Environ. Sci. Technol. 2004, 38, 6277-6282
Modulation of the Hepatic CYP1A1 System in the Marine Fish Gobius niger, Exposed to Xenobiotic Compounds FRANCESCA MARADONNA,† VALERIA POLZONETTI,‡ STELVIO M. BANDIERA,§ BEATRICE MIGLIARINI,† AND O L I A N A C A R N E V A L I * ,† Dipartimento di Scienze del Mare, Universita` Politecnica delle Marche, 60131 Ancona, Italy, Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Universita` di Camerino, Camerino (MC), Italy, and Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, British Columbia V6T 1Z3, Canada
Anthropogenic chemicals in the aquatic environment are known to cause reproductive disturbances in vertebrate and invertebrate organisms by interfering with the endocrine systems. Large efforts have recently been devoted to dissect the mechanisms of action of xenobiotics in aquatic species, with the ultimate aim of detecting and controlling the effects of chemical exposure on the aquatic ecosystem and humans. In the present paper, males of a marine species, the black goby (Gobius niger), were treated with estrogenic and dioxin-like compounds commonly discharged into the environment from industry, agriculture, and urban waste such as nonylphenol (NP) and β-naphthoflavone (β-NF). Their effects were compared with those induced by estradiol (E2), analyzing the expression of biomarkers commonly used in ecotoxicological studies such as vitellogenin (VTG) and cytochrome P4501A1. The treatment with NP induced the synthesis of the female specific protein VTG in males, showing its estrogenic activity. NP and E2 lowered cytochrome P4501A1 basal levels while β-NF determined a significant rise of its expression. The detoxification pathway was investigated, and the most relevant finding of this paper was the evidence that cytochrome P4501A1 inhibition by estrogen and estrogen-like compounds is mediated through the activation of the aryl hydrocarbon receptor repressor.
Introduction It has been known for some time that the normal operation of the endocrine (hormonal) system can be disrupted by a number of anthropogenic and naturally occurring chemicals, thereby affecting physiological processes under hormonal control (1). There are several ways in which chemicals can affect the endocrine system: for example, they can bind to hormone receptors and either mimic or inhibit the action of * Corresponding author telephone: +39-071-2204990; fax: +39071-2204650; e-mail:
[email protected]. † Universita ` Politecnica delle Marche. ‡ Universita ` di Camerino. § University of British Columbia. 10.1021/es049786h CCC: $27.50 Published on Web 09/25/2004
2004 American Chemical Society
natural hormones, or they can affect their synthesis and metabolism. Research to date has focused mainly on xenoestrogens or estrogen-like compounds, including nonylphenol, bisphenol A, and biphenyls (PCBs) (2-5), that mimic the biological activities of the female endogenous hormone estradiol (E2), inducing effects including the production of vitellogenin (VTG), the precursor of the egg yolk proteins. The presence of VTG in the plasma of a male fish is therefore considered a sensitive biomarker of exposure to an estrogenic chemical (6). In the past few years, attention has focused on industrial compounds and byproducts of industrial and combustion processes (polycyclic aromatic hydrocarbons, halogenated aromatic hydrocarbons including polychlorinated dibenzop-dioxins (PCDDs), dibenzofurans (PCDFs), PCBs, and their metabolites) that have been identified in almost every component of the global ecosystem including fish, wildlife, human adipose tissue, milk, and serum. Exposure of vertebrates to such compounds has been shown to cause liver damage, growth retardation, certain types of cancer, birth defects, depression of immunological function, and reproductive problems in several species (7). Halogenated aromatic hydrocarbons are metabolized very slowly and are highly persistent in lipophilic tissues (8). Furthermore, these compounds often biomagnify up the aquatic food chain. The biotransformation of lipophilic chemicals is a requisite for their detoxification and excretion in invertebrates and in fish. With this regard, it is well-known that the induction of hepatic cytochrome