics. The expression profiles of certain selected genes in each tissue type were further validated using real-time quantitative polymerase chain reaction assays and a similar tissue-specific trend was seen.
Conclusions:
The extensive sequence data generated from this study will provide a valuable molecular resource for facilitating environmental studies with P. viridis, and highlight the importance of tissue-specific approaches in the future.
... These were described for fish (Arnot et al., 2008) and, when accounting for the reported uncertainty (De Laender et al., 2011a), were directly applicable for the fish species in the current study. For M. edulis and P. borealis, we assumed that the biotransformation rates corresponded to 10% of the fish biotransformation rates because invertebrate biotransformation rates are generally considered lower than vertebrate biotransformation rates (Livingstone, 1998;Sole and Livingstone, 2005). Despite the Monte Carlo approach used here and the uncertainty range on fish biotransformation rates that was accounted for (De Laender et al., 2011a), we cannot exclude the possibility that the biotransformation of some PAHs was modeled inaccurately for M. edulis and P. borealis. ...
... However, it is expected that the sampling rates of passive samplers under agitated test conditions are faster than invertebrates which have a lower surface area to volume ratio (Bayen et al., 2009). In addition, crustaceans may metabolize hydrocarbons thereby reducing tissue residues (Sole and Livingstone, 2005). Thus, passive sampler measurements would appear to provide a conservative basis for quantifying the freely dissolved concentrations of key hydrocarbon constituents that are hypothesized to contribute to aquatic toxicity in acute toxicity test exposures while conveniently excluding components that are expected to be too insoluble to invoke effects (Abernethy et al., 1988). ...
... Hepatic CbE was about four times lower in M. brachydactyla than in the other two contrasted crustacean mentioned above, whereas muscular esterases are similar (Antó et al. 2009). In another comparative study contrasting xenobiotic metabolism activities in hepatopancreas in marine invertebrates including this species (referred as M. squinado) from UK waters, Solé and Livingstone (2005) also revealed that total cytochrome P450 and some associated reductases were lower in M. brachydactyla than in other crustaceans. Present results and those from the literature evidence a low hepatic xenobiotic metabolism in this species. ...
Adults from spider crab, Maja brachydactyla, Balss, 1922 were kept in captivity for up to 2 years. Haemolymph of ten females was extracted monthly for monitoring several metabolites and the presence of Vibrionacea bacteria. A neurotoxic marker and hepatic metabolic and digestive enzyme capacities were contrasted between wild specimens and those reared in captivity. As a result, no differences were observed in hepatosomatic index (HSI) between males and females, but captive animals presented lower HSI than the wild ones. After 2 years of confinement, no changes in quantities of haemolymph microflora and metabolites were observed and this was considered indicative of adaptation of the broodstock to confinement. Spider crab also showed a large plasticity in their hepatopancreatic digestive capabilities suggested by the adaptation to the food supplied in captivity. Moreover, captivity conditions did not seem to negatively alter their antioxidant defences, xenobiotic capacity or neuromuscular activity, as no enhanced oxidative stress damage was shown in either males or females. These results indicate that although the confinement conditions used in this study do not mimic those from the wild, they could be considered as adequate for rearing M. brachydactyla in captivity, at least with regard to the set of parameters assessed.
... Previous evidence for the presence of CYP enzymes in cnidarians (Solé and Livingstone 2005), as well as UGT and GST, has been presented . However, this study is the first to demonstrate both protein presence and activity, and describe changes over time in the lunar reproductive cycle for both Phase I (CYPR, CYP2E1) and Phase II (UGT, GST, b-glucuronidase, and ASC) enzymes. ...
The continued deterioration of coral reefs worldwide demonstrates the need to develop diagnostic tools for corals that go beyond general ecological monitoring and can identify specific stressors at sublethal levels. Cellular diagnostics present an approach to defining indicators (biomarkers) that have the potential to reflect the impact of stress at the cellular level, allowing for the detection of intracellular changes in corals prior to outright mortality. Detoxification enzymes, which may be readily induced or inhibited by environmental stressors, present such a set of indicators. However, in order to apply these diagnostic tools for the detection of stress, a detailed understanding of their normal, homeostatic levels within healthy corals must first be established. Herein, we present molecular and biochemical evidence for the expression and activity of major Phase I detoxification enzymes cytochrome P450 (CYP450), CYP2E1, and CYP450 reductase, as well as the Phase II enzymes UDP, glucuronosyltransferase (UGT), β-glucuronidase, glutathione-S-transferase (GST), and arylsulfatase C (ASC) in the coral Pocillopora damicornis. Additionally, we characterized enzyme expression and activity variations over a reproductive cycle within a coral’s life history to determine natural endogenous changes devoid of stress exposure. Significant changes in enzyme activity over the coral’s natural lunar reproductive cycle were observed for CYP2E1 and CYP450 reductase as well as UGT and GST, while β-glucuronidase and ASC did not fluctuate significantly. The data represent a baseline description of ‘health’ for the expression and activity of these enzymes that can be used toward understanding the impact of environmental stressors on corals. Such knowledge can be applied to address causes of coral reef ecosystem decline and to monitor effectiveness of mitigation strategies. Achieving a better understanding of cause-and-effect relationships between putative stressors and biological responses in corals, and other marine invertebrates, can guide and evaluate mitigation and conservation approaches for marine ecosystem protection.
... Since many environmental pollutants such as heavy metals and PAHs are known to be strongly pro-oxidant, we focused our research on the study of biomarkers reflecting oxidative stress. Bivalves have evolved an extensive battery of antioxidant defences [10], including heat shock proteins [40,41], components of the cytochrome P450 (CYP)-dependent monooxygenase system [42], metallothioneins [43,44] and variations in these biomarkers were found to be useful in environmental monitoring. In our study, an immunopositivity to CYP1A, HSP70, HNE and NT antibodies was detected by immunohistochemistry in various tissues: mantle, intestine, digestive gland and gills. ...
