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Protective effects of genistein on cytochrome P-450 and vitellogenin expression in liver of zebrafish after PCB-126 exposure
Science of the Total Environment 674 (2019) 71–76
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Protective effects of genistein on cytochrome P-450 and vitellogenin expression in liver of zebrafish after PCB-126 exposure Patrizia Licata a,⁎, Giuseppe Piccione a, Francesco Fazio a, Eugenia Rita Lauriano b, Margherita Calò a a b
Department of Veterinary Science, University of Messina, Polo SS Annunziata, 98168 Messina, Italy Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Via F. Stagno d'Alcontres 31, 98166 Messina, Italy
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Action of Vtg and P-4501A1 following coexposure to genistein and PCB-126 in zebrafish • A marked immunoreactivity of Vtg at 12 h in liver than the control with only PCB-126 • Reduction in the estrogenic effect after treatment with genistein and PCB-126 at 12 h • Low levels of genistein decrease the marked P450 expression induced by PCB-126.
a r t i c l e
i n f o
Article history: Received 1 February 2019 Received in revised form 25 March 2019 Accepted 30 March 2019 Available online 31 March 2019 Editor: Henner Hollert Keywords: PCB-126 Immunoreactivity Danio rerio CYP1A1 Vitellogenin
a b s t r a c t The objective of the research is to study the action of Vitellogenin and P-4501A1 following coexposure at different times to genistein and PCB-126 using zebrafish as a model system. Polychlorinated biphenyls are ubiquitous substances in environment. The genistein is a phytoestrogen extracted from soybeans and it's contained in food for humans and animals. For this study, 200 adult zebrafish were used. Our findings show a marked immunoreactivity of Vtg at 12 h in liver than the control with only PCB-126. Regarding effects of PCB-126 on Vtg after pretreatment with genistein in fishes, the immunohistochemistry results show a minor increase at 12 h. After 24 h the immunoreactivity is lower than 12 h and then slightly increased at 72 h with only PCB-126 and PCB-126 and genistein together. CYP1A1 progressively increases from 12 h to 72 h in all groups with minor immunoreactivity when we treated fish with genistein and PCB-126. We show a reduction in the estrogenic effect when the fishes were treated with genistein and PCB-126 together at 12 h than the group treated with only PCB-126. Moreover, low concentrations of genistein decrease the marked P450 expression induced by PCB-126. This shows that genistein decreases the expression of P450 target genes mediated by AhR. © 2019 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author. E-mail address: [email protected] (P. Licata).
https://doi.org/10.1016/j.scitotenv.2019.03.467 0048-9697/© 2019 Elsevier B.V. All rights reserved.
A very important aspect for food health is the content of chemical contaminants in food. Chemical contamination is still today an extremely complex problem, which is due to the economic, industrial, urban, and not always correct use of xenobiotic substances, that a food
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can be polluted at different distinct periods of growth or development in the life of an animal (Thompson and Darwish, 2019). Among the various organic contaminants to which the fish is exposed there are the polychlorinated biphenyls (PCBs) as a resulting from industrial sources (AMAP, 2009; WHO, 2007). PCBs are ubiquitous pollutants in environment as a result of emission and atmospheric deposition. These toxic substances have a high chemical stability and persistence and their content in the environment is a clear indication of anthropogenic pollution (Licata et al., 2004). PCBs