The interference of nitro- and polycyclic musks with endogenous and xenobiotic metabolizing enzymes in carp: an in vitro study.

Synthetic musks are widely used as perfuming agents in products, such as cosmetics, detergents, and soaps. The increased detection of these substances in the aquatic environment and their high bioconcentration potential raises concerns about potential effects on aquatic species. This work aimed at assessing the interactions of the most widely used musks: nitromusks (musk xylene, musk ketone) and polycyclic musks (celestolide, galaxolide, and tonalide) with fish enzymatic systems involved in both xenobiotic and endogenous metabolism. Therefore, CYP catalyzed pathways were investigated in carp liver microsomes (CYP1A, CYP3A), ovarian microsomes (CYP19) and testicular mitochondria (CYP17 and CYP11beta) using standard substrates. Phase II activities (UDP-glucuronosyltransferases and sulfotransferases) were determined in carp liver microsomes and cytosol, respectively. Polycyclic musks (galaxolide and tonalide) were stronger inhibitors of CYP3A- (IC(50): 68-74 microM), CYP17- (IC(50): 213-225 microM), CYP11beta- and CYP19-catalyzed activities than nitromusks, while the latter showed higher ability to interfere with CYP1A (IC(50): 35-37 microM). The sulfation of estradiol was also significantly inhibited by tonalide and galoxolide (IC(50): 140-294 microM). Overall, polycyclic musks showed the highest potential to interfere with those activities involved in the synthesis and metabolism of steroids while nitromusks mainly interfered with xenobiotic metabolism (CYP1A-catalyzed reactions). The obtained data suggest that CYP isoforms are potentially sensitive targets of synthetic musk substances in fish.

Endocrine alteration and other biochemical responses in juvenile turbot exposed to the Prestige fuel oil.

Juvenile turbot (Scophthalmus maximus) were exposed to different concentrations of the Prestige fuel oil through the diet. The effects on hepatic biotransformation enzymes, namely, 7-ethoxyresorufin O-deethylase (EROD), catalase, and phase II activities - UDP-glucuronosyltransferases (UGT) and cytosolic sulfotransferases (SULT) -, were monitored after 42-day exposure. Additionally, potential alterations on the endocrine system of juvenile turbot were assessed by measuring circulating levels of testosterone and 17beta-estradiol in plasma, together with gonadal P450 aromatase activity and the glucuronidation of testosterone by liver microsomal fractions. Fluorescent aromatic compounds (FACs) in bile were determined as an indicator of exposure to polycyclic aromatic hydrocarbons (PAHs). EROD activity increased in a dose dependent manner, and a positive correlation between levels of FACs in bile and EROD activity was observed. A significant increase in UGT activity was observed in fish exposed to medium and high fuel doses, whereas SULT cytosolic activity was not responsive to exposure. Exposure to the Prestige fuel oil sharply reduced circulating levels of testosterone in plasma. The obtained results suggest the ability of the Prestige fuel oil to alter hepatic biotransformation enzymes and to disrupt endogenous hormone levels in juvenile turbot, with unknown consequences in terms of sexual differentiation and reproduction.

Sulfotransferase activities towards xenobiotics and estradiol in two marine fish species (Mullus barbatus and Lepidorhombus boscii): characterization and inhibition by endocrine disrupters.

We have characterized hepatic phenol sulfotransferase (SULT) activities in two benthic fish species, Mullus barbatus and Lepidorhombus boscii, using p-nitrophenol, dopamine, 17beta-estradiol, 4-nonylphenol, and 1-naphthol as substrates. High affinity sulfation of 17beta-estradiol was observed in both species (Km=28-75 nM), suggesting the presence of a specific estrogen sulfotransferase that catalyzes the formation of estradiol-3 sulfate. Among the tested compounds, 1-naphthol was the most effective substrate for sulfation, with Vmax/Km ratios several hundred-fold higher than the other substrates examined. Both species sulfated the tested compounds, but only M. barbatus was able to sulfate dopamine. We also tested the inhibitory effects of common marine pollutants with estrogenic (4-nonylphenol) and androgenic (tributyltin, triphenyltin) properties on p-nitrophenol and 17beta-estradiol SULT activities. 4-Nonylphenol and triphenyltin inhibited sulfation of both substrates at micromolar concentrations in both species. However, tributyltin was only effective against SULTs from L. boscii, again at micromolar concentrations. The data indicate that M. barbatus and L. boscii are able to sulfate a range of xenobiotics and endogenous compounds, and inhibition of these activities by environmental pollutants may contribute to the known toxic effects of these compounds.

Endocrine alteration in juvenile cod and turbot exposed to dispersed crude oil and alkylphenols.

Juvenile Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) were exposed for 3 weeks in a continuous water flow to 0.5 ppm of dispersed North Sea crude oil, 0.5 ppm of dispersed North Sea crude oil spiked with 0.1 ppm of a mixture of alkylphenols (offshore oil production), and 30 ppb of nonylphenol (NP). As potential markers of endocrine alteration, key enzymatic activities involved in both synthesis (17beta-hydroxysteroid dehydrogenases and P450 aromatase) and metabolism (liver UDP-glucuronosyltransferases (UGT) and sulfotransferases) of steroids were assessed together with circulating levels of testosterone and estradiol in plasma. NP-exposed turbot had lower ovarian P450 aromatase, lower levels of testosterone and estradiol in plasma, and lower glucuronidation rates of sex steroids than those from the control group. In contrast, higher liver UGT-testosterone, and a trend towards higher P450 aromatase was detected in oil-exposed specimens. Those exposed to the combination oil+alkylphenols had lower levels of estradiol in plasma than controls, and no significant effects on any the enzymatic activities tested was observed. All these alterations were more evident in turbot than in cod. In fact, apart from a higher glucuronidation rate of estradiol detected in the liver of NP-exposed cod, no significant differences were observed between control and exposed cod.

The combined use of chemical and biochemical markers to assess water quality along the Ebro River.

Carp (Cyprinus carpio) and barbel (Barbus graellsii) were collected from five sites along the Ebro River. The study was designed to assess levels of persistent organic pollutants and metals bioaccumulated by fish, and some biochemical responses (cytochrome P450 system, phase II activities, and metallothioneins) against those pollutants. The highest levels of PCBs and DDTs were detected in carp from industrialised areas, which also showed high levels of mercury and cadmium in the liver, and high levels of nonylphenol in bile. Significant alterations in some biochemical markers were observed and associated to combined exposure to pollutants. The activity 7-ethoxyresorufin O-deethylase ranged from 69 pmol/min/mg protein in carp from the reference site to 415 pmol/min/mg protein in those from polluted sites. Carp from the Ebro Delta, an agricultural area, had depressed acetylcholinesterase in muscle tissue. Metallothionein concentrations were positively correlated with copper residues, but no significant differences among sampling points were observed.

Evidence of endocrine alteration in the red mullet, Mullus barbatus from the NW Mediterranean.

Red mullet (Mullus barbatus) were collected from different sampling sites (NW Mediterranean) in spring and autumn, with the aim of assessing potential alterations of the endocrine system. Alkylphenols were measured in fish bile as an indicator of estrogenic exposure. Key enzymatic activities involved in both synthesis (ovarian 17beta-hydroxysteroid dehydrogenases and P450 aromatase) and metabolism of steroids were assessed together with histological alterations of the gonads. During the spring sampling, delayed gamete maturation, intersexuality, fibrosis, and depressed ovarian P450 aromatase activity were observed in organisms from the most polluted sites. During the autumn sampling, those effects were less evident, indicating that fish might be more susceptible to endocrine disrupting chemicals during the reproductive period. Nonetheless, enhanced glucuronidation of testosterone and estradiol was observed. Overall, this work provides first evidences of significant alterations in the endocrine system of red mullet from highly impacted areas in the NW Mediterranean.