Comparative Biochemistry and Physiology, Part C 142 (2006) 356 – 364 www.elsevier.com/locate/cbpc Vitellogenin induction in the endangered goodeid fish Girardinichthys viviparus: Vitellogenin characterization and estrogenic effects of polychlorinated biphenyls☆ Armando Vega-López a,⁎, Laura Martínez-Tabche a , Maria Lilia Domínguez-López b , Ethel García-Latorre b , Eva Ramón-Gallegos c , Alejandra García-Gasca d a c Laboratorio de Toxicología Acuática, Escuela Nacional de Ciencias Biológicas, IPN. Prol. Carpio y Plan de Ayala s/n, Col. Plutarco Elías Calles “Casco de Santo Tomás”, D.F. CP 11340, México b Laboratorio de Inmunoquímica, Escuela Nacional de Ciencias Biológicas, IPN. Prol. Carpio y Plan de Ayala s/n, Col. Plutarco Elías Calles “Casco de Santo Tomás”, D.F. CP 11340, México Laboratorio de Citopatología Ambiental. Escuela Nacional de Ciencias Biológicas, IPN. Prol. Carpio y Plan de Ayala s/n, Col. Plutarco Elías Calles “Casco de Santo Tomás”, D.F. CP 11340, México d Laboratorio de Biología Molecular, CIAD Mazatlán. Mazatlán Sinaloa, México Received 23 June 2005; received in revised form 24 October 2005; accepted 1 November 2005 Available online 27 December 2005 Abstract Vitellogenin (VTG) is a widely used biomarker in studies of endocrine disruption induced by xenobiotics such as polychlorinated biphenyls (PCBs). This study evaluates the estrogenic effects of these compounds on the black-fin goodeid Girardinichthys viviparus, an endangered fish species in Mexico with a reduced range of distribution due to pollution of its natural environment. Adult fish born in the laboratory were exposed to half the LC0 of Inerteen® commercial PCB mixture. VTG was determined through an inhibition enzyme-linked immunosorbent assay (ELISA) using a homologous–heterologous system. Male and female fish were killed after 1, 2, 4, 8 and 16 days of exposure. The distal third of each specimen was used for analysis. VTG was obtained from cultured hepatocytes and blood serum of males previously exposed to 17β-estradiol. VTG molecular mass was 348 kDa. PCBs were found to elicit greater estrogenic effects on VTG induction in males than in females (p b 0.05) and sex differences were noted. Time-dependent VTG induction kinetics in males and a stationary phase in females were also observed. © 2005 Elsevier Inc. All rights reserved. Keywords: Endocrine disruption; Girardinichthys viviparus; Goodeid fish; Vitellogenin; PCBs; Waterborne exposure; Sex differences 1. Introduction Vitellogenin (VTG) is a characteristic protein in females, associated with egg production (Tata and Smith, 1979; Wiley et al., 1979; Nagler and Idler, 1990) in both oviparous and viviparous species, and it is used as a biomarker for monitoring xenobiotics that mimic estrogen (Fukada et al., 2003). Synthesis ☆ This paper is part of a special issue of CBP dedicated to “The Face of Latin American Comparative Biochemistry and Physiology” organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Tania ZentenoSavín (Mexico) and the editors of CBP. This issue is in honour of Cicero Lima and the late Peter W. Hochachka, teacher, friend and devoted supporter of Latin American science. ⁎ Corresponding author. Tel.: +52 55 57 29 63 00x62343. E-mail address: avegadv@terra.com (A. Vega-López). 1532-0456/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.11.009 of this biomolecule is regulated by the endocrine control axis, where external stimuli condition internal stimuli and there is a feedback from the latter to the axis (Nagler and Idler, 1990; Singh and Singh, 1991; Arukwe and Goksøyr, 2003). Despite endocrine control by the organism, certain man-made and natural substances are capable of altering these processes (García et al., 1997; Bowman et al., 2000; Nicolas, 2001; Spengler et al., 2001; Corsi et al., 2003; MacLatchy et al., 2003). Such compounds are known as endocrine disruptors (Nicolas, 1999; Arukwe and Goksøyr, 2003). In vitro and in vivo studies have shown that polychlorinated biphenyls (PCBs) in commercial mixtures elicit estrogenic and anti-estrogenic effects, and toxic response is related to the number of chlorine atoms in the PCB molecule (Gierthy et al., 1997; Vakharia and Gierthy, 