are transported mainly through the atmosphere and that accumulated through the food pyramid. Negative effects of PCBs in fish were egg shell alteration which results in embryo mortality, edema and teratogenic dysfunction (Maisano et al., 2016; Nugegoda and Kibria, 2017; Zhao et al., 2017). It has been seen by numerous studies carried out in vivo and in vitro that the exposure to PCBs is to serious toxic effects, like weight loss, edema, hepatotoxicity, immunotoxicity, decreased reproductive success, teratogenicity, promotion of cancer, and enzyme induction (Ahlborg et al., 1994; Calò et al., 2017; Cammilleri et al., 2017; Di Bella et al., 2010; Körber et al., 2017). Polychlorinated biphenyls are recognized physiological stressors to fish which over time may impair individual performance and perhaps fitness by inducing changes that could have population-level consequences. PCB-126 (3,3′,4,4,5pentachlorobiphenyl) accumulates in lipids and can subsequently be released into the bloodstream during periods of high activity that involve the mobilization of stored fuels to meet with increasing energy demands (Bellehumeur et al., 2016). Previous studies indicate that PCB-126 effects are mediated through the aryl hydrocarbon receptor (AhR), a ligand-activated member of the bHLH-PAS family of transcription factors (Chopra and Schrenk, 2011; Calò et al., 2014). PCB-126 is activated by cytochrome P450 (CYP) 1A1/CYP1B1 enzymes to reactive intermediates that bind to DNA, a crucial step in the initiation of carcinogenesis. Aryl-hydrocarbon receptor (AhR) plays a critical activity in the induction of CYP1 enzymes by PCB-126 of which is AhR ligands. Numerous studies showed that PCB126 may interact with estrogen (ERs) and exhibited either estrogenic or antiestrogenic responses (Ambolet-Camoit et al., 2010; Di Paolo et al., 2015; Kais et al., 2017). Particularly, cytochrome P450s, which catalyze various Phase 1 metabolism reactions for lipophilic compounds in the body. Molecular mechanisms and their consequences of AhRmediated regulation of mammalian cytochrome P450 enzymes by chemical substances like polycyclic aromatic hydrocarbons and dioxins, have been investigated extensively (Birnbaum, 1994; Calò et al., 2009, 2012). Binding of such ligand to AhR leads to transcriptional activation of CYP1A1, CYP1A2 and CYP1B1. These enzymes catalyze oxdative detoxication or activation of most ligands. AhR binds a host of chemicals, such as bilirubin, biliverdin, 7-ketocholesterol, and structurally diverse phytochemicals. The phytochemicals (flavonoids, resveratrol, carotenoids) known weak AhR agonists and are chemoprotective. In this work, we investigate a flavonoid (genistein) that is the most used in the diet of humans and animals as it is a soy-derived. Genistein was found to alter reproductive processes, along with steroid hormone secretion in pigs, rabbits and humans (Tiemann et al., 2007). Several studies have established that genistein binds to both of the ERα and has more mighty transcriptional activity in cells transfected with ERβ and moreover, it has been demonstrated to inhibit cell growth in breast and prostate cancers by regulating genes responsible for the cell proliferation, apoptosis and transcriptional regulations (Froyen and Steinberg, 2016). There are phytoestrogens alter CYPs through binding to AhR, acting as either AhR agonists or antagonists. Some authors disagree on the effects of long-term genistein. Vitellogenin (Vtg) is a phospho-lipo-glycoprotein produced by oviparous animals in response to estrogen receptor (ER) binding. The presence of Vtg in juvenile and male fish liver and plasma has been used as biomarker to evaluate levels of environmental contaminants as dioxin and PCBs (Sumpter and Jobling, 1995).