P4501A1 facilitates the excretion of lipophilic chemicals, and considering that polycyclic aromatic hydrocarbons (PAHs) and related compounds, such as β-naphthoflavone (β-NF) and dioxins induce cytochrome P4501A1 synthesis, its high level is a useful biomarker and an early warning signal of exposure in fish to these compounds. The gene codifying for the cytochrome P4501A1 is commonly indicated as CYP1A1 and its transcription is mediated by activation of the aryl hydrocarbon receptor (AhR) pathway. Xenobiotics initially bind to the cytosolic AhR, a protein that contains a basic helix-loop-helix motif and the Per-ARNT-Sim (PAS) domains (9). Upon ligand binding, AhR translocates into the nucleus and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein (10). The heterodimer binds to the xenobiotic response element (XRE) and alters the expression of genes controlled by XREs. Enhancer XREs are found in the promoter regions of several genes involved in the metabolism of xenobiotics including CYP enzymes (11), glutathione-Stransferase Ya (12), and NAD(P)H-quinone oxidoreductase (13). In 1999, Mimura et al. (14) isolated cDNA clones that encoded a polypeptide with high similarity to the sequence of AhR. This polypeptide was found to repress the transcription activity of AhR by competing with AhR in forming a heterodimer with ARNT and binding with XRE sequence and is thus designated AhRR or AhR repressor. AhRR-ARNT complexes bind to AhR responsive elements but are transcriptionally inactive. Furthermore, the expression of AhRR is induced by the AhR/ARNT heterodimer through binding to the enhancer sequence XRE, upstream of the AhRR gene. Thus the AhR functions are regulated by the feedback inhibition of AhRR. While the involvement of AhRR in mammalian physiology is well-known, only a few concerns have been raised about its role in teleosts. In addition, while induction of CYP enzymes in response to PAH compounds has been well-studied and documented (15, 16), data on the regulation of CYP enzymes by estrogenic VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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compounds are still limited (17). Steroids such as E2 and xenoestrogenic compounds such as nonylphenol (NP) act by binding to the estrogen receptor (ER) protein, which is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. The E2-ER complex interacts with the estrogen responsive elements (ERE) and activates the transcription of estrogen-regulated genes (18). A number of reports have indicated that the content of CYP1A in the liver depends on the sex and on the physiology of the fish. In both naturally maturing female fish with elevated levels of the reproductive steroid, E2, as well as in fish treated experimentally with E2, suppression of hepatic CYP1A mRNA, protein, and CYP1A-associated ethoxyresorufin O-deethylase (EROD) activity has been observed (19). For this reason, we focused our attention on the expression of CYP1A1 not only as a biomarker of exposure to hydrocarbons but also as a potentially sensitive bioindicator for the exposure of fish to xenoestrogenic compounds. Although most of the in vivo studies conducted to date have been on freshwater fish from polluted areas, recently the interest on the effects of these pollutants in marine fish is increasing (19-21). Moreover, as CYP1A1 expression can be influenced by environmental factors, reproduction, and starvation and in order to avoid the complexity of hormone effects in vivo, in vitro cultures of hepatocytes (22) or immature fish (22) were used as experimental models. In the present study a marine teleost the black goby, Gobius niger, was used. This marine fish is a small coastal species widespread over the Mediterranean area (23); it is a bottom-dweller that gathers in coastal water at spawning time and moves in the circumlittoral mud in winter (24). The aim of the present study was to assess the in vivo effect of estrogenic (NP and E2) and dioxin-like (β-NF) chemicals on black goby (G. niger) CYP1A1, a key molecule of phase I of detoxification system. Particular focus was placed on the analysis of AhRR gene expression as a new biomarker of contamination and its possible links with CYP1A1 gene expression.
Materials and Methods Fish Sampling and Treatments. Male black goby (G. niger) weighing 30-35 g were captured in the Adriatic Sea during the reproductive period (April-May) and maintained in tanks in a closed system with a salinity of 35 ppt (parts per thousand) with a natural photoperiod and at a constant temperature of 18 °C. After a recovery period of 3 weeks, the fish were divided into groups of 10 and treated. Doses and time of exposure were determined on the basis of the highest biological response obtained as previously described in ref 25: C (control): injected i.p. with corn oil and sacrificed after 72 h NP1: injected with NP (50 µg/kg) and sacrificed after 72 h NP2: injected with NP (500 µg/kg) and sacrificed after 72 h E2: injected with E2 (3.5 µg/kg) and sacrificed after 72 h β-NF: injected with β-NF (60 mg/kg) and sacrificed after 72 h NP, E2, and β-NF were dissolved in corn oil. At the end of exposure periods, fish were anesthetized with MS222 (Sigma); plasma samples were collected and kept at -20 °C; liver samples were taken and immediately frozen in liquid nitrogen and kept at -80 °C until assayed. NP was obtained from Fluka (Buchs, Switzerland). NP is a mixture of isomers with differently branched nonyl side chains and contains approximately 85% para isomers. The major impurities are 2-nonylphenol (ortho isomers), dodecylphenol, and dinonylphenol, which together comprise approximately 10% 6278
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of the 4-NP mixture. E2 and β-NF were purchased from Sigma (St. Louis, MO). Histology. Testis were removed and fixed for 6 h in Bouin’s solution for histological examination. Tissues were rinsed with 70% ethanol and then dehydrated in a graded series of alcohol concentrations. Tissues were embedded in paraffin, and serial sections of 6 µm thickness were cut. Sections were stained with hematoxylin and eosin (H&E) for examination by light microscopy (LM). Vitellogenin Analysis. Plasma samples were prepared and electrophoresed as previously described in ref 26. Briefly samples were electrophoresed on SDS-PAGE and electroblotted onto a Bio-Rad filter using a Bio-Rad mini trans-blot electrophoretic transfer cell. The transfer was carried out at 7 V/cm overnight at 4 °C using a 25 mM Tris base, 192 mM glycine, and 20% methanol as electrode solution. The nitrocellulose membrane was soaked in 5% Nonidet-P40 for 1 h to remove SDS and incubated with 2% bovine serum albumin (BSA; Sigma) in PBS buffer. The primary antibody (anti-VTG S. aurata) diluted 1:1000 in a solution containing BSA and 0.01% NaN3 in PBS was incubated for 2 h at room temperature (about 20 °C) and rinsed 3 times with PBS plus 0.05% Tween 20. The second antibody solution (HRPconjugated anti-rabbit IgG; Bio-Rad) diluted 1:1000 in 2% BSA in PBS buffer was incubated for 1 h. The filter was rinsed again with PBS without Tween 20. The blot was developed using as substrate ECL + Plus Western Blotting Detection System (Amersham Biosciences, UK). RNA Extraction and RT-PCR. Total RNA and first strand cDNA synthesis were performed as already described in ref 25. Briefly, total RNA was extracted from 50 mg of liver using TRIzol RNA isolation reagent (Invitrogen) based on the acid guanidinium thiocyanate-phenol-chloroform extraction (27) and following the manufacturer’s protocol. A total amount of 5 µg of RNA was used for cDNA synthesis, employing 0.5 mg of oligo d(T) + adapter primer, 5′GACTGCAGTCGACATCGATTTTTTTTTTTTTTTTTT-3′, in a buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM of each dNTP and 200 units of Superscript II RT (Invitrogen, Life Technologies, Milan, Italy) with incubation at 42 °C for 50 min. CYP1A1 and AhRR primers were designed on the basis of the homology among the sequences available in GenBank. CDNA was amplified with 5 units of Taq DNA polymerase (Dynazyme) in 20 µL of master mix containing 1× PCR buffer, 1.5 mM MgCl2, 2.5 mM dNTPs, and CYP1A1 primers (forward: 5′-CCCTGCAGACTTCATCCC-3′, reverse: 5′-TTTGTGCTTCATTGTGAGACC-3′) (50 pmol) or AhRR primers (forward: 5′-CATCTCCAAGCTGGACAA-3′, reverse 5′-GAT GGA AGC CCA GAT AGT C-3′). PCR amplification of CYP1A1 was carried out for 27 cycles with the following profile: denaturation at 94 °C for 1 min, primer annealing at 54 °C for 90s, and primer extension at 72 °C for 1 min. PCR amplification of AhRR was carried out for 25 cycles with the following profile: denaturation at 94 °C for 30 s, primer annealing at 52 °C for 30 s, and primer extension at 72 °C for 45 s. The PCR amplification of β-actin was carried out for 20 cycles using specific β-actin primers (forward: 5′-TTCCTCGGTATGGAGTCCT-3′, reverse: 5′TGGGGCAATGATCTTGATCTT-3′) with the following termocycler profile: 20 s at 94 °C, primer annealing at 56 °C for 30 s, and primer extension at 72 °C for 30 s. Cloning and Sequencing. PCR products obtained with specific primers were cloned as already described in ref 25. Briefly, the single PCR products obtained with CYP1A1 and AhRR specific primers were purified using the PCR purification kit (QIAGEN) and then cloned into the p-GEM T easy vector (Promega), following the manufacturer’s protocol. The plasmid was transformed into DH5R cells by the TransformAid kit (MBI Fermentas). Several positive clones were analyzed by PCR and restriction cutting in order to
FIGURE 1. Representative hematoxilin and eosin stained transverse section through the testis of male black goby, G. niger, to illustrate the organization of mature testis. Mature sperm (M) and spermatocytes cysts (SC) were indicated in the picture. verify the presence of the insert and then sequenced using an ABI model 310 DNA sequencer (Perkin-Elmer, Oak Brook, IL). Southern Blot. The DNA fragments from PCR were loaded onto a 1.5% TAE agarose gel, blotted onto a nylon membrane (Nytran Super charge, Schleicher & Schuell), and hybridized with homologous probes as described in ref 25. DNA probes were labeled with digoxygenin by a random primed reaction using the DIG DNA labeling kit (Roche). Gene Expression Quantification. The variation in CYP1A1 and AhRR mRNA expressions was evaluated by semiquantitative PCR using β-actin as an internal standard. At the amplification conditions described above, the parallel amplification efficiency was obtained at 27, 25, and 20 cycles for CYP1A1, AhRR, and β-actin, respectively. After homologous hybridization of Southern blots (standard buffer: 5× SSC, 1× blocking reagent, 0.1% sarkosyl, 0.02% SDS, at maximum stringency conditions), the PCR products were visualized by chemiluminescent detection and autoradiography. The films were scanned using a laser scanner (Sharp Electronics, Milan, Italy) and then subjected to densitometric analysis by ImageQuant software version 1.2 (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA). CYP1A1 Immunoblot Analysis. Whole tissue homogenates were prepared as previously described in ref 28. Briefly, 25-50 mg of liver tissue was homogenized in 125 µL of icecold buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1% TritonX100, 10% glycerol, 1 mM EGTA, 1.5 mM MgCl2, 0.1 mg/mL AEBSF (4-(2-aminoethhyl)benzensulfonylfluoride), 20 µg/ mL trypsin inhibitor, 4.46 µg/mL leupeptin, and 1.9 µg/mL aprotinin) for 5 × 10 s at 2000 rev/min using a 1-mL glass Teflon Potter-Elvehjem homogenizer. Homogenates were transferred to 1.5-mL microcentrifuge tubes and gently rotated for 1 h at 4 °C to complete cellular disruption. Cellular debris was removed by centrifugation at 10000g for 25 min at 4 °C, and the supernatant was collected and stored at -80 °C until used for immunoblots. Proteins from whole liver homogenates were separated by SDS-PAGE using a 7.5% acrylamide gel. For analysis of CYP1A, 100 µg of hepatic whole homogenate protein was loaded per lane. The gel was
subjected to electrophoresis at 32 mA/gel and then electrophoretically transferred onto nitrocellulose membranes (0.45 µm pore size; Bio-Rad) at 250 mV for 3 h at 4 °C. CYP1A was detected with a polyclonal rabbit anti-rainbow trout CYP1A peptide antibody (29) at a concentration of 10 µg/ mL. Protein concentration was measured by the Coomassie Brilliant Blue method (30) using BSA as standard. EROD Activity Determination. Liver slices (200 ( 10 mg) from each fish were homogenized with 1 mL of ice-cold homogenizing buffer (50 mM TRIS, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 150 mM NaCl) using a Potter-Elvehjem automatic homogenizer set at 4000 rpm. The homogenates were then transferred to a lidded polyethylene Eppendorf tube and stored on ice. Homogenates were centrifuged at 10000g for 20 min at 4 °C. Post-centrifugation supernatants were kept on ice and assayed within 30 min. EROD activity was measured using the modified assay of ref 31 with continuous fluorimetric detection. Fluorescence was measured at an emission wavelength of 580 nm and at an excitation wavelength of 535 nm. The reaction mixture, containing 8 µM ethoxyresorufin, 100 mM potassium phosphate buffer, 100 mM NaCl, pH 7.5, and an aliquot of the liver homogenate in a final volume of 495 µL, was incubated at 25 °C. The reaction was initiated by the addition of 5 µM NADPH. A standard curve of resorufin, dissolved in DMSO, was determined in the range of 0-0.5 mM. Data Analysis. Data presented in this paper are in the form mean ( SD of means. Results were examined by oneway ANOVA followed by Student-Newman-Keuls test or the Student’s t-test as appropriate, using a statistical software package, Stat View 512+TM (Brain Power Inc., USA). A p-value of 0.05 was used as the limit of statistical significance.
Results Testis Histology. Histological analysis revealed the testis in active spermatogenesis (Figure 1). The analysis was performed both on the control and the treated gonad, and no differences due to the chemical treatments were detected. Vitellogenin Immunoblot Analysis. Western blot analysis using antibody against the purified seabream VTG (26) VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Western blot analysis of vitellogenin in plasma tissue probed with anti-Sparus VTG (diluted 1:1000). C?, control female; C/, control male; NP1, nonylphenol 50 µg/kg; NP2, nonylphenol 500 µg/kg; E2, 17β-estradiol; β-NF, β-naphthoflavone.
FIGURE 4. Immunoblot analysis of CYP1A1 in liver tissue probed with rabbit anti-rainbow trout CYP1A peptide IgG. C, control male; NP1, nonylphenol 50 µg/kg; NP2, nonylphenol 500 µg/kg; E2, 17βestradiol; β-NF, β-naphthoflavone.
FIGURE 3. Southern blot analysis of CYP1A mRNA expression. Data, normalized using β-actin, are means of 10 independent fish. Data are expressed as mean ( SD; values with different letters indicate statistical significance (P < 0.05). C, control male; NP1, nonylphenol 50 µg/kg; NP2, nonylphenol 500 µg/kg; E2, 17β-estradiol; β-NF, β-naphthoflavone. detected the presence of a single VTG band of about 100 KDa both in the plasma of control females, NP and E2 treated males, whereas there was no detectable VTG in the plasma of control males or β-NF treated males (Figure 2). These data clearly evidenced the well-known estrogenic action of NP and E2, while β-NF seems to be a nonestrogenic compound. CYP1A1 and AhRR Sequence. A single fragment of 838 bp for CYP1A1 was obtained. Its sequence (Accession No. AF520610) provided a 82% match with the CYP1A1 sequences of Seriola quinqueradiata and Dicentrarchus labrax (data not shown). A 273 bp fragment for AhRR (Bankit 581307) 6280
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displaying 84% homology with Fundulus heteroclitus AhRR was obtained. CYP1A1 mRNA Expression. The densitometric analysis of the Southern blot hybridized with homologous CYP1A1 probe showed considerable variation (P < 0.05) in hepatic CYP1A1 gene expression among the different experimental groups. All treatments produced significant (P < 0.05) changes in CYP1A1 gene expression relative to the control group. Treatment with E2 and the estrogen-like compound NP produced a decrease of CYP1A1 mRNA levels, while treatment with β-NF, increased the transcription of CYP1A1 with respect to the control. As expected, β-NF induced CYP1A1 gene. The data shown in Figure 3 represent the means of 10 different fish for each treatment. Each analysis was repeated three times and normalized against β-actin. P450 Immunoblot Analysis. As anti-trout CYP1A IgG has been reported to specifically recognize the orthologous CYP1A1 enzyme in diverse species (29, 32), we used it to probe the hepatic homogenates of black goby, G. niger. The hepatic level of CYP1A1 protein was increased significantly following treatment with β-NF, and this protein was not evident in the control fish. In the E2- and NP-treated fish, no band corresponding to the CYP1A1 protein was detected (Figure 4). EROD Activity. The ethoxyresorufin O-deethylase assay (EROD) serves as a sensitive method to measure CYP1A1 catalytic activity. A significant increase in this activity was observed following the β-NF treatment. In contrast, treatment with estrogenic compounds, E2 and NP, both caused a reduction of EROD activity that was now at undetectable levels. The enzymatic activity levels presented, correlated well with the protein expression observed by immunoblot analysis and those of gene expression quantification, indi-
FIGURE 5. Standard curve of resorufin determined in the range 0-0,5 mM (A). EROD activity expressed as U/mg. A unit (U) of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of product (resorufin) per min at 25 °C and pH 7.5 (B). Values with different letters indicate statistical significance (P < 0.05). nd, not detected; C, control male; NP1, nonylphenol 50 µg/kg; NP2, nonylphenol 500 µg/kg; E2, 17β-estradiol; β-NF, β-naphthoflavone.
FIGURE 6. Southern blot analysis of AhRR mRNA expression. Data, normalized using β-actin, are means of 10 independent fish. Data are expressed as mean ( SD; values with different letters indicate statistical significance (P < 0.05). C, control male; NP1, nonylphenol 50 µg/kg; NP2, nonylphenol 500 µg/kg; E2, 17β-estradiol; β-NF, β-naphthoflavone. cating that the suppression observed at the level of gene transcription provoked a dramatic activity reduction of this enzyme in the hepatic tissue (Figure 5). Data shown represent the means of 10 different fish for each treatment. Each analysis was repeated three times. AhRR mRNA Expression. Densitometric analysis of the Southern blot hybridized with homologous AhRR probe showed considerable variation in hepatic AhRR gene expression among the different experimental groups relative to the control group. Treatment with E2 and NP produced an increase in AhRR mRNA levels, while treatment with β-NF decreased (P < 0.05) the transcription of AhRR respect to the control group. The data shown in Figure 6 represent the means of 10 different fish for each treatment. Each analysis was repeated three times and normalized against β-actin.
Discussion Considerable progress has been made in understanding the mechanisms by which environmental compounds may act to disturb normal physiological functioning in fish. While compounds that interact with endogenous hormone receptors (i.e., the estrogen receptor) and their biological effects still dominate the literature, other potential mechanisms induced by environmental contaminants continue to be
investigated. In many cases, these mechanisms are linked to effects observed in wild fish populations including compromised growth and reproduction, altered development, abnormal behavior, and alterations in hormone biosynthesis demonstrating a wide range of effects from the molecular to the whole animal level. From a mechanistic perspective, this paper evidenced some of the popularized effects of estrogenic chemical such as the induction of VTG in liver male treated with NP and the modulation of the P4501A1 system by compounds with contrasting potencies. Involvement of AhRR in the regulation of P4501A1 by estrogenic and dioxin like compounds was introduced to explore a novel pathway. In the present study, reproductive male black goby (G. niger) were treated with compounds commonly discharged in the environment, whose effects are of great interest for human and wildlife health: NP, E2, and β-NF. The effects of these compounds were examined following a molecular toxicological approach, using biomarkers such as cytochrome P4501A1 and VTG. The main goal of the study, however, was to investigate the effects of the above-mentioned compounds on a new target gene, the AhRR. This novel molecule had been evidenced for the first time in 1999 by Mimura et al. (14), who found that in mammals the AhRR protein is able to negatively regulate the transcriptional activity of the PAH receptor (the AhR) by its competition for the XRE binding. Taking into account this recent knowledge, the working hypothesis of the present study was the possible involvement of AhRR in the modulation of the cytochrome P4501A1 system under estrogenic and dioxin-like chemicals exposure. As a first step, by Western blot, the estrogenicity of NP was compared with that of E2 using VTG as an indicator. This female-specific protein was induced in the liver of both NPand E2-treated males, evidencing the estrogenic potency of NP in the species studied here. In comparison, as no VTG band was detected in the plasma of black goby males under β-NF treatment, this pollutant did not show any estrogenic action. In addition, the estrogenic compounds were found to suppress CYP1A1 gene expression and its associated activity, and the effect was related to the dose considered; the lower dose of NP produced a lower suppression of gene expression. Looking at P4501A1 protein, using a heterologous immunoblot assay, the protein was undetectable in the control and in NP- and E2-treated fish. These data are in agreement with previous in vitro studies showing that, in Salmo salar (33) and in cultured trout hepatocytes (34), both E2 and NP significantly suppressed hepatic CYP1A1 mRNA levels (34) and EROD activity (33, 34) and decreased P4501A1 protein (33). On the contrary, the β-NF treatment, as already demonstrated in Sparus aurata by Pretti et al. (35), produced an increase of CYP1A1 mRNA, protein, and activity. Several hypotheses have been advanced to explain the CYP1A1 down-regulation by E2. Steroids can bind the P4501A1 protein (36), and through this binding, E2 or the metabolites generated from E2 may inhibit the catalytic activity of P4501A1 protein (37). Navas and Segner (34) hypothesized that the inhibitory action of E2 could be mediated, at least in part, through the hepatic estradiol receptor: the ER-E2 complex can interfere with the CYP1A1 gene directly or alternatively may interact with the AhR, thereby indirectly regulating CYP1A1 gene expression through binding the XRE. In addition, β-NF is also able to control the ERs recruitment; in fact, besides activating the detoxification pathway, it was recently shown to exert its effects by activating the AhR/ARNT heterodimer, which is able to interact with the unliganded ER, leading to induction of estrogenic pathway (38). VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Although genes regulated by the AhR/XRE often present XRE sequences in the promoter regions, but whether a given XRE sequence will function as a transcriptional active enhancer or silencer may depend on the context of a specific promoter, and this needs to be experimentally tested in each case. In our experimental model, the down-regulation of CYP1A1 by compounds with estrogenic activity indicated that the XRE sequence may function as a transcriptional active silencer. However, the most relevant finding was that the modulation of P4501A1 system by NP, E2, and β-NF is clearly mediated by AhRR gene expression. In summary, the present results show that, in black goby (G. niger), the reduction of the cellular detoxification capability determined by estrogenic compounds may be explained not only through the involvement of the ER pathway (34) as indicated by the VTG synthesis induction but also by their ability to prevent AhR activation, through the transcription of the AhRR. The inhibition of the CYP1A1 gene and the reduction of the cellular detoxification capability by estrogenic compounds together with their capacity to induce VTG (a femalespecific protein in male liver) led us to speculate that the fitness of organisms living in such contaminated habitats may be compromised. In conclusion, this study may be extremely important in the determination of new biomarkers necessary to clarify the up- or down-regulation capacity of individual pollutants with respect to the targets commonly used to evaluate environmental health. To better understand the effect exerted from the different pollutants on organisms and populations, it is of great importance to focus on how each chemical acts at molecular level. This will provide evidence and knowledge to build an integrated model to use in further studies focused on an environmental relevant contest and possibly to fill up the lack of evidence to sustain that many of the molecular changes are associated with ecologically relevant effects in terms of their impact on population.
Acknowledgments We thank Dr. Ike Olivotto, Dr. Giorgia Gioacchini, and Dr. Cristina Cionna for helping with animal care; Prof. Charles Tyler for critically reading the manuscript; and Dr. Ronny Van Aerle for the technical support in histological studies. We also thank Angelo Maradonna for kindly supplying the fish. This study was supported by “Fondi di Ateneo 2003, Universita` Politecnica delle Marche” grant awarded to O.C.
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Received for review February 11, 2004. Revised manuscript received July 21, 2004. Accepted July 23, 2004. ES049786H
