Gemfibrozil modulates cytochrome P450 and peroxisome proliferation-inducible enzymes in the liver of the yellow European eel (Anguilla anguilla).

The human lipid regulator gemfibrozil (GEM) has been shown to induce peroxisome proliferation in rodents leading to hepatocarcinogenesis. Since GEM is found at biological active concentrations in the aquatic environment, the present study investigates the effects of this drug on the yellow European eel (Anguilla anguilla). Eels were injected with different concentrations of GEM (0.1 to 200 µg/g) and sampled 24- and 96-h post-injection. GEM was shown to inhibit CYP1A, CYP3A and CYP2K-like catalytic activities 24-h post-injection, but at 96-h post-injection, only CYP1A was significantly altered in fish injected with the highest GEM dose. On the contrary, GEM had little effect on the phase II enzymes examined (UDP-glucuronyltransferase and glutathione-S-transferase). Peroxisome proliferation inducible enzymes (liver peroxisomal acyl-CoA oxidase and catalase) were very weakly induced. No evidence of a significant effect on the endocrine system of eels was observed in terms of plasmatic steroid levels or testosterone esterification in the liver.

Use of effect-directed analysis for the identification of organic toxicants in surface flow constructed wetland sediments.

Wetlands constitute one of the most efficient ecosystems with a great capacity to recycle the organic matter and able to attenuate or mitigate the chemical pollution. However, limited information exists on the ecotoxicological effects that may be caused due to the presence of these pollutants in wetland sediments. In this work, a bioassay-directed approach was used to identify toxicologically active compounds retained in sediments from a surface flow constructed wetland located in the North-Eastern of Spain. Sediment fractionation was accomplished by pressurized-liquid extraction (PLE) followed by semipreparative normal phase high performance liquid chromatography (NP-HPLC). During the extraction procedure, different solvents were sequentially applied in order to selectively extract the compounds as a function of their polarity. The cytotoxicity of the resulting fractions was assessed on the fish hepatoma cell line PLHC-1 by using the thiazolyl blue tetrazolium bromide (MTT) assay, while the presence of CYP1A inducing agents was determined by measuring the activity 7-ethoxyresorufin-O-deethylase (EROD) in exposed cells. Identification of the compounds was performed by gas chromatography coupled to mass spectrometry (GC/MS). Compounds such as polycyclic aromatic hydrocarbons (PAHs), non-steroidal anti-inflammatory drugs (NSAIDs), polycyclic musk fragrances and pesticides were identified in the most toxic fractions.

UDP-glucuronosyltransferase 1A6 overexpression in breast cancer cells resistant to methotrexate.

Methotrexate is a chemotherapeutic agent used in breast cancer treatment, but the occurrence of resistance limits its therapeutic use. A microarrays analysis between sensitive and methotrexate resistant MCF7 and MDA-MB-468 breast cancer cells pointed out the UDP-glucuronosyltransferase 1A (UGT1A) family as a common deregulated node in both cell lines. This family of genes is involved in Phase II metabolism. UGT1A6 was the main isoform responsible for UGT1A family overexpression in these cells. Its overexpression was not due to gene amplification. Transfection of a vector encoding for UGT1A6 in sensitive cells counteracted the cytotoxicity caused by methotrexate. Methotrexate increased the transcriptional activity from a luciferase reporter driven by the UGT1A6 promoter and induced UGT1A6 mRNA and enzymatic activity. Promoter analysis suggested that UGT1A6 induction by methotrexate could be driven by the transcription factors ARNT (HIF-1) and AhR/ARNT. Cells incubated with anticancer drugs susceptible to glucuronidation, such as tamoxifen or irinotecan, together with methotrexate, showed a lesser degree of cytotoxicity, due to UGT1A6 induction. The pharmacological effect of this induction should be taken into account when combining methotrexate with other drugs that are glucuronidated.

Assessment of metabolic capabilities of PLHC-1 and RTL-W1 fish liver cell lines.

Metabolic capabilities of PLHC-1 and RTL-W1 cell lines were investigated since to date, cytochrome P450 (CYP) 1A and glutathione-S-transferase have been almost the unique biotransformation enzymes reported in these cells. Functionality of CYP3A-, CYP2M- and CYP2K-like enzymes was assessed by studying the hydroxylation of testosterone (T) and lauric acid (LA), and glucuronidation and sulfation capacity was assessed by looking at 1-naphthol (1-N) and T conjugation. Only PLHC-1 cells showed the ability to hydroxylate T at 6beta-position (a CYP3A-like catalysed pathway) and LA at (omega-1)-position (a CYP2K-like catalysed pathway). Hydroxysteroid dehydrogenase and steroid reductase enzymes showed comparatively higher activities than CYPs: 5alpha-dihydrotestosterone, androstenedione and 3beta-androstanediol were the major metabolites of T detected in both cell lines. Regarding phase II activities, both cell lines metabolised 1-N to glucuronide and sulfate conjugates. In contrast, when using T as substrate, RTL-W1 formed the glucuronide, whilst PLHC-1 formed the corresponding sulfate. Overall, the observed enzymatic activities are much lower (up to 17.5 x 10(3) times) than those reported in primary cultures of fish hepatocytes. The present study highlights the need of developing new fish cell lines that could be used as alternative in vitro tools for studying xenobiotic metabolism and toxicity in fish.

Effects of fibrates, anti-inflammatory drugs and antidepressants in the fish hepatoma cell line PLHC-1: cytotoxicity and interactions with cytochrome P450 1A.

Effects of 11 pharmaceuticals belonging to three therapeutic classes (lipid regulators from the fibrate group, non-steroidal anti-inflammatory drugs and anti-depressives from the selective serotonin reuptake inhibitors group) were assessed in the fish hepatoma cell line (PLHC-1) by looking at cytotoxicity and interactions with cytochrome P450 1A (CYP1A) function. Among the tested pharmaceuticals, fluoxetine and paroxetine exerted cytotoxic effects, cell viability decreased to 52% and 6% after 24 h of exposure to 20 microM fluoxetine and paroxetine, respectively. The cytotoxicity of both compounds was modulated by cytochrome P450 inhibitors and was dramatically reduced when culture medium was supplemented with reduced glutathione and vitamin E succinate. Additionally, exposure of PLHC-1 cells to some pharmaceuticals led to an early and transient induction of ethoxyresorufin O-deethylase (EROD) activity: bezafibrate and antidepressants induced EROD activity at a concentration of 1 microM whereas clofibrate, ibuprofen and naproxen acted as inducers at a higher concentration (10 microM). These effects might be of toxicological concern since alterations of CYP1A may affect xenobiotic metabolism and toxicity.

The interference of pharmaceuticals with endogenous and xenobiotic metabolizing enzymes in carp liver: an in-vitro study.

The interactions of fibrate (clofibrate, fenofibrate, bezafibrate, gemfibrozil), antiinflammatory (ibuprofen, diclofenac, naproxen, ketoprofen), and anti-depressive (fluoxetine,fluvoxamine, paroxetine) drugs with CYP catalyzed pathways (CYP1A, CYP3A-, CYP2K-, and CYP2M-like) and Phase II activities (UDP-glucuronosyltransferases and sulfotransferases), involved in both xenobiotic and endogenous metabolism in fish, were investigated in-vitro by incubating carp liver subcellular fractions in the presence of the substrate and the selected drug. Anti-depressive drugs were strong inhibitors of CYP1A (92-94% inhibition), CYP3A-like (69-80% inhibition), and CYP2K-like (36-69% inhibition) catalyzed activities, while antiinflammatory drugs were potent CYP2M-like inhibitors (32-74% inhibition). Among the lipid regulators, gemfibrozil strongly inhibited CYP2M-catalyzed activity (91% inhibition) and other CYP isoforms (CYP1A and CYP3A-like). Additionally, glucuronidation of naphthol and testosterone were targeted by antiinflammatory drugs, and to a lesser extent, by fibrate drugs (48-78% inhibition). No significant alteration on sulfotransferase activities was observed, apart from a minor inhibitory effect of clofibrate, gemfibrozil, and fluoxetine on the sulfation of estradiol. Overall, gemfibrozil, diclofenac, and the three anti-depressive drugs appear to be the pharmaceuticals with the highest potential to interfere with fish metabolic systems.

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.

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.

Effects of 17beta-estradiol exposure in the mussel Mytilus galloprovincialis: a possible regulating role for steroid acyltransferases.

Mussels (Mytilus galloaprovincialis) were exposed to different concentrations of estradiol (20, 200, and 2000 ng/L) in a semi-static regime (1-day dosing intervals) for up to 7 days in an attempt to see how mussels deal with exogenous estrogenic compounds. Whole tissue free-estradiol levels were only significantly elevated at the high exposure dose, whereas total-estradiol (free+esterified) sharply increased in a dose-dependent manner, from 2 ng/g in controls to 258 ng/g at the high exposure group. Neither free nor esterified testosterone levels showed significant differences between control and exposed organisms. The results suggest the existence of mechanisms that allow mussels to maintain their hormonal levels stable, with the exception of the high exposure dose, and the important role that fatty acid esterification, e.g. palmitoyl-CoA:estradiol acyltransferases, may play within those mechanisms. Additionally, the activity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 5alpha-reductase, P450-aromatase, and estradiol-sulfotransferases were investigated in digestive gland microsomal and cytosolic fractions. All these activities were differently affected by estradiol exposure. Overall, the study contributes to the better knowledge of molluscan endocrinology, and defines new mechanisms of regulation of free steroid-levels in mussels.

Effects of 17beta-estradiol exposure in the mussel Mytilus galloprovincialis.

Mussels Mytilus galloprovincialis were exposed to different concentrations of estradiol (20, 200, and 2000 ng/l) in a semi-static regime (1-day dosing intervals) for up to 7 days in an attempt to see how mussels dealt with exogenous estrogenic compounds. Sex hormone levels were determined in whole tissue. Free-estradiol was only significantly elevated at the highest exposure dose (up to 10-fold). Most of the estradiol was in the tissues as fatty acid esters (> 78%), which sharply increased in a dose-dependent manner (from 4 ng/g in controls to 258 ng/g at the high exposure group). In contrast, neither free nor esterified testosterone levels showed significant differences between control and exposure groups. The results suggest the existence of mechanisms that allow mussels to maintain their hormonal status, and the important role that fatty acid esterification may play within those mechanisms. Synthesis and conjugation rates of estradiol were further investigated by measuring the activity of P450 aromatase, and palmitoyl-CoA:estradiol acyltransferase, in digestive gland microsomal fractions. Overall, the study contributes to the better knowledge of molluscan endocrinology, and defines new mechanisms of regulation of free steroid-levels in mussels.

First evidence of endocrine disruption in feral carp from the Ebro River.

Feral carps (Cyprinus carpio) were collected in spring 2001 from five sites along the lower course of Ebro River (Spain) with the aim of investigating the existence of endocrine-disrupting effects. Several findings (low gonadosomatic index (GSI), plasmatic vitellogenin (VTG), depressed levels of testosterone, and histological alterations in gonads) detected in male carps downstream of Zaragoza's sewage treatment plant (STP) strongly suggest that the concentration of sewage effluent in the area is a major causal factor leading to the detected estrogenic effects. Important alterations (viz. delayed maturation in females, indications of arrested spermatogenesis in males) were detected in carps from Flix, a heavily industrialized area. Low ovarian P-450 aromatase and reduced glucuronidation of testosterone and estradiol in males were observed in Zaragoza and Canal Imperial de Aragón-an agricultural area-which suggest decreased estrogen synthesis, and possibly, reduced sex hormone excretion in those organisms. These results were related to some in vitro assays aimed to assess the interference of model compounds (atrazin, vinclozolin, diuron, pp'-DDE, dicofol, triphenyltin, nonylphenol, and fenarimol) with the glucuronidation of testosterone and estradiol by liver microsomal fractions. The fungicide fenarimol (10-20 microM) and nonylphenol (50 microM) were found to significantly inhibit (20%) both activities at relatively low doses. Overall, this work provides the first evidence of the existence of significant alterations of the endocrine system of carps from the medium-low course of the Ebro River and demonstrates the ability of several chemicals to modulate the inactivation of endogenous steroids.

Effects of endocrine disrupters on sex steroid synthesis and metabolism pathways in fish.

The interactions of estrogenic (nonylphenol, dicofol, atrazine), androgenic (organotins, phthalates, fenarimol) and anti-androgenic compounds (vinclozolin, diuron, p,p'-DDE) with key enzymatic activities involved in both synthesis and metabolism of sex hormones was investigated. Carp testicular microsomes incubated in the presence of androstenedione and different xenobiotics evidenced higher sensitivity of 5alpha-reductase activity than 17beta-hydroxysteroid dehydrogenase activity towards those chemicals. Dicofol, organotins and phthalates were among the most effective inhibitors. In contrast, ovarian synthesis of maturation-inducing hormones (20alpha- and 20beta-hydroxysteroid dehydrogenase activities) were enhanced by nonylphenol, dicofol, fenarimol and p,p'-DDE. Metabolic clearance pathways of hormones were also affected. Fenarimol, nonylphenol and triphenyltin inhibited the glucuronidation of testosterone and estradiol at concentrations as low as 10, 50 and 100 microM, respectively. Triphenyltin, tributyltin and nonylphenol were also inhibitors of estradiol sulfation with IC(50) values of 17, 18 and 41 microM. Overall, the data indicates the interaction of selected chemicals with key enzymatic pathways involved in both synthesis and metabolism of sex hormones. This interference might be one of the underlying mechanisms for the reported hormonal disrupting properties of the tested compounds, and might finally affect physiological processes such as gamete growth and maturation.

Residues of 14C-4n-nonylphenol in mosquitofish (Gambusia holbrooki) oocytes and embryos during dietary exposure of mature females to this xenohormone.

In order to assess in fish the maternal transfer of alkylphenolic compounds to the progeny, the identification and quantification of the labelled compounds present in oocytes and embryos was conducted after dietary exposure of mature female mosquitofish to 14C-4n-nonylphenol during vitellogenesis and embryogenesis respectively. Radioactivity found in bile and liver extracts accounted for 0.9-0.6 and 0.2-0.1% of ingested radioactivity for females exposed during vitellogenesis and embryogenesis respectively. The amount of extractable radioactivity present in oocytes and embryos was 0.19 and 0.07% of the ingested dose respectively. The radio-HPLC profiles obtained from bile, liver, oocyte and embryo extracts were similar. They showed the presence of 4n-NP-glucuronide as the major metabolite and traces of unchanged 4n-NP. The other metabolites corresponded to 8-hydroxynonylphenol, 9-(4-hydroxyphenyl)-nonanoic acid and para-hydroxybenzoic acid which is the final product of the alkyl chain oxidation. Our results indicate that exposure of ovoviviparous female fish to 4-NP during vitellogenesis and embryogenesis leads to the contamination of the progeny by 4-NP and its metabolites.