According to some authors it causes the tumor while others say it has anticancer effects. Many works on the exposure of AhR ligands, such as dioxins and PCB, and genistein have been examined on various cells but have not investigated yet using an in vivo animal model (Piasecka-Srader et al., 2016; Zhang et al., 2003). Aim of this work is to study the possible protective effect of genistein on cytochrome P-450 and vitellogenin expression in liver of zebrafish after PCB-126 exposure. 2. Materials and methods 2.1. Experimental protocol For this study, 200 adult zebrafish (Danio rerio) are used (body weight about 3 g) raised and kept at standard laboratory conditions of 28 °C, 14:10 dark/light photoperiod as to standard zebrafish breeding protocols. Zebrafish are obtained from the breeding colony at the C.I.S.S. (Centre of Experimental Ichthyopathology of Sicily, Department of Veterinary Science, University of Messina). In accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals are all procedures used. Procedures are in accordance with the Guidelines of Italian Health Minister and approved by the local Ethic Committee. The animals do not exhibit anomalies and infestations after external examination. The fish are subjected to an acclimatization period for 10 days in 25 L tanks. The static water system is used to conduct the fish experiment. 30-l tanks are utilized considering an animal per liter of water. The zebrafish are fed with freshly hatched Artemia. 48 h before the experiments, feeding is stopped (Jönsson et al., 2002, 2003). The liver is stored at −20 °C for 2 days before analysis. Four groups of 50 zebrafish are made and each and allocated in separate tanks; the first group (A) used as control, receive the vehicle, dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) used to dissolve PCB-126, 1.10 g/ml; the second group (B) receive PCB-126 (10−8 M) dissolved in DMSO; the third group (C) receive genistein and PCB-126 (RPC-102) (Ultra Scientific-North Kingstown, RI) (192 μg/L and 1.10 g/ml dissolved in DMSO); the fourth group (D) receive genistein at 192 μg/L. At the start of the experiment, groups C and D are pre-treated for 6 consecutive days with genistein and at the end of the sixth day, B and C groups receive the PCB-126. PH and oxygen dissolved in water are continuously checked. The physicochemical parameters of the water are: 20 °C, salinity 38%, dissolved oxygen 7 (mg/l) and pH 7.8. Animals from each group are anaesthetised (tricaine methan sulfonate; MS-222 Sigma, 200 mg/l of water) and sacrifice after 12, 24, and 72 h. Furthermore the livers are taken and stored for analysis. 2.2. Immunochemistry The histological examination is carried out according to the method reported by Calò et al. (2012). To perform the immunochemistry analysis we use the indirect method of peroxidase based on incubation of the liver sections with a primary antibody specific for Vtg (1:200; Biosense Bergen, Norway) and for CYP1A1 (1:250; Biosense Bergen, Norway). The immunohistochemical investigations are carried as described by Woods et al. (2009) and Calò et al. (2009). 2.3. Determination of Vtg and CYP1A1 by Western-blot analysis in liver Liver samples are treated as reported by method of Calò et al. (2012). Tween for 1 h at room temperature, washed three times for 10 min each in TBS-0.15% Tween, and incubated both with a primary Vtg antibody (Biosense Bergen, Norway) and with a primary CYP1A1 antibody (Biosense Bergen, Norway) in TBS–0.1% Tween at 4 °C overnight. After being washed three times for 10 min each in TBS–0.15% Tween, the
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membranes were incubated with a secondary peroxidase-conjugated antibody (Pierce, UK) for 1 h at room temperature. After washing, the membranes are analyzed by the enhanced chamiluminescence's system according to the manufacture's protocol (Amersham, UK). The protein signal is quantified by scanning densitometry using a bio-image analysis system (C-DiGit Blot Scanner, with Image Studio software; Lincoln, NE, United States) and the results are expressed as relative integrated intensity compared to controls.
Fig. 1C shows the effects of PCB-126 on immunnohistochemical detection of Vtg after pretreatment with genistein in fishes. The immunohistochemistry results demonstrate an increase of immunoreactivity at 12 h in liver of treated fishes than the control. After 24 h this immunoreactivity is lower than 12 h and then slightly increased at 72 h. Regarding effects of only genistein on immunoistochemical detection of Vtg, its immunoreactivity progressively increases from 12 h to 72 h (Fig. 1D).
2.4. Statistical analysis
3.1.2. CYP1A1 immunoreactivity CYP1A1 progressively increases from 12 h to 72 h in all groups (Fig. 2A, B, C, D). This increase in immunoreactivity emerges more in the hepatic parenchyma of fish treated with PCB-126 instead it has a smaller increase when the fish is treated with genistein and PCB-126 together.