2000; Bonefeld-Jørgensen et al., 2001). Toxicity and biotransformation are inversely proportional A. Vega-López et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 356–364 to the number of chlorine atoms. There is evidence that octa-, nona- and decachlorobiphenyls are not biotransformed (Groten et al., 1999) and endocrine disruption potential is species-dependent. When VTG is synthesized due to false stimuli, a number of important alterations may follow. For instance, the energy investment involved in reproducing outside normal periods may be enormous (Stancel et al., 1995). Males bear the corresponding genes for VTG synthesis and a series of complications such as reduced fertility and feminization arise due to presence of this phospholipoprotein (Tata and Smith, 1979; Maitre et al., 1984, 1986; Le Guellec et al., 1988; Mori et al., 1998; Okoumassoun et al., 2002). In extreme cases, this large-sized protein may lead to death from kidney failure (Pajor et al., 1990; Sultan et al., 1995; Schultz et al., 2003). Although endocrine disruption studies have been conducted on several indicator species, few of them involve species of intrinsic ecological importance such as the black-fin goodeid Girardinichthys viviparus (Bustamante). The latter is a live-bearing fish endemic to Mexico as well as an endangered species with a reduced range of distribution (NOM-059-ECOL, 1994). G. viviparus lives near the largest urban population center in Mexico and must endure the impact of domestic inputs and industrial wastewater in its natural environment, along with the general disappearance of the latter (Díaz-Pardo and OrtízJiménez, 1985). An electric power plant that makes routine use of PCBs is located near its habitat. It is thus essential to carry out several environmental risk-assessment studies to protect this species. In the case of the present study, our goals have been to assess the toxic effects of PCBs at sublethal concentrations, by waterborne exposure to PCBs, on vitellogenin induction in the black-fin goodeid G. viviparus as well as to find any sex-linked differences in these responses and characterize the protein in this particular species. 2. Methodology 2.1. Fish Since the present study involves a protected species, collection of the parent group of fish was conducted subsequent to evaluation and authorization by Mexican authorities (Oficio No./SGPA/DGVD/02750). Fish were collected from reservoirs near Lake Texcoco in the State of Mexico. Pollutant impact in these reservoirs is relatively small as they are filled by deep-well waters. Fish were kept in the laboratory at 25 °C under natural daylight with fresh food (Daphnia pulex and Artemia salina) or pellets available ad libitum, in 200-L glass aquariums at a density of 0.5 g fish/L water until reproduction occurred. All specimens in the study were 8-month-old adults born in the laboratory. Four months prior to beginning tests, fish were separated by sex, a task made easier by their sexual dimorphism, to ensure a state of rest in female gonad development through elimination of the visual stimuli associated with courtship. 2.2. Lethal toxicity tests Tests followed the criteria established by USEPA (OPPTS 850.1075): five concentrations were performed in triplicate in a 357 glass aquarium with seven fish in static medium at a density of 0.8 g fish/L. Dimethyl sulphoxide (DMSO, Sigma) was used as a vehicle for the PCBs. Pure DMSO dissolved in semi-hard synthetic water was used for the control group. The LC50 was estimated after 96 h by the Probit method. LC0 was calculated using a semi-logarithmic method since the latter is capable of determining extreme concentrations. 2.3. Sublethal toxicity tests Nominal PCBs concentrations was dosed at half the LC0 in DMSO as well as the solvent control were prepared in semihard synthetic water in semi-static medium that was completely renewed every 4 days. Three males and three females were selected at random after 1, 2, 4, 8 and 16 days of exposure. Fish were anesthetized with xylocaine (30 mg/L) for several seconds and killed via fast freezing to − 70 °C followed by cervical dislocation. Because of the small size of this species (males: 25.54 ± 2.8 mm LP and 0.39 ± 0.02 g; females: 39.55 ± 4.6 mm LP and 1.20 ± 0.018 g), the distal third of each specimen (without fins) was homogenized in phosphate buffered saline (PBS, pH 7.5) with protease inhibitor (Aprotinin, 3 mg/mL Sigma), centrifuged at 1500×g (5 min) and stored at − 70 °C until VTG analysis. The Inerteen® chromatographic profile was obtained using a CG Varian 3400 coupled to a Saturn II mass detector with programmed temperatures, in a 0.25-mmdiameter, 30-m-long Restec capillary column preheated to 40 °C, with the injector set at 200 °C and detector at 280 °C. 2.4. VTG quantification VTG purification: VTG was obtained from blood serum of male fish exposed to 17β-estradiol (E2) in aqueous medium (1.0 μg/L) that was completely renewed every 2 days (Sherry et al., 1999; Lattier et al., 2003; Van den Belt et al., 2003). After 2 weeks, fish were anesthetized and completely bled through the caudal vein. VTG was purified via triple differential precipitation (Sherry et al., 1999) and filtration in a diethyl-amino-ethyl– cellulose (DEAE–cellulose) column in a KCl gradient (0.01– 0.5 M) as suggested by Wiley et al. (1979). VTG fractions were obtained at KCl concentrations of 0.2–0.25 M and concentrated by dialysis in 12,000 Da bags. Protein concentrations were determined according to Bradford (1976) using bovine serum albumin (Merck) as a standard. VTG was also obtained in vitro from cultured hepatocytes. Anesthetized adult males were killed by cervical dislocation. Perfusion, separation, and cell culture were performed following the techniques proposed by several authors (Maitre et al., 1986; Kordes et al., 2002; Rouhani et al., 2002) with certain modifications. Livers were rinsed in sterile PBS containing a 2% antibiotic mix (10,000 U/mL penicillin, 10,000 U/mL streptomycin, 25 μg/mL amphotericin) and dissected. Liver fractions were shaken for 25 min in 0.05% type IV collagenase (Sigma) and filtered through sterile cloth. Then the collagenase was neutralized with the same amount of culture medium and hepatocytes were centrifuged and re-suspended in culture medium. A total of 3 × 105 hepatocytes were placed per well 358 A. Vega-López et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 356–364 in 24-well flat-bottom plates. Cell cultures were kept at 25 °C in phenol red-free DMEM (Dulbecco's modification of Eagle's medium) with 2% bovine fetal serum. After 16 h, the culture medium was changed and after 48 h, a medium with E2 dissolved in ethanol at a concentration of 1 × 10− 10 M was added. This culture medium was changed after 96, 144, 192, 240 and 288 h, and VTG obtained each time from the harvested medium. VTG was purified via triple differential precipitation and filtration in a DEAE–cellulose column as previously described. VTG obtained from the cultured hepatocytes and blood sera was denatured by heating with 2-mercaptoethanol (0.0143 M) and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions (Korsgaard and Ladegaard, 1998; Kordes et al., 2002; Van den Belt et al., 2003). Electrophoretic separation was performed using a spacing gel and a 20% running gel with molecular mass markers of 14,200 to 66,000 Da (Sigma). As it is difficult to obtain purified VTG from G. viviparus in sufficient amounts for immunization and since cross-reactivity has been previously observed with other species (Huggeu et al., 2003; Mylchreest et al., 2003; Nilsen et al., 2004), the rainbow trout Oncorhynchus mykiss was selected as a substitute. Rainbow trout weighing 300–400 g were used to obtain VTG for immunization and ELISA protocol. VTG was induced via two intraperitoneal injections of E2 in saline solution (5.0 mg/kg mass) administered on days 0 and 5. On day 10, fish were anesthetized and partially bled through the caudal vein. Blood samples were taken and VTG purified as previously described. Fish were revived by filling the mouth and gills with cold running water until they recovered from the anesthetic. 2.5. ELISA protocol for VTG 2.5.1. Polyclonal anti-VTG serum A young male New Zealand rabbit was immunized with rainbow trout vitellogenin (omVTG) according to the following procedure. First dose: 500 μg omVTG in 500 μL complete Freund's adjuvant, given subcutaneously at various sites. Second dose: an equal amount but in incomplete Freund's adjuvant, administered 15 days later. Third, fourth, fifth and sixth doses (days 30, 31, 32 and 39): 250 μg omVTG in saline solution via intramuscular injection. On day 46, an anesthetic (20 mg/kg ketamin + 5 mg/kg xilacin) was applied and blood drawn via intracardiac puncture. The polyclonal anti-omVTG serum was obtained by centrifugation and stored at − 70 °C. 2.5.2. Inhibition of hybrid ELISA A calibration curve was done as follows. 96-well flat-bottom plates were used. The wells were coated with 7.0 ng of omVTG (homologous) in 100 μL of carbonate buffer pH 9.0–9.5 (1.5 g Na2CO3 + 2.93 g NaHCO3/1000 L) and incubated overnight at 4 °C. Wells were washed two times with PBS–0.05% Tween 20 and blocked for 1 h at 37 °C with a 0.25% gelatin (Sigma) in PBS. Different amounts of the heterologous G. viviparus VTG (gvVTG) (1, 2, 4, 8 15, and 30 ng) were mixed with 100 μL of the polyclonal serum anti-omVTG diluted 1:100 in PBS– gelatin solution in a 1.5-mL microcentrifuge tube, agitated in a vortex, added to the wells and incubated for 1 h at 37 °C. The wells were washed three times (5 min) with PBS–0.05% Tween 20 and 100 μL of the conjugate (peroxidase-conjugated goat anti-rabbit gamma globulin, Sigma) at a dilution of 1:4000 was added. Incubation and washing process were done as above. 100 μL of the substrate (10 mg of o-phenylendiamine + 10 μL of H2O2 in 25 mL citrate solution pH 5.0) was added. The reaction was stopped with 30 μL of 7 M H2SO4 after 25 min. The color intensity was inversely proportional to the amount of gvVTG at A490. For the gvVTG analysis in the different samples, 5 μL of homogenized fish sample was mixed with 100 μL of the polyclonal serum anti-omVTG diluted 1:100. The procedure was done as described for the calibration curve. To validate the detection limit (MDL) of the inhibition of hybrid ELISA method the test was run with 2.0 ng of gvVTG, for 3 consecutive days, 24 wells each time. All calibration curves and samples were run six times during 3 consecutive days. Accuracy of the method, was determined by spiked samples (adding three known concentrations of gvVTG to the samples) and proceeding according to the described methodology. 2.6. Statistics MDL was estimated using the formula: MDL = 2 * αn−1 absorbance * CAC / Average absorbance; where 2 = 95.45% reliability, αn−1 = the standard deviation of the samples, and CAC = the mean sample concentration derived from the calibration curve. The method quantification limit (MQL) was obtained by means of the formula: MQL = MDL * tn−1; where t = the “t” score of tables with 95% confidence at n − 1. Method precision was estimated through the coefficient of variation: % CV = (αn−1replicates / mean) * 100. Accuracy was determined by the recovery average: %R = [(ECA) − (C) / (A)] * 100; where ECA = the experimental concentration of the spiked sample; C = sample concentration; and A = the concentration of the gvVTG used to spike samples (CENAM, 1993). Results were subjected to a two-way ANOVA and the means compared using a two-way Student's t-test. Minimum probability criterion for significant differences was set at p ≤ 0.05. 3. Results More than 46 different compounds were detected in the Inerteen® analysis (4 mono-, 5 di-, 7 tri- and 31 tetrachlorobiphenyls). Tri-and tetrachlorobiphenyls with different chlorination patterns were predominant (data not shown). Penta-, hexa- and heptachlorobiphenyls were also observed. An LC50 equivalent to 17.04 mg PCBs/L (y = − 4.3644 + 7.6082 * X; χ2 = 0.3563, p b 0.05) was observed. The LC0 in the black-fin goodeid was 1.84 mg PCBs/L. Output of gvVTG was less than 2.0 mg in both cell cultures and blood sera, but larger amounts were obtained in vivo. The gvVTG elusion pattern in the DEAE–cellulose column (Fig. 1) shows two well-defined peaks within the KCl gradient (0.2– A. Vega-López et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 356–364 359 600 500 gvVTG µg protein 400 300 200 100 0 0.2 -100 0.2 0.2 0.225 0.225 0.225 0.225 0.225 0.25 0.25 0.25 0.25 0.25 0.275 KCl concentration (M) Fig. 1. DEAE–cellulose column chromatographic patterns of VTG obtained from Girardinichthys viviparus exposed to 17β-estradiol. Protein concentration obtained by the UV method (280 nm) via linear regression with a bovine albumin curve. [Note: Negative values are due to the effect of statistical ones (slope and origin) in the calibration curve]. 0.25 M) reported as typical of VTG (Wiley et al., 1979; De Vlaming et al., 1980; Parks et al., 1999). SDS–PAGE electrophoresis showed seven polypeptide bands of 64,000, 62,000, 60,000, 59,000, 46,000, 43,000 and 14,000 Da adding up to 348,000 Da (Fig. 2). Inhibition of polyclonal anti-omVTG serum by heterologous gvVTG was observed from 27.07% for 1 ng of gvVTG to 71.52% for 30 ng of gvVTG in the calibration curve of the hybrid ELISA. The following values were determined: CV = 28.87% (25.60–32.15%), MDL = 0.049 ng gvVTG (p b 0.05) and MQL = 0.09 ng gvVTG. Accuracy was within the range %R = 86 to 107. This method is suitable based on statistical parameters due to the correlation coefficient obtained and the level of significance of the measurements in the calibration curve (r2 = 0.983, p b 0.001). In the control group, females had average base values of 0.009 pg gvVTG/mg protein/g tissue/g fish. The protein was not detected in control males. PCBs elicited estrogenic effects in males and females at significant levels from day 2 of exposure. Average gvVTG was up to 10 times greater (p b 0.05) in males Fig. 2. SDS–PAGE (20% running gel) of G. viviparus VTG under reducing conditions, stained with Coomassie Brilliant Blue R250. Lane 1–4: G. viviparus VTG at different concentrations (1.5, 1.0, 0.5 and 0.3 μg, respectively). Lane m: molecular mass markers (molecular masses on left side are those of the commercial markers). Arrowheads indicate the three main polypeptides of VTG (64,000, 43,000 and 14,000 Da). than females (0.96 vs. 0.09 pg VTG/mg protein/g tissue/g fish, respectively). Figs. 3 and 4 show the induction kinetics of this biomolecule. PCBs-treated females showed significant differences from controls from day 2 to day 16 of exposure relative to control. PCBs-treated males exhibited differences starting on day 1 relative to control. Estrogenic effects in males were clearly time-dependent after day 2, and showed significant differences between all days of exposure (Fig. 4). In contrast, induction was more or less constant in females after day 2 (Fig. 3). CV was 24.5% (21.2–27.8%, p b 0.05) in females and 19.4% (14.2–24.7%, p b 0.05) in males. Accuracy of spiked samples was within the range %R = 71 to 114. 4. Discussion The two peaks in the elusion profile derived from the DEAE–cellulose column indicate that the two VTG polypeptide chains (α and β) are present. Based on their position, we deduced that the α chain is lighter in this species, as has been reported by several authors for different species such as the fathead minnow Pimephales promelas, sheepshead minnow Cyprinodon variegatus, zebrafish Danio rerio, white perch Morone america, and rainbow trout O. mykiss (Parks et al., 1999; Bowman et al., 2000; Fenske et al., 2001; Hiramatsu et al., 2002; Van den Belt et al., 2003). Results found by the former authors about the VTG mass agree with our observations regarding the black-fin goodeid VTG (348 kDa). Though similarities in the VTG of G. viviparus and other species are undoubtedly interesting, it is more noteworthy that the presence of this biomolecule in the Goodeidae has been established for the first time in this study as well as confirmed by us after partial sequencing of the VTG mRNA and registration in GenBank Accession No. AY845859 (data reported elsewhere). Other authors have found higher VTG molecular mass (De Vlaming et al., 1980; Maitre et al., 1984; Korsgaard and Ladegaard, 1998; Fukada et al., 2003). In view of the 360 A. Vega-López et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 356–364 pg VTG/mg proteins/g tissue/g fish 0.16 0.14 Control D* A* PCBs 0.12 C* B* 0.1 0.08 0.06 0.04 0.02 0 1 2 4 8 16 Exposure time (days) Fig. 3. Vitellogenin induction kinetics in female G. viviparus exposed to PCBs [half the LC0 (0.92 mg/L)] during a 16-day period and DMSO control in semi-hard synthetic water. Significant differences were not found after day 2 of exposure: (A) day 2 vs. day 1, p b 0.05; (B) day 4 vs. day 2, ns; (C) day 8 vs. day 4, ns; (D) day 16 vs. day 8, ns. *Significant differences relative to control. ns = not significant. Results were subjected to a two-way ANOVA and the set of triplicates compared using a two-ways Student's t-test. Minimum probability criterion for significant differences was set at p ≤ 0.05. differences in the values reported by these two groups of authors, the fact that this protein shows significant variations in molecular weight must be further examined. For instance, it is estimated that VTG can weigh anywhere between 250 to 600 kDa (Norberg and Haux, 1985; Arukwe and Goksøyr, 2003). Such disparities are a clear indication of little evolutionary conservatism in this biomolecule, even among members of the same family, as observed by us in VTG of the black-fin goodeid and the butterfly split-fin goodeid Ameca splendens (379 kDa). MacLatchy et al. (2003) discuss VTG variations in different species as well as loss of the primary sequence of this protein as noted by us. In the present study, gvVTG was quantified by means of a hybrid ELISA in an inhibition format showing suitable detection and quantification. To be able to compare our data with previously reported results, we used similar units to estimate limits (MDL = 0.5 ng/mL and MQL = 0.9 ng/mL gvVTG). Different detection limits have been reported in 2 D*** Control pg VTG/mg proteins/g tissue/g fish 1.8 PCBs 1.6 C** 1.4 1.2 1 B** 0.8 ** A* 0.6 0.4 0.2 0 1 2 4 8 16 Exposure time (days) Fig. 4. Vitellogenin induction kinetics in male G. viviparus exposed to PCBs [half the LC0 (0.92 mg/L)] during a 16-day period and DMSO control in semi-hard synthetic water. [Note: VTG not detected in the control]. Significant differences were found between all days of exposure: (A) day 2 vs. day 1, p b 0.05; (B) day 4 vs. day 2, p b 0.01; (C) day 8 vs. day 4, p b 0.01; (D) day 16 vs. day 8, p b 0.05. Significant differences relative to control: * p b 0.05, **p b 0.01, ***p b 0.001. Results were subjected to a two-way ANOVA and the set of triplicates compared using a two-ways Student's t-test. Minimum probability criterion for significant differences was set at p ≤ 0.05. A. Vega-López et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 356–364 validation of various ELISAs using a sandwich format based on monoclonal and polyclonal antibodies. Nilsen et al. (2004) report MDLs of 0.1 ng/mL and 0.6 ng/mL in the fathead minnow, P. promelas, and the Japanese medaka, Oryzias latipes, respectively. Brion et al. (2002) recorded 0.4 ng/mL MDL in D. rerio, while Holbech et al. (2001) found 0.2 ng/mL in this same species. Using polyclonal antibodies, Fenske et al. (2001) reported 2–3 ng/mL, again in D. rerio. In contrast, it has been observed that VTG quantification with different ELISA formats is suitable as long as the MDL is within the range of concentration of the samples (Nilsen et al., 2004). In regard to the working range of the calibration curve (1–30 ng gvVTG), it is similar to the ranges reported by previously mentioned authors. The intra- and inter-assay coefficient of variation was slightly higher than those reported by Brion et al. (2002), Holbech et al. (2001) and Fukada et al. (2003). However, this deviation may be due to reduced sensitivity during the Ag–Ab hybrid reaction (Mylchreest et al., 2003). We consider inhibition of polyclonal antibodies with heterologous VTG in solution to be a more precise method since we know the amounts of protein used, and the uncertainty related to wellcoating is eliminated. A further aspect of method validation is accuracy. Our results show suitable recovery averages, since during this type of biological reactions an equal bimolecular reactivity cannot be expected due to several sources of variation associated with matrix interference. Certain quality control aspects observed during our study lead us to conclude that a hybrid ELISA in an inhibition format is sufficiently sensitive, accurate and precise for VTG analysis in small-sized species, especially when not enough purified VTG is available. It is nonetheless necessary to test for cross-reactivity between the homologous and heterologous systems. A weak endocrine potential from commercial PCB mixtures has been considered (Gierthy et al., 1997; Arcaro et al., 1998; Bonefeld-Jørgensen et al., 2001; Carlson and Williams, 2001). Our results differ from those of previous reports in that the estrogenic effects elicited by the commercial mixture used in this study were significantly different from the control. For example, the amount of PCB-induced VTG on day 16 was up to 15 times more in females, than in the females control group. In the PCBs-treated males, this protein was detected in the range of 2.0 pg VTG/mg protein/g tissue/g fish whereas in the male control fish, VTG was not detected. These results are comparable to the amount elicited by the most potent VTG inducers in fish: 17α-ethynylestradiol (EE2) and E2 (Holbech et al., 2001; Brion et al., 2002; Panter et al., 2002; Andersen et al., 2003; Mylchreest et al., 2003; Versonnen and Janssen, 2004). These findings establish the endocrine disruption potential of Inerteen® in this goodeid fish under experimental conditions. Unfortunately, detailed action mechanisms involved in egg yolk protein induction have not been fully studied (Nicolas, 1999; Arukwe and Goksøyr, 2003). However, it is necessary to consider that VTG is not the only oogenic protein; for instance, Fujita et al. (2005) comment that the choriogenin levels (H and L types) in serum of masu salmon Oncorhynchus masou were higher than VTG in pre-vitellogenic growth phase; but on the vitellogenic phase, the VTG were the most abundant oogenic 361 protein coincident with the higher E2 levels. Also, the former authors comment that the initiation of choriogenin and VTG synthesis and their inducibility appear to be species-dependent. Sex differences in VTG production are associated with steroid metabolism and cytochrome P450 activity. Some PCBs and dioxins have both estrogenic and anti-estrogenic potential (Spink et al., 1990) mediated by an aryl hydrocarbon receptor (AhR). Such interactions include a down-regulation of estrogen receptors (ER), ER ligand–E2 interference with response genes in DNA, and/or cytochrome P450 induction, specifically of the enzymes CYP1A1 and CYP1A2 involved in E2 metabolism (Bonefeld-Jørgensen et al., 2001). For instance, in females, increased estrogen levels have been found to lead to a decrease in mixed oxidase function (MOF) enzymes, as in the case of the brook trout Salvelinus fontinalis (Stegeman et al., 1982), the winter flounder Pleuronectes americanus and the scup Stenotomus chrysops (Gray et al., 1991). However, sex differences may also be explained by genetic regulation, since MOF activity is higher in males and testosterone 6β-hydroxylase activity is higher in females, as observed in the winter flounder (Gray et al., 1991). These observations show that the comparatively higher activity of CYP1A in male G. viviparus (unpublished data) leads to formation of PCBs metabolites with greater efficiency and possibly estrogenic potential (Figs. 3 and 4). In addition, suppression of P450 catalytic activity elicited by E2 is suggested to take place at pre-transcriptional levels (mRNA for P4501A) while endogenous regulation may overcome the exogenous regulation exerted by high concentrations of inductors of this enzyme superfamily (Elskus et al., 1992). Another aspect contributing to increased blood levels of VTG induced by PCB mixtures may be the lack of target organs in males. Relative to VTG induction kinetics by PCBs exposure, Petit et al. (1997) found in a primary culture of rainbow trout hepatocytes, an induction of gene expression of VTG after 2 days exposure to Aroclor 1221 and Aroclor 1248. Flouriot et al. (1995) inform an induction of gene expression of VTG in rainbow trout hepatocytes after over 30 days of xenobiotics exposure. In juvenile zebrafish, D. rerio, a VTG half-life of 2.4 days has been observed when induced by a single exposure to EE2 (Andersen et al., 2003). These reports are in agreement with our observations, since VTG blood levels in female G. viviparus remained relatively constant throughout the exposure to xenobiotics. This supports the assumption of a fast uptake of induced amounts of this protein by oocytes as observed by Fujita et al. (2005). In contrast, VTG induction in male P. promelas has been reported to have two different-length stages (Schmid et al., 2002), the first stage lasting approximately 2 days, and the second one, 21 days. These findings support our observations, since this protein increased with time in male G. viviparus reaching maximum levels on day 16 of observation. VTG induction kinetics elicited by PCBs in male G. viviparus also appeared to be two-staged, although the first stage took place during the first 24 h while the second one continued for at least 16 days. One of the most unsettling characteristics of PCBs endocrine disruption is related to the structure and biotransformation of these xenobiotics and to ER–AhR interactions. Groten et al. 362 A. Vega-López et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 356–364 (1999) find that phase I (oxidation by MOF) takes place in liver microsomes by way of an arene to form an intermediate metabolite (arene oxide or epoxide), and the consequence of this biotransformation is a biphenylol. This oxidation is enhanced in meta and para positions (Bonefeld-Jørgensen et al., 2001). Gierthy et al. (1997) found the estrogenic effects elicited by PCB metabolites in human adenocarcinoma MCF-7 cells are enhanced by para-OH and ortho substitution, and these events are related to the affinity of these biphenylols for ER. The authors explained this process as an effect of the structural similarity between biphenylols and E2, or the conformational restriction derived from ortho substitution (Bonefeld-Jørgensen et al., 2001). In MCF-7 cells, ortho-OH and meta-OH metabolites do not compete with ER, while para-OH metabolites do so depending on their concentration (Vakharia and Gierthy, 2000). The Inerteen® chromatographic profile shows all these related compounds are present in the commercial mixture used in our study, di-ortho and ortho substituted compounds being predominant. These compounds are capable of interacting with ER. Metabolite formation due to MOF activity may also be conjectured. Metabolites have been shown to form in the presence of liver microsomes and NADPH, although chromatographic confirmation is still lacking (Vakharia and Gierthy, 2000). Such events lead us to assume that the black-fin goodeid has the ability to biotransform these xenobiotics into metabolites of enhanced estrogenic capability. Bonefeld-Jørgensen et al. (2001) mention that evaluation of commercial PCB mixtures is important since it allows prediction of toxic, particularly estrogenic, effects. These authors mention that di-ortho substitutions also have an affinity for ER. However, when mixing different di-ortho biphenyls (PCB # 138, 2,2′,3,4,4′,5-hexachlorobiphenyl; PCB # 153, 2,2′,4,4′,5,5′-hexachlorobiphenyl; and PCB # 180, 2,2′,3,4,4′,5,5′-heptachlorobiphenyl) at 3μM concentrations, anti-estrogenic effects at ER level were observed in MCF-7 cells. In addition to the concentration used, these effects may be further explained by the affinity of PCB # 138 to link to androgen receptors. Gierthy et al. (1997) report anti-estrogenic effects elicited by 3,4′,5-trichlorobiphenyl, while none were observed for its metabolite (3,4′,5-trichloro-4-biphenylol). It is thus evident that the in vivo interactions of commercial PCB mixtures are quite complex in G. viviparus; nonetheless, we were able to establish presence of estrogenic effects in this species. Although the toxic effects of the commercial PCB mixture were estrogenic in their manifestation in both G. viviparus sexes, the possibility of anti-estrogenic and anti-androgenic interactions has not been ruled out, since all these interactions are considered endocrine disruptions. Nevertheless, the ecological significance of these disorders must be evaluated since endocrine disruption is closely tied to physiological and hormonal cycles and shows strong sex-linked seasonal variation. Detoxification schemes to get rid of these xenobiotics must also be considered, as such schemes may be speciesdependent. Toxic response modifications are vitally important to assess risk of exposure to endocrine disruptors in the natural environment. 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