The aim of this study was to evaluate the histomorphology and the stress response in the bivalve Venerupis philippinarum sampled in four differently polluted sites of the Venice Lagoon (Palude del Monte, Marghera, Ca' Roman and Val di Brenta). This species is often used as bioindicator of environmental pollution since it can bioaccumulate a large variety of pollutants because of its filter feeding. Chemical analyses for heavy metals (Cd, Cu, Hg, Pb) and polycyclic aromatic hydrocarbons (PAHs) were performed on whole soft tissues of V. philippinarum. The histological evaluation of clams revealed the presence of Perkinsus sp. infection in animals from all sites, although a very high prevalence of parasites was evidenced in clams from Ca' Roman. Perkinsus sp. were systemically distributed in the mantle, in the intestine and digestive gland, in gonads and gills. The trophozoites of Perkinsus sp. were found isolated or in cluster surrounded by a heavy hemocitical response. Hemocytes always exhibited an immunopositivity to citochrome P4501A (CYP1A), heat shock protein 70 (HSP70), 4-hydroxy-2-nonenal (HNE) and nitrotyrosine (NT) antibodies. The digestive gland of animals from Palude del Monte showed the highest malondialdehyde (MDA) concentration, whereas clams from Ca' Roman exhibited the highest quantity of metallothioneins.
... A battery of multiple biomarkers was applied on mussel specimens in two key organs, the digestive gland and gills, including cytochrome P450 (Peters et al. 1999;Solé & Livingstone 2005;Cappello et al. 2013a), esterases (Moreira & Guilhermino 2005), lysosomal membrane stability (Da Ros et al. 2002;Castro et al. 2004;Pisoni et al. 2004), micronucleus test and comet assay (Rank et al. 2007;Banni et al. 2010). ...
Lake Faro (Sicily, Italy) is a natural confined brackish environment particularly subject to anthropogenic impact, resulting in a mixture of xenobiotic substances, i.e. heavy metals and polycyclic aromatic hydrocarbons (PAHs), and characterised by low hydrodynamics. In order to assess the water quality status of this pond, a multi-biomarker approach was applied on mussel Mytilus galloprovincialis (Lamarck, 1819) both inhabiting the lake and from a control site (Goro). Different biomarkers were investigated on mussel digestive glands and gills, including biomarkers of exposure (cytochrome P450 4, CYP4), neurotoxicity (acetylcholinesterase, AChE; choline acetyltransferase, ChAT), general stress (lysosomal membrane stability, LMS), and genotoxicity (micronucleus and comet assays). The results suggest significant responses related to the selected area. A statistically significant inhibition (P < 0.0001) of CYP4 in the digestive gland, and of AChE and ChAT in the gills, was found in specimens collected from Faro compared with the control. The lysosomal membrane stability of mussels from Lake Faro was lower than the reference site, while the DNA damages were significantly higher in mussels from the brackish area compared to the control. This study represents the first time the quality status of Lake Faro is assessed using a multi-biomarker approach on the mussel M. galloprovincialis, which proved to be suitable to identify the effects of environmental pollutants at molecular and cellular levels.
... A battery of multiple biomarkers was applied on mussel specimens in two key organs, the digestive gland and gills, including cytochrome P450 (Peters et al. 1999;Solé & Livingstone 2005;Cappello et al. 2013a), esterases (Moreira & Guilhermino 2005), lysosomal membrane stability (Da Ros et al. 2002;Castro et al. 2004;Pisoni et al. 2004), micronucleus test and comet assay (Rank et al. 2007;Banni et al. 2010). ...
Lake Faro (Sicily, Italy) is a natural confined brackish environment particularly subject to anthropogenic impact, resulting in a mixture of xenobiotic substances, i.e. heavy metals and polycyclic aromatic hydrocarbons (PAHs), and characterised by low hydrodynamics. In order to assess the water quality status of this pond, a multi-biomarker approach was applied on mussel Mytilus galloprovincialis (Lamarck, 1819) both inhabiting the lake and from a control site (Goro). Different biomarkers were investigated on mussel digestive glands and gills, including biomarkers of exposure (cytochrome P450 4, CYP4), neurotoxicity (acetylcholinesterase, AChE; choline acetyltransferase, ChAT), general stress (lysosomal membrane stability, LMS), and genotoxicity (micronucleus and comet assays). The results suggest significant responses related to the selected area. A statistically significant inhibition (P < 0.0001) of CYP4 in the digestive gland, and of AChE and ChAT in the gills, was found in specimens collected from Faro compared with the control. The lysosomal membrane stability of mussels from Lake Faro was lower than the reference site, while the DNA damages were significantly higher in mussels from the brackish area compared to the control. This study represents the first time the quality status of Lake Faro is assessed using a multi-biomarker approach on the mussel M. galloprovincialis, which proved to be suitable to identify the effects of environmental pollutants at molecular and cellular levels.
... A dose-and time-dependent manner of B[a]P uptake was confirmed in scallop's ovary. The prominent use of bivalve mollusks in pollution monitoring programs has prompted numerous efforts to find some CYP genes, proteins or catalytic activities that would provide a similar marker of exposure in these organisms, but few was successful due to lack of knowledge of specific CYP sequences, probes, or substrates (Monari et al., 2007(Monari et al., , 2009Peters et al., 1998;Porte et al., 2001;Shaw et al., 2002;Sole and Livingstone, 2005;Wootton et al., 1995). ...
In this study, we isolated a CYP3A-like gene from ovary of the scallop (Chlamys farreri). High levels of CYP3A-like gene expression occur in the digestive gland and gonad, which suggested their role in the metabolism of steroids and xenobiotics. Scallops were exposed to a polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (B[a]P) for 10 days. The CYP4 and CYP3A-like gene can be up-regulated by B[a]P in a dose-dependent manner after 10 days exposure. But no induction of the CYP3A-like was observed in 10 μg/L B[a]P group. The CYP1A-like expression can only be induced by 0.025 μg/L B[a]P. 0.5 and 10 μg/L B[a]P caused significant DNA damage and 10 μg/L B[a]P can also lead to oxidative damage. These results demonstrate that the mollusk CYPs can be modulated by environmental pollutant, and the blocked induction of CYP3A-like and CYP1A-like expression probably results from the high genotoxicity and oxidative damage partly.
... The reactive BaP quinones (BaP metabolites) are oxidant agents that can cause the generation of reactive oxygen species (ROS) (Akcha et al., 2000;Solé and Livingstone, 2005), which can lead to a direct or indirect damage of actin by altering the protein redox status (Dalle-Donne et al., 2001). Hence, protein alterations (reversible or irreversible) that occur after ROS action may result in protective (redox regulation) or non-protective (function failure) protein inactivation (Ghezzi and Bonetto, 2003). ...
In natural waters, chemical interactions between mixtures of contaminants can result in potential synergistic and/or antagonic effects in aquatic animals. Benzo(a)pyrene (BaP) and copper (Cu) are two widespread environmental contaminants with known toxicity towards mussels Mytilus spp. The effects of the individual and the interaction of BaP and Cu exposures were assessed in mussels Mytilus galloprovincialis using proteomic analysis. Mussels were exposed to BaP [10μgL(-1) (0.396μM)], and Cu [10μgL(-1) (0.16μM)], as well as to their binary mixture (mixture) for a period of 7 days. Proteomic analysis showed different protein expression profiles associated to each selected contaminant condition. A non-additive combined effect was observed in mixture in terms of new and suppressed proteins. Proteins more drastically altered (new, suppressed and 2-fold differentially expressed) were excised and analyzed by mass spectrometry, and eighteen putatively identified. Protein identification demonstrated the different accumulation, metabolism and chemical interactions of BaP, Cu and their mixture, resulting in different modes of action. Proteins associated with adhesion and motility (catchin, twitchin and twitchin-like protein), cytoskeleton and cell structure (α-tubulin and actin), stress response (heat shock cognate 71, heat shock protein 70, putative C1q domain containing protein), transcription regulation (zinc-finger BED domain-containing and nuclear receptor subfamily 1G) and energy metabolism (ATP synthase F0 subunit 6 protein and mannose-6-phosphate isomerase) were assigned to all three conditions. Cu exposure alone altered proteins associated with oxidative stress (glutathione-S-transferase) and digestion, growth and remodelling processes (chitin synthase), while the mixture affected only one protein (major vault protein) possibly related to multi drug resistance. Overall, new candidate biomarkers, namely zinc-finger BED domain-containing protein, chitin synthase and major vault protein, were also identified for BaP, Cu and mixture, respectively.
... A single study verified that freshwater sponge removes 60% of anthracene and pyrene from water after 6 h of exposure, suggesting fast uptake of hydrocarbons by these organisms (Glyzina et al., 2002). Moreover, the existence of components of the cytochrome P450dependent monooxygenase system and the NADPH-dependent benzo[a]pyrene hydroxylase activity at low levels in the wholebody of sponges suggest lower biotransformation capacities of pollutants, since these enzyme systems are involved in the catalyzes of a wide range of xenobiotic (Mansuy, 1998;Sóle and Livingstone, 2005). Here, H. heliophila sampled from several stations exhibited ability to accumulate PAH, however, a high variability was observed within populations. ...
Chicoreus torrefactus, which has a typical crossed lamellar structure, was utilized as an ideal species to investigate the structure and function of the mantle in shell formation. Field emission scanning electron microscopy was used to study the microstructure of the gastropod shell. Histological methods, histochemical methods and transcriptome sequencing were used to understand the roles of central mantle and mantle edge in crossed lamellar structure formation. In the central mantle and mantle edge, acidic-neutral mucopolysaccharides were observed. The Gene Ontology (GO) functional enrichment analyses of differentially expressed genes (DEGs) between the mantle edge and foot, as well as between the central mantle and foot, were mainly associated with chitin binding and calcium ion binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs revealed that 15 pathways were significantly enriched in both the mantle edge and foot, as well as in the central mantle and foot, with 12 pathways being shared. In the domain annotation, comparisons were made between the five most abundant domains in the mantle edge and foot, central mantle and foot, three of which were shared. A total of 86 DEGs were detected between the mantle edge and central mantle. Our study could provide a preliminary basis for studying the formation mechanism of crossed lamellar shell structure.
The bioconcentration of dimethyl phthalate (DMP) diethyl phthalate (DEP) dibutyl phthalate (DBP) butyl benzyl phthalate (BBzP), di-(2-ethy hexyl) phthalates (DEHP), mono-butyl phthalate (MBP), mono-benzyl phthalate (MBzP), mono-(2-ethy hexyl) phthalate (MEHP) in the soft corals Coelogorgia palmosa, Sinularia sp., Sarcophyton glaucum, and Lobophytum sp. was investigated. Specimens were cultured in a microcosm environment built-up at the Genova Aquarium and analyses were carried out by in vivo SPME-LC-MS/MS. The distributions of the phthalates among the four surveyed species resulted significantly different. Calculated bioconcentration factors (BCFs) showed values spanning over two orders of magnitude, from a minimum of log10 BCFDEP = 1.0 in Sarcophyton glaucum to a maximum of log10 BCFDBP = 3,9 calculated for Coelogorgia palmosa. Moreover, the calculated BCFs of the long chain phthalates resulted up to three orders of magnitude lower than theoretically predicted (from logKow), whereas BCF of short chain phthalates resulted higher. This, together with the detection of phthalic acid monoesters, suggests the presence of species-specific different metabolic transformation among the surveyed soft coral species that involve DEHP.
Azole fungicides are known inhibitors of the important enzyme class cytochrome P450 monooxygenases (CYPs), thereby influencing the detoxification of co-occurring substances via biotransformation. This synergism in mixtures containing an azole has mostly been studied by effect measurements, while the underlying mechanism has been less well investigated. In this study, six azole fungicides (cyproconazole, epoxiconazole, ketoconazole, prochloraz, propiconazole and tebuconazole) were selected to investigate their synergistic potential and their CYP inhibition strength in the aquatic invertebrate Gammarus pulex. The strobilurin fungicide azoxystrobin was chosen as co-occurring substrate, and the synergistic potential was measured in terms of internal concentrations of azoxystrobin and associated biotransformation products (BTPs). Azoxystrobin is biotransformed by various reactions, and 18 BTPs were identified. By measuring internal concentrations of azoxystrobin and its BTPs with high resolution tandem mass spectrometry in the presence and absence of azole fungicides followed by toxicokinetic modeling, we showed that inhibition of CYP-catalyzed biotransformation reactions indeed played a role for the observed synergism. However, synergism was only observed for prochloraz at environmentally realistic concentrations. Increased uptake rate constants, an increase in the total internal concentration of azoxystrobin and its BTPs, in vivo assays for measuring CYP activities, and G. pulex video-tracking suggested that the 2-fold increase in bioaccumulation, and, thereby, the raised toxicity of azoxystrobin in the presence of prochloraz is not only caused by inhibited biotransformation but even more by increased azoxystrobin uptake induced by hyperactivity.
Marine ciliate Euplotes crassus, a single-cell eukaryote, and has been considered as a model organism for monitoring of environmental pollutions in sediments. Cytochrome P450 (CYP450) monooxygenase are phase I enzyme involved in detoxification of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs). However, little information on CYP450 family genes in ciliate is available. In the present study, acute toxicity of PAH, benzo[a]pyrene (B[a]P) and PAH-like model compound, beta-naphthoflavone (β-NF), was investigated; full-length cDNA sequences and genomic structure of five CYP450 genes (CYP5680A1, CYP5681A1, CYP5681B1, CYP5682A1, and CYP5683A1) were analyzed; and finally their activities and transcriptional changes were measured after exposure to PAHs for 48h. According to the results, B[a]P exposure showed a negative effect on E. crassus survival, whereas β-NF exposure showed no significant effect. The 8 h-LC50 value of B[a]P was determined to be 2.449 μM (95%-C.L., 7.726–3.619 μM). Five genes belonging to the CYP450 family had conserved domains and clustered with those of ciliate group, as revealed in phylogenetic analysis. CYP activity did not change after exposure to B[a]P, whereas it was slightly, but significantly, induced after exposure to β-NF. The mRNA expression of five CYP450 genes was significantly modulated in a concentration- and time-dependent manner after exposure to both the chemicals. Our findings suggest that CYP450 genes in E. crassus may be involved in detoxification of B[a]P and β-NF. This study would give a better understanding about the mode of action of B[a]P and β-NF in marine ciliates at the molecular level.
This study aimed to evaluate the possible remedial effects of three marine benthic annelids on organically polluted sediments from the waters of Hatsukaichi Marina, Hiroshima, Japan. Two polychaetes, Perinereis nuntia and Capitella cf. teleta, and an oligochaete, Thalassodrilides sp., were incubated in sediments for 50 days. Their effects on physicochemical properties such as organic matter (loss on ignition), redox potential (Eh), acid volatile sulfides (AVS), and degradation of polycyclic aromatic hydrocarbons (PAHs) were assessed. The polychaetes P. nuntia and C. cf. teleta significantly increased Eh level and decreased AVS level compared with the oligochaete Thalassodrilides sp. and control (without benthic organisms). Total PAH concentration significantly decreased from the initial level with all three groups; Thalassodrilides sp. had a marked ability to reduce PAHs in sediment. These results indicate that benthic organisms have species-specific remediation properties and ecological functions in organically polluted sediments.
Estuarine organisms were impacted by the Deepwater Horizon oil spill which released ∼5 million barrels of crude oil into the Gulf of Mexico in the spring and summer of 2010. Crassostrea virginica, the American oyster, is a keystone species in these coastal estuaries and is routinely used for environmental monitoring purposes. However, very little is known about their cellular and molecular responses to hydrocarbon exposure. In response to the spill, a monitoring program was initiated by deploying hatchery-reared oysters at three sites along the Alabama and Mississippi coast (Grand Bay, MS, Fort Morgan, AL, and Orange Beach, AL). Oysters were deployed for 2-month periods at five different time points from May 2010 to May 2011. Gill and digestive gland tissues were harvested for gene expression analysis and determination of aliphatic and polycyclic aromatic hydrocarbon (PAH) concentrations. To facilitate identification of stress response genes that may be involved in the hydrocarbon response, a nearly complete transcriptome was assembled using Roche 454 and Illumina high-throughput sequencing from RNA samples obtained from the gill and digestive gland tissues of deployed oysters. This effort resulted in the assembly and annotation of 27,227 transcripts comprised of a large assortment of stress response genes, including members of the aryl hydrocarbon receptor (AHR) pathway, Phase I and II biotransformation enzymes, antioxidant enzymes and xenobiotic transporters. From this assembly several potential biomarkers of hydrocarbon exposure were chosen for expression profiling, including the AHR, two cytochrome P450 1A genes (CYP1A-like 1 and CYP1A-like 2), Cu/Zn superoxide dismutase (CuZnSOD), glutathione S-transferase theta (GST theta) and multidrug resistance protein 3 (MRP3). Higher expression levels of GST theta and MRP3 were observed in gill tissues from all three sites during the summer to early fall 2010 deployments. Linear regression analysis indicated a statistically significant relationship between total PAH levels in digestive gland tissue samples with CYP1A-like 2, CuZnSOD, GST theta and MRP3 induction. These observations provide evidence of a potentially conserved AHR pathway in invertebrates and yield new insight into the development of novel biomarkers for use in environmental monitoring activities.
The alerted presence in recent decades of pharmaceuticals has become an issue of environmental concern, and most of the mechanisms of biotransformation and biochemical and physiological responses to them in fish are still unknown, as well as the influence of water temperature in their ability to cope with them. This study aims to detect the main effects of two of the most widespread drugs on a set of physiological and biochemical markers in Solea senegalensis. Sole juveniles acclimatized at 15 and 20 °C were administered an intraperitoneal injection of the non-steroidal anti-inflammatory drug ibuprofen (IB; 10 mg/kg) and the anti-convulsant drug carbamazepine (CBZ; 1 mg/kg). Two days after the injection, liver, muscle and plasma were sampled. Liver enzymatic activities of 15 °C acclimated fish were more responsive to pharmaceuticals than those acclimated at 20 °C, especially for CYP450-related activities (7-ethoxyresorufin (EROD), 7-methoxyresorufin (MROD), 3-cyano-7-ethoxycoumarin (CECOD) and 7-benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD)) and uridine diphosphate glucuronosyltransferase (UDPGT). Cytosolic anti-oxidant enzyme activities and glutathione S-transferase (GST) did not show a clear effect of temperature. Glucose and transferase activities in plasma were not affected by the treatments, while ammonium, osmolality and lactate were affected by both pharmaceuticals. Plasma triglycerides were affected in a temperature-dependent manner, and creatinine was only responsive to CBZ injection. HSP70 levels in muscle were only affected by CBZ injection. Some of the physiological identified responses to IB and CBZ are proposed as endpoints in further chronic studies.
In this study we examined the behaviour of biomarkers in corals combining the reproductive status of colonies and reef sites with different levels of contamination. The content of P 450 and P 420 and the activities of NADPH c reductase, glutathione S-transferase (GST), catalase (CAT) and superoxide dimutase (SOD) were measured in the coral Siderastrea siderea at two marine parks. Parque Nacional Morrocoy (PNM) encompasses a continental marine ecosystem largely influenced by anthropogenic impacts. In contrast, Parque Nacional Archipielago Los Roques (PNALR) is 160 km offshore and relatively less impacted. As these enzymes are also affected by reproduction, samples were taken in both parks during the reproductive and non-reproductive seasons. In both parks, NADPH c reductase and GST were higher in the reproductive than in the non-reproductive season. Differences in enzymatic content or activity in corals from the two parks were usually more prominent during the reproductive season. Hence, P 450 and P 420 were undetectable during the non-reproductive season, while during the reproductive season, P 420 was highest in colonies from the most impacted park (PNM). Activities of NADPH c reductase, CAT and SOD were also higher at PNM than at PNALR, while GST was an exception to this trend. Our results show that the aforementioned enzymes act as biomarkers in the coral S. siderea, changing in relation to its reproductive status and with the habitat quality.
A survey to evaluate the impact of organic contaminants on the mussel Mytilus galloprovincialis in the Venice Lagoon, Italy was carried out in May 1993. M. galloprovincialis were sampled from putative moderately contaminated (Alberoni, Lio Grande, Crevan), urban (Salute) and industrial (CVE) sites in the Venice Lagoon, and from a clean reference site (Plataforma) in the adjacent Adriatic Sea. Measurements comprised (i) whole-tissue body burdens of aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and other organochlorines (DDTs, hexachlorocyclohexanes and hexachlorobenzene); and (ii) digestive gland microsomal cytochrome P450 (CYP)-dependent monooxygenase system (i.e. total CYP and cytochrome P4501A (CYP1A)-immunopositive protein levels, benzo(a)pyrene hydroxylase (BPH) activity) as a specific biomarker of impact by organic contaminants. Chemical analysis identified a contaminant gradient with Plataforma as the cleanest and CVE followed by Salute as the most contaminated extremes. No elevation of total CYP content or CYP1A- immunopositive protein level was seen at any of the lagoon sites compared with Plataforma. In contrast, BPH activity and BPH turnover (i.e. BPH activity per amount total CYP) were respectively 1- and 2.5-fold higher at CVE than Plataforma (P<0.05), and indicated to be higher (up to 1-fold) at all the other lagoon sites compared with Plataforma. Correlation was seen between BPH activity and tissue levels of total aliphatic hydrocarbons (r=0.94-0.98), but not between the former and total PAHs or PCBs. The results are consistent with other studies in the area and indicate greatest biological impact of contaminants was at CVE followed by the other lagoon sites, with a possible genotoxic role for the elevated BPH activity in the formation of bulky DNA-adducts.
Organic contaminants are continually entering aquatic environments and thence the tissues of resident biota. Mussels and other molluscs are used worldwide as sentinels in pollution monitoring. Recent years have seen the development of biological measurements (biomarkers) as tools for use in monitoring and environmental impact assessment, such biomarkers being indicative of contaminant exposure and/or impact. This paper describes established and developmental biomarkers in mussels responsive to exposure to organic contaminants, including some indicative of damage to DNA ('comet' assay) putative induction of biotransformation enzymes (CYP1A-like protein), contaminant removal (MXR-like protein), lysosomal membrane damage and impairment of membrane function.
The phylum Mollusca consists of 8 classes; Chaetodermomorpha, Neomeniomorpha, Monoplacophora, Polyplacophora, Scaphopoda, Cephalopoda, Gastropoda, and Bivalvia. The latter two classes contain 98% of the known molluscan species. This chapter describes the characteristics of the properties and regulation of molluscan (bivalve, gastropod, cephalopod, chiton) cytochrome P450 monooxygenase system, including enzymological, antibody-recognition and molecular biological information on multiple forms of cytochrome P450 (CYP1A, CYP2B, CYP2E, CYP3A, CYP4A, CYP10, CYP11A). It discusses the evidence for the presence of an inducible CYP1A-like enzyme and presents field data on the application of the cytochrome P450 monooxygenase system and a CYP1A-like enzyme as biomarkers of organic pollution for an oil spill (“Aegean Sea” incident off Spain), long-term industrial pollution (Venice Lagoon, Italy) and transplant studies (Skagerrak, North Sea). To identify the induction of specific CYPs including any CYP1A-like enzymes in molluscan species, it is necessary to design molecular probes that will hybridize with molluscan CYP nucleic acid sequences. To date, such robust molecular probes are not readily available. However procedures such as RT-PCR and other molecular techniques have enabled the indication or elucidation of CYP 1 A-like sequences in Mytilus sp.
Literature on the properties, forms and regulation of cytochrome P450 (CYP) in digestive gland of Mytilus sp., including studies indicating the existence of an organic contaminant-inducible CYP1A-like protein, are briefly reviewed. Laboratory and field studies show increases in digestive gland microsomal CYP1A-immunopositive protein levels and/or benzo[a]pyrene hydroxylase (BPH) activity (i.e. metabolism of benzo[a]pyrene to phenols) with exposure of Mytilus sp. to certain polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls. In order to examine further the relationship between these two parameters, M. edulis were collected 25 and 130 days after the release of oil following the grounding of the tanker 'Sea Empress' in South Wales, UK (15 February 1996); and M. galloprovincialis were sampled from sites in south-western France and south-eastern Spain during a cruise aboard the IFREMER Research Vessel 'L'Europe' (2-18 August 1996). In both studies, sites with higher levels of CYP1A-immunopositive protein showed higher levels of BPH activities. Positive correlations were observed between the two measurements-R=0.65 (M. edulis) and 0.68 (M. galloprovincialis), and both fitted linear regression models (P<0.05). The CYP1A-immunopositive protein levels and BPH activities tended to be highest at sites with greatest PAH body burden for the Mediterranean study. It is concluded that development of the CYP1A-like protein into a robust biomarker of exposure to organic contaminants will depend upon sequencing of the gene/protein and the subsequent production of Mytilus-specific cDNA and antibody probes. Such probes will then allow a full characterization of the enzyme's properties and gene regulation.
Since their heterotrophic origin, animals have been faced with a continual input of foreign compounds, so-called xenobiotics, from their food sources, e.g. polybromomethanes and alkyl halides in seaweeds (Gschwend et al. 1985) and defensive toxins in other animals (Bakus and Kawaguchi 1984); and from the attacks of predators, e.g. venoms of certain gastropods and cephalopods (Fange 1984). For example, “animal-plant warfare” is considered to be a major selective pressure in driving the evolution of some of the gene families of the biotransformation enzyme, cytochrome P-450 (Nebert et al. 1989b). Over time, additional sources of xenobiotics have included erosion of hydrocarbon-containing shales, and oil seepage from natural reservoirs (started at least 100,000 years ago) (Farrington 1985), hostile environments such as the high sulphide levels around deep sea hydrothermal vents (Vetter et al. 1987), and, in more recent years, man’s multifarious industrial and other activities (Mix 1984). Central to the defense against such an enormous and diverse number of potentially toxic compounds is an impressive array of enzymes, which function ideally to detoxify and eliminate xenobiotics from an organism. The biological significance of biotransformation enzymes is increased by the inducibility of some of them by xenobiotics, and by their metabolism of certain xenobiotics to molecular species more toxic, mutagenic or carcinogenic than the parent compound. Elaboration of the qualitative and quantitative aspects of organic xenobiotic metabolism in marine invertebrates, and the function and regulation of the enzymes involved, is important for several reasons, viz. predicting and modelling the fate and toxicity of xenobiotics in marine organisms and ecosystems (e.g. Harris et al. 1984); development of specific indices of biological effect for use in pollution monitoring and impact assessment (e.g. Malins et al. 1985; Kleinow et al. 1987; Payne et al. 1987; Bayne et al. 1988); and understanding the evolutionary relationships of the biotransformation pathways in different phylogenetic groups, and the use of the pathways in invading, or exploiting, ecological time and space to create new niches.
Literature on the properties, forms and regulation of cytochrome P450 (CYP) in digestive gland of Mytilus sp., including studies indicating the existence of an organic contaminant-inducible CYP1A-like protein, are briefly reviewed. Laboratory and field studies show increases in digestive gland microsomal CYP1A-immunopositive protein levels and/or benzo[a]pyrene hydroxylase (BPH) activity (i.e. metabolism of benzo[a]pyrene to phenols) with exposure of Mytilus sp. to certain polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls. In order to examine further the relationship between these two parameters, M. edulis were collected 25 and 130 days after the release of oil following the grounding of the tanker 'Sea Empress' in South Wales, UK (15 February 1996); and M. galloprovincialis were sampled from sites in south-western France and south-eastern Spain during a cruise aboard the IFREMER Research Vessel 'L'Europe' (2-18 August 1996). In both studies, sites with higher levels of CYP1A-immunopositive protein showed higher levels of BPH activities. Positive correlations were observed between the two measurements - R=0.65 (M. edulis) and 0.68 (M. galloprovincialis), and both fitted linear regression models (P < 0.05). The CYP1A-immunopositive protein levels and BPH activities tended to be highest at sites with greatest PAH body burden for the Mediterranean study. It is concluded that development of the CYP1A-like protein into a robust biomarker of exposure to organic contaminants will depend upon sequencing of the gene/protein and the subsequent production of Mytilus-specific cDNA and antibody probes. Such probes will then allow a full characterization of the enzyme's properties and gene regulation.
A survey to evaluate the impact of organic contaminants on the mussel Mytilus galloprovincialis in the Venice Lagoon, Italy was carried out in May 1993. M. galloprovincialis were sampled from putative moderately contaminated (Alberoni, Lio Grande, Crevan), urban (Salute) and industrial (CVE) sites in the Venice Lagoon, and from a clean reference site (Plataforma) in the adjacent Adriatic Sea. Measurements comprised (i) whole-tissue body burdens of aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and other organochlorines (DDTs, hexachlorocyclohexanes and hexachlorobenzene); and (ii) digestive gland microsomal cytochrome P450 (CYP)-dependent monooxygenase system (i.e. total CYP and cytochrome P4501A (CYP1A)-immunopositive protein levels, benzo(a)pyrene hydroxylase (BPH) activity) as a specific biomarker of impact by organic contaminants. Chemical analysis identified a contaminant gradient with Plataforma as the cleanest and CVE followed by Salute as the most contaminated extremes. No elevation of total CYP content or CYP1A-immunopositive protein level was seen at any of the lagoon sites compared with Plataforma. In contrast, BPH activity and BPH turnover (i.e. BPH activity per amount total CYP) were respectively 1- and 2.5-fold higher at CVE than Plataforma (P < 0.05), and indicated to be higher (up to 1-fold) at all the other lagoon sites compared with Plataforma. Correlation was seen between BPH activity and tissue levels of total aliphatic hydrocarbons (r = 0.94-0.98), but not between the former and total PAHs or PCBs. The results are consistent with other studies in the area and indicate greatest biological impact of contaminants was at CVE followed by the other lagoon sites, with a possible genotoxic role for the elevated BPH activity in the formation of bulky DNA-adducts.
Since the last review of this topic, further insight has been gained into the presence and functions of cytochrome P450 proteins in the hepatopancreas and other organs of aquatic crustacean species, although progress has been slow relative to the advances in other species. Recent studies with several lobster, shrimp, crab and crayfish species suggest that cytochromes P450 in the 2 and 3 families are the most abundant forms in hepatopancreas microsomes. Substrates normally metabolized by CYP2 and CYP3 family members are monooxygenated more rapidly by crustacea than substrates normally metabolized by CYP1 family enzymes, e.g. erythromycin, testosterone and aminopyrine are much more rapidly monooxygenated than ethoxyresorufin. Some progress has been made in cloning and sequencing crustacean P450 forms. CYP2L1 and CYP2L2 cDNA sequences have been cloned from spiny lobster hepatopancreas libraries, and there was evidence for at least two more cytochromes P450 in spiny lobster hepatopancreas. An area of continued interest, but of no consensus or general findings, relates to the presence and inducibility of CYP1 family members in crustacea. Some studies indicate weak induction of total cytochrome P450 and increased turnover of substrates normally associated with CYP1, while others show no effect of the classic inducers that act at the Ah receptor in vertebrates. A few studies of the roles of cytochromes P450 in the biosynthesis and degradation of steroids, including ecdysteroids, have been published. Further studies are needed to understand the regulation and normal function of the crustacean cytochromes P450.
Organic contaminants are continually entering aquatic environments and thence the tissues of resident biota. Mussels and other molluscs are used worldwide as sentinels in pollution monitoring. Recent years have seen the development of biological measurements (biomarkers) as tools for use in monitoring and environmental impact assessment, such biomarkers being indicative of contaminant exposure and/or impact. This paper describes established and developmental biomarkers in mussels responsive to exposure to organic contaminants, including some indicative of damage to DNA (''comet'' assay) putative induction of biotransformation enzymes (CYP1A-like protein), contaminant removal (MXR-like protein), lysosomal membrane damage and impairment of membrane function.
The presence and nature of immunoreactive proteins to several cytochrome P450 (CYP) antibodies in the columnar or whole body microsomal fraction of five sea anemone species (Anthopleura elegantissima, Aiptasia pallida, Anthopleura xanthogrammica, Bunodosoma cavernata and Condylactis gigantea) were probed by Western blot analysis. Anthopleura elegantissima, A. xanthogrammica, B. cavernata and C. gigantea columnar microsomes contained proteins that crossreacted with monoclonal anti-scup CYP 1A1 (~ 70 kDa), polyclonal anti-trout CYP 2K1 (~ 40 kDa) and polyclonal anti-trout CYP 3A1 (~ 30 kDa). Aiptasia pallida whole body microsomes contained various proteins that cross-reacted with scup CYP 1A1, trout CYP 2K1 and trout CYP 3A1 antibodies. 3-Methylcholantrene (3MC, 20 mg/kg) did not induce proteins recognized by anti-CYP 1A1 in A. elegantissima. Our results indicate the presence in anemone microsomes of various proteins with epitope regions recognized by several CYP antibodies; the most strongly recognized proteins differ from the 50–60 kDa molecular weights which are typically ascribed to P450s of vertebrate microsomes. In all anemones, proteins in the 50–60 kDa range are very weakly recognized even at relatively high protein concentrations.
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.
1. Sex differences in the MFO system were minimal.2. Coincident increases in contents of cytochrome P-450, the “416-peak” and total haemoprotein occurred in summer and early winter.3. Changes in NADH-dependent reductase activities were similar, and distinct from those of NADPH-cytochrome c reductase and cytochrome b5.4. NADPH-independent 7-ethoxycoumarin O-deethylase activity changed from undectable in summer to a maximum in early winter. The increase coincided with a peak in P-450 content, the appearance of a 51.5 Kd protein (SDS-PAGE), and a blue shift in the λmax of P-450.5. The results are interpreted in terms of isozymes of P-450 and a seasonal role in endogenous monooxygenation.bl]
Exposure to tributyl tin (TBT) at concentrations as low as 0.5 ng/liter has been associated with disrupted sexual physiology (imposex) in 40–50 species of marine gastropod. Imposex is a state of pseudohemaphrodism in which females exhibit nonfunctional secondary male characteristics. It has been suggested that the mechanism underlying imposex involves disrupted metabolism of endogenous sex hormones and in particular inhibition of cytochrome P450-dependent aromatization of androgens to estrogens. In the current study, the effects of TBT on the ability of the Periwinkle (Littorina littorea) to metabolize the androgenic sex steroid testosterone was examined in vitro and in vivo. Microsomes were prepared from ‘digestive gland’ (visceral complex including the gonads) and combined kidney and gill fractions. CO-ligated reduced difference spectra contained peaks at 459 nm and calculated P450 contents of 0.3 and 0.05 nmol/mg. Digestive gland microsomes were found to be capable of oxidative metabolism of testosterone in either the presence or absence of NADPH. In the absence of NADPH, testosterone was metabolized to androstenedione, 6β-, 6α- and 15β-hydroxytestosterone, 17β-estradiol and an unidentified metabolite possibly 3α-androstene-17β-diol. In the presence of NADPH the same products were produced at a higher rate but the major products formed were the products of the NADPH-dependent steroid 5α-reductase-3α(β)hydroxysteroid dehydrogenase pathway; dihydrotestosterone (DHT) and 3α(β)androstane-17β-diols (DHT diols). TBT, even at high concentrations up to 100 μM, had only modest effect on P450-dependent testosterone metabolism in vitro, producing increases in androstenedione formation and only 30–40% inhibition of aromatase. In vivo, [14C] testosterone was injected directly into the cephalopedal sinus of adult snails and animals maintained in sea water at 15 °C for 42 h in the presence of 0, 0.5 and 5 mM TBT. Testosterone was almost completely metabolized during this time, predominantly to water-soluble sulfur conjugates of testosterone, the 5α-reduced products and 6α-hydroxytestosterone. However, with increasing concentrations of TBT, more radioactivity was retained within the animal and was increasingly associated with organic extractable unmetabolized testosterone and its phase I products androstenedione, dihydroandrostenedione (DHA), DHT and DHT-diols. Thus it appears that, in vivo, TBT inhibits sulfur conjugation of testosterone and its phase I metabolites and their excretion resulting in a build-up of pharmacologically active androgens in the tissues. This data are consistent with the hypothesis that TBT-induced imposex in sensitive gastropods, such as stenoglossans, may arise from peturbations in sex steroid metabolism. However, the major biochemical targets of TBT appear to be steroid conjugation and excretory transport mechanisms rather than P450-dependent oxidative pathways such as aromatase.
Levels of cytochrome P-450 and related enzyme activities in Octopus pallidus were investigated for the first time to assess their potential for use as biomarkers of xenobiotic exposure. Spectral analysis revealed cytochrome P-450 in the digestive gland microsomes. Enzyme assays 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase were optimised with respect to temperature, pH and incubation time and NADPH cytochrome c reductase assays were performed. Corn oil was demonstrated to have an inhibitory effect on octopus cytochrome P-450 system. The study with Aroclor 1254 suspended in glycerol (100 mg/kg) revealed an elevation of 7-ethoxycoumarin O-deethylase activity, which would indicate cytochrome P-450 was induced.
Cytochrome P-450 and xenobiotic metabolizing enzymes have been detected in several marine invertebrate species, but have not been reported in corals. Low but detectable quantities of cytochrome P-450 (0.09 ± 0.16 pmol˙mg−1 microsomal protein), and the activities of several microsomal enzymes (benzo[a]pyrene hydroxylase (BaPH) (1.26 ± 0.97 pmol˙min˙mg−1), epoxide hydrolase (14.1 ± 6.9 nmol˙min˙mg−1) and UDP-glucuronosyltransferase (UDPGT) (2.73 ± 1.72 pmol˙min˙mg−1)) and a cytosolic enzyme (glutathione-S-transferase (GST) (15.2 ± 7.8 nmol˙min˙mg−1 soluble protein)) were found in the animal tissue of the scleractinian coral, Favia fragum. With the exception of GST, enzyme activities were low or equivalent to those found in other marine invertebrates. GST activities, which exceed those found in most invertebrates, may be related to protection from high oxygen tensions in coral tissues originating from photosynthesis by zooxanthellae. Cytochrome P-450, microsomal and soluble protein content, and the activities of several xenobiotic metabolizing enzymes were examined in colonies from three different reefs, along an inshore-offshore gradient, which varied in the amount of anthropogenic inputs. The levels of enzyme activity for BaPH and UDPGT were significantly higher in corals from the inshore site suggesting prior induction possibly by terrigenously derived compounds. Cytochrome P-450 content and xenobiotic metabolizing enzyme activities from colonies collected on a fourth reef over a lunar reproductive cycle varied greatly over the 1 month sampling period and did not exhibit any obvious patterns.
We investigated the ability of the edible crab (Cancer pagurus) to biotransform polycyclic aromatic hydrocarbon (PAH) compounds in vivo and in vitro. In vivo the metabolism of benzo(a)pyrene (BaP) was analysed by direct fluorimetry of hepatopancreas cytosol, demonstrating the formation of water-soluble metabolites of this PAH compound. A pyrene hydroxylase assay was developed for in vitro studies. The activity was dependent upon artificially supported NADPH-cytochrome P450 reductase, but no induction effect was observed in crabs treated with BaP (5 mg/kg) up to 30 d after treatment.
Enzymic studies showed that cholesterol is converted by mitochondrial preparations from the hepatopancreas of male Carcinus maenas into 7-ketochoIesterol, 7α and 7β-hydroxycholesterol, the compounds being identified by thin-layer chromatography and capillary gas chromatography-mass spectrometry. The enzyme system involved was significantly inhibited by polycyclic aromatic hydrocarbons, especially benzo[a]pyrene, as well as by cytochrome P-450 inhibitors such as carbon monoxide and SKF 525A. In addition, 7β-hydroxycholesterol and other steroid diols partially inhibited benzo [a] pyrene metabolism in microsomal preparations from the antennary glands of Carcinus. These interrelationships of cholesterol and benzo[a]pyrene metabolism in Carcinus are discussed with particular reference to possible biotransformations involving an initial peroxidation of cholesterol. © 1985, Marine Biological Association of the United Kingdom. All rights reserved.
Fenitrothion (O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate) is readily taken up from the water by the crayfish Procambarus clarkii. The bioconcentration factor, however, is lower than expected on the basis of its octanol/water partition coefficient. The ability of P. clarkii to metabolize fenitrothion was evaluated in vitro by using 12,000-g supernatant or microsomal fractions isolated from hepatopancreas and the results pointed out the existence of both oxidative and glutathione-mediated metabolism. Several biomarkers of exposure viz., the microsomal mixed-function oxygenase system, a phase II enzyme [glutathione S-transferase], and acetylcholinesterase inhibition, were studied in organisms exposed to 20 μg/L of fenitrothion for 48 h. Cytochrome P450 content, NADPH cytochrome c(P450) reductase, and 7-ethoxyresorufin O-deethylase activity showed significant induction the second day of treatment, whereas glutathione S-transferase activity appeared to be unaffected. Acetylcholinesterase activity in the neuromuscular tissue of P. clarkii was a good indicator of exposure to this compound.
Seasonal variations in the mixed-function oxygenase (MFO) system components (cytochrome P450, “418” peak, and NADPH-cytochrome c[P450] reductase) and antioxidant enzymes (catalase, superoxide dismutase [SOD], glutathione peroxidase [GPX], and DT-diaphorase) of the mussel Mytilus galloprovincialis have been evaluated. Its relation with contaminant body burden (PAHs, PCBs, DDTs, and lindane) as well as environmental parameters (water temperature, salinity, oxygen concentrations, and suspended matter) was determined. As a general trend, low MFO and antioxidant enzyme activities were detected in February-March, a peak in late April, and a gradual decrease with a minimum in June. This pattern was similar to tissue concentrations of PAHs, PCBs, DDTs, and lindane normalized to lipid weight. Cytochrome P450 content, however, exhibited a steady decrease from February to June. The observed seasonal variations are presumably related to the metabolic status of the animal, itself dependent on such factors as gonadal ripening, food availability, and the hydrological cycle, which regulates productivity in the area.
Freshwater pond mesocosms were used to validate xenobiotic-metabolizing enzymes as biomarkers of contamination by atrazine and hexachlorobenzene (HCB) in a basommatophoran gastropod, Lymnaea palustris (Müller). Over long-term (21-d) exposure to 5, 25, and 125 μg/L atrazine and to 0.5, 1.25, and 5 μg/L HCB, the uptake and internal concentration of both pesticides were followed, and the activities of benzo[a]pyrene hydroxylase (BaPH) and glutathione S-transferases (GSTs) of pesticide-exposed snails were compared with those of control animals maintained in untreated mesocosms. Internally recovered HCB concentrations were much higher than internal atrazine concentrations, but the uptake of atrazine was faster than that of HCB. Although it affected the integrity of microsomal membranes, HCB had no relevant effects on BaPH and GST activities at concentrations which affected growth and fecundity, thus confirming the low inducibility of mollusc xenobiotic-metabolizing enzymes by chlorinated compounds. In contrast, atrazine markedly inhibited BaPH and both postmitochondrial and cytosolic GSTs at the same concentrations, which had no effects on growth or reproduction. Enzyme inhibition was negatively correlated with the maximal internal amount of atrazine and positively correlated with the bioconcentration factor, suggesting that effects on xenobiotic-metabolizing enzymes may affect pharmacokinetics of atrazine within the snail body. Correlation between the bioconcentration factor and enzyme inhibition may serve as a descriptor of the physiological status of animals and can also be used to indirectly estimate the pesticide concentration in the environment. Laboratory data were considered for the interpretation of results obtained in the mesocosms. In the biomarker context, BaPH and GST activities are proposed, along with other biochemical markers already identified in atrazine- and HCB-exposed L. palustris, as elements of a multiparametric approach of the ecotoxicological effects of pesticides on freshwater ecosystems.
A comparative study on mixed-function oxygenase (MFO) systems was carried out on four echinoderm species: the asteroidsAsterias rubens andMarthasterias glacialis, a holothurianHolothuria forskali and an echinoidEchinus esculentus. Cytochromes P-450 andb
5 and the MFO system-associated NADH-ferricyanide reductases NADH-cytochromec reductase, NADPH-cytochromec reductase and benzo(a)pyrene (BP) hydroxylase activities were present in microsomal fractions of pyloric caeca ofA rubens andM. glacialis and in the haemal plexus ofH. forskali. In contrast, cytochrome P-450 and BP hydroxylase activity were not detectable in the gonads ofE. esculentus. The tissue and subcellular distribution of the MFO system was studied inA. rubens. MFO system components were found in the stomachs and gonads, although detection of cytochrome P-450 in the latter tissue was difficult. Sex-related differences were not significant. The contents of the MFO system components and BP hydroxylase activities were highest in the microsomal (100 000 g) fractions, but MFO system components were also found in the mitochondrial (12 000 g) and cytosolic fractions. The BP hydroxylase activity in pyloric caeca microsomes ofA. rubens was NADPH-dependent and was inhibited by several agents known to be inhibitors of vertebrate cytochromes P-450. In the former respect, the characteristics of the MFO system were more like those of vertebrates and crustaceans than that of molluscs.