All data are expressed as mean values standard deviation (SD). Data are analyzed by analysis of variance (ANOVA) for multiple comparison of results. To compare group means, the Bonferroni's test is conducted. A P ≤ 0.05 is considered statistically significant. The data are analyzed using STATISTICA 7.5 software package (Stat Soft Inc.). 3. Results 3.1. Immunohistochemical investigation 3.1.1. Vitellogenin immunoreactivity Fig. 1A shows the control sample of Vtg at 12, 24 and 72 h. In Fig. 1B, where only PCB-126 is administered, a marked immunoreactivity of Vtg at 12 h than the control is observed. This immunoreactivity decrease only at 24 h and it increases slightly at 72 h than the control as reported in our previous work by Calò et al. (2012).
3.1.3. Western-blot analysis Immunohistochemical results confirm Western-blot analysis. Fig. 3 shows effect of time on immunoreactivity detection of Vtg and CYP1A1 after treatment with PCB-126 and/or genistein in zebrafish. In particularly, the control group of Vtg shows a statistical significance at 12, 24 and 72 h. In PCB-126 group is observed a marked immunoreactivity of Vtg at 12 h compared to control group. This immunoreactivity decrease only at 24 h and it increases slightly at 72 h respect to control as reported in previous work by Calò et al., 2012. The immunoreactivity of Vtg in Genistein group progressively increases from 12 h to 72 h. The fishes treated with PCB-126 and genistein (Group C) show a lower
VITELLOGENIN A
CONTROL
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C PCB126+GEN ..
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GENISTEIN
12h CONTR. 12h Vtg
PCB 12h Vtg
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PCB+ G. 24h Vtg
G.24h Vtg
72h
CONTR. 72h Vtg
PCB 72h Vtg
PCB + G.72h Vtg
G. 72h Vtg
Fig. 1. Effects of PCB-126, genistein+PCB-126 and genistein on immunohistochemical detection of vitellogenin in fish. Representative images in control fishes (Group A); representative images of vitellogenin in fishes exposed to PCB-126 (Group B), to PCB-126 + genistein (Group D) and genistein (Group C) (original magnification, ×400) Scale Bars 50 μm.
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CYP1A1 A
12h
CONTROL
CONTR. CYP1A
B
PCB126
PCB12h-CYP1A
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G24h-CYP1A
24h CONTR. CYP1A
PCB24h-CYP1A
72h CONTR. CYP1A
PCB72h-CYP1A
PCB+G72h-CYP1A
G72h-CYP1A
Fig. 2. Effects of PCB-126, genistein+PCB-126 and genistein on immunohistochemical detection of P-450 CYP1A1 in fish. Representative images in control fishes (Group A); representative images of vitellogenin in fishes exposed to PCB-126 (Group B), to PCB-126 + genistein (Group D) and genistein (Group C) (original magnification, ×400) Scale Bars 50 μm.
immunoreactivity at 12 h compared to PCB-126 (Group B). After 24 h this immunoreactivity is lower than 12 h with a slightly increase at 72 h. CYP1A1 progressively increases from 12 h to 72 h in all groups. The increase in immunoreactivity emerges more in the hepatic parenchyma of fish treated with PCB-126 instead it has a smaller increase when the fish is treated with PCB-126 and genistein. 4. Discussion PCBs as ubiquitous and persistent in the environment, they can give many toxic effects on the reproduction, nervous, endocrine system development and immunological function of animals. The toxicity of PCBs are largely based on the similarity in chemical structure and toxic mode of action with dioxin. Many steps of their effects are known to be ER-mediated by acting as estrogen mimics or as estrogen antagonists (Calò et al., 2010). Phytoestrogens such as genistein and resveratrol, contrary to chemically synthesized substances such as EDC, showed chemopreventive effects on specific cancers by contending with ER binding and regulating normal ER action in target structure of zebrafish. In this study adult zebrafish (Danio rerio) are used to show a significant increase of vitellogenin at 12 h in all groups treated than the control group (Lecomte et al., 2017). We show a reduction in the estrogenic effect when the fishes are treated with PCB-126 and genistein together at 12 h than the group treated with only PCB-126. Infact the most important result is the reduction of the estrogenic effect of PCB-126 when genistein is added antagonizing the effects. Moreover, we also demonstrate that low
concentrations of genistein decrease the marked P450 expression induced by PCB-126 as demonstrated in a work by Calò et al. (2009). This shows that genistein decreases the expression of P450 target genes mediated by AhR at 24 h. Also Piasecka-Srader et al. (2016) established the TCDD and genistein administered together decreased ERβ (48 h) and AhR (24 h) protein expression in the cells in contrast to the individual treatments. Messina and Wood (2008) showed that soyfoods are essentially a unique dietary source of isoflavones, compounds which bind to estrogen receptors and exhibit weak estrogen-like effects under certain experimental conditions. This work also demonstrates that higher levels of the isoflavone genistein inhibits the development of tumor cells and then breast cancer. Genistein has a biphasic effect in MCF-7 cells in vitro, cells proliferate when a low concentration of genistein is given, on the contrary an inhibition occurs when a high concentration is administered. In ovariectomized athymic mice implanted with MCF-7 cells, both genistein and soy protein stimulate tumor growth in a dose-dependent manner. In intact mice fed with estrogens and genistein, the latter inhibits tumor growth compared to mice fed only with estrogens. (Messina and Loprinzi, 2001). However, other research is necessary in the long term at lower doses and mixtures of phytoestrogens are required to demonstrate that dietary supplements do have beneficial rather than adverse effects on cancer. Moreover, the many targets in cancer cells and their ability to modulate epigenetic events associated with cancer and prevention may lead to new, non-toxic therapeutic approaches with the study of highly specific and long-acting analogues of phytoestrogens (Bilal et al., 2014).
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References
Fig. 3. Effect of time on immunoreactivity detection of Vtg and CYP1A1 after treatment with PCB-126 and/or genistein.
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Protective effects of genistein on cytochrome P-450 and vitellogenin expression in liver of zebrafish after PCB-126 exposure Patrizia Licata a,⁎, Giuseppe Piccione a, Francesco Fazio a, Eugenia Rita Lauriano b, Margherita Calò a a b
Department of Veterinary Science, University of Messina, Polo SS Annunziata, 98168 Messina, Italy Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Via F. Stagno d'Alcontres 31, 98166 Messina, Italy
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Action of Vtg and P-4501A1 following coexposure to genistein and PCB-126 in zebrafish • A marked immunoreactivity of Vtg at 12 h in liver than the control with only PCB-126 • Reduction in the estrogenic effect after treatment with genistein and PCB-126 at 12 h • Low levels of genistein decrease the marked P450 expression induced by PCB-126.
a r t i c l e
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Article history: Received 1 February 2019 Received in revised form 25 March 2019 Accepted 30 March 2019 Available online 31 March 2019 Editor: Henner Hollert Keywords: PCB-126 Immunoreactivity Danio rerio CYP1A1 Vitellogenin
a b s t r a c t The objective of the research is to study the action of Vitellogenin and P-4501A1 following coexposure at different times to genistein and PCB-126 using zebrafish as a model system. Polychlorinated biphenyls are ubiquitous substances in environment. The genistein is a phytoestrogen extracted from soybeans and it's contained in food for humans and animals. For this study, 200 adult zebrafish were used. Our findings show a marked immunoreactivity of Vtg at 12 h in liver than the control with only PCB-126. Regarding effects of PCB-126 on Vtg after pretreatment with genistein in fishes, the immunohistochemistry results show a minor increase at 12 h. After 24 h the immunoreactivity is lower than 12 h and then slightly increased at 72 h with only PCB-126 and PCB-126 and genistein together. CYP1A1 progressively increases from 12 h to 72 h in all groups with minor immunoreactivity when we treated fish with genistein and PCB-126. We show a reduction in the estrogenic effect when the fishes were treated with genistein and PCB-126 together at 12 h than the group treated with only PCB-126. Moreover, low concentrations of genistein decrease the marked P450 expression induced by PCB-126. This shows that genistein decreases the expression of P450 target genes mediated by AhR. © 2019 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author. E-mail address: [email protected] (P. Licata).
https://doi.org/10.1016/j.scitotenv.2019.03.467 0048-9697/© 2019 Elsevier B.V. All rights reserved.
A very important aspect for food health is the content of chemical contaminants in food. Chemical contamination is still today an extremely complex problem, which is due to the economic, industrial, urban, and not always correct use of xenobiotic substances, that a food
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can be polluted at different distinct periods of growth or development in the life of an animal (Thompson and Darwish, 2019). Among the various organic contaminants to which the fish is exposed there are the polychlorinated biphenyls (PCBs) as a resulting from industrial sources (AMAP, 2009; WHO, 2007). PCBs are ubiquitous pollutants in environment as a result of emission and atmospheric deposition. These toxic substances have a high chemical stability and persistence and their content in the environment is a clear indication of anthropogenic pollution (Licata et al., 2004). PCBs are transported mainly through the atmosphere and that accumulated through the food pyramid. Negative effects of PCBs in fish were egg shell alteration which results in embryo mortality, edema and teratogenic dysfunction (Maisano et al., 2016; Nugegoda and Kibria, 2017; Zhao et al., 2017). It has been seen by numerous studies carried out in vivo and in vitro that the exposure to PCBs is to serious toxic effects, like weight loss, edema, hepatotoxicity, immunotoxicity, decreased reproductive success, teratogenicity, promotion of cancer, and enzyme induction (Ahlborg et al., 1994; Calò et al., 2017; Cammilleri et al., 2017; Di Bella et al., 2010; Körber et al., 2017). Polychlorinated biphenyls are recognized physiological stressors to fish which over time may impair individual performance and perhaps fitness by inducing changes that could have population-level consequences. PCB-126 (3,3′,4,4,5pentachlorobiphenyl) accumulates in lipids and can subsequently be released into the bloodstream during periods of high activity that involve the mobilization of stored fuels to meet with increasing energy demands (Bellehumeur et al., 2016). Previous studies indicate that PCB-126 effects are mediated through the aryl hydrocarbon receptor (AhR), a ligand-activated member of the bHLH-PAS family of transcription factors (Chopra and Schrenk, 2011; Calò et al., 2014). PCB-126 is activated by cytochrome P450 (CYP) 1A1/CYP1B1 enzymes to reactive intermediates that bind to DNA, a crucial step in the initiation of carcinogenesis. Aryl-hydrocarbon receptor (AhR) plays a critical activity in the induction of CYP1 enzymes by PCB-126 of which is AhR ligands. Numerous studies showed that PCB126 may interact with estrogen (ERs) and exhibited either estrogenic or antiestrogenic responses (Ambolet-Camoit et al., 2010; Di Paolo et al., 2015; Kais et al., 2017). Particularly, cytochrome P450s, which catalyze various Phase 1 metabolism reactions for lipophilic compounds in the body. Molecular mechanisms and their consequences of AhRmediated regulation of mammalian cytochrome P450 enzymes by chemical substances like polycyclic aromatic hydrocarbons and dioxins, have been investigated extensively (Birnbaum, 1994; Calò et al., 2009, 2012). Binding of such ligand to AhR leads to transcriptional activation of CYP1A1, CYP1A2 and CYP1B1. These enzymes catalyze oxdative detoxication or activation of most ligands. AhR binds a host of chemicals, such as bilirubin, biliverdin, 7-ketocholesterol, and structurally diverse phytochemicals. The phytochemicals (flavonoids, resveratrol, carotenoids) known weak AhR agonists and are chemoprotective. In this work, we investigate a flavonoid (genistein) that is the most used in the diet of humans and animals as it is a soy-derived. Genistein was found to alter reproductive processes, along with steroid hormone secretion in pigs, rabbits and humans (Tiemann et al., 2007). Several studies have established that genistein binds to both of the ERα and has more mighty transcriptional activity in cells transfected with ERβ and moreover, it has been demonstrated to inhibit cell growth in breast and prostate cancers by regulating genes responsible for the cell proliferation, apoptosis and transcriptional regulations (Froyen and Steinberg, 2016). There are phytoestrogens alter CYPs through binding to AhR, acting as either AhR agonists or antagonists. Some authors disagree on the effects of long-term genistein. Vitellogenin (Vtg) is a phospho-lipo-glycoprotein produced by oviparous animals in response to estrogen receptor (ER) binding. The presence of Vtg in juvenile and male fish liver and plasma has been used as biomarker to evaluate levels of environmental contaminants as dioxin and PCBs (Sumpter and Jobling, 1995).
According to some authors it causes the tumor while others say it has anticancer effects. Many works on the exposure of AhR ligands, such as dioxins and PCB, and genistein have been examined on various cells but have not investigated yet using an in vivo animal model (Piasecka-Srader et al., 2016; Zhang et al., 2003). Aim of this work is to study the possible protective effect of genistein on cytochrome P-450 and vitellogenin expression in liver of zebrafish after PCB-126 exposure. 2. Materials and methods 2.1. Experimental protocol For this study, 200 adult zebrafish (Danio rerio) are used (body weight about 3 g) raised and kept at standard laboratory conditions of 28 °C, 14:10 dark/light photoperiod as to standard zebrafish breeding protocols. Zebrafish are obtained from the breeding colony at the C.I.S.S. (Centre of Experimental Ichthyopathology of Sicily, Department of Veterinary Science, University of Messina). In accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals are all procedures used. Procedures are in accordance with the Guidelines of Italian Health Minister and approved by the local Ethic Committee. The animals do not exhibit anomalies and infestations after external examination. The fish are subjected to an acclimatization period for 10 days in 25 L tanks. The static water system is used to conduct the fish experiment. 30-l tanks are utilized considering an animal per liter of water. The zebrafish are fed with freshly hatched Artemia. 48 h before the experiments, feeding is stopped (Jönsson et al., 2002, 2003). The liver is stored at −20 °C for 2 days before analysis. Four groups of 50 zebrafish are made and each and allocated in separate tanks; the first group (A) used as control, receive the vehicle, dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) used to dissolve PCB-126, 1.10 g/ml; the second group (B) receive PCB-126 (10−8 M) dissolved in DMSO; the third group (C) receive genistein and PCB-126 (RPC-102) (Ultra Scientific-North Kingstown, RI) (192 μg/L and 1.10 g/ml dissolved in DMSO); the fourth group (D) receive genistein at 192 μg/L. At the start of the experiment, groups C and D are pre-treated for 6 consecutive days with genistein and at the end of the sixth day, B and C groups receive the PCB-126. PH and oxygen dissolved in water are continuously checked. The physicochemical parameters of the water are: 20 °C, salinity 38%, dissolved oxygen 7 (mg/l) and pH 7.8. Animals from each group are anaesthetised (tricaine methan sulfonate; MS-222 Sigma, 200 mg/l of water) and sacrifice after 12, 24, and 72 h. Furthermore the livers are taken and stored for analysis. 2.2. Immunochemistry The histological examination is carried out according to the method reported by Calò et al. (2012). To perform the immunochemistry analysis we use the indirect method of peroxidase based on incubation of the liver sections with a primary antibody specific for Vtg (1:200; Biosense Bergen, Norway) and for CYP1A1 (1:250; Biosense Bergen, Norway). The immunohistochemical investigations are carried as described by Woods et al. (2009) and Calò et al. (2009). 2.3. Determination of Vtg and CYP1A1 by Western-blot analysis in liver Liver samples are treated as reported by method of Calò et al. (2012). Tween for 1 h at room temperature, washed three times for 10 min each in TBS-0.15% Tween, and incubated both with a primary Vtg antibody (Biosense Bergen, Norway) and with a primary CYP1A1 antibody (Biosense Bergen, Norway) in TBS–0.1% Tween at 4 °C overnight. After being washed three times for 10 min each in TBS–0.15% Tween, the
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membranes were incubated with a secondary peroxidase-conjugated antibody (Pierce, UK) for 1 h at room temperature. After washing, the membranes are analyzed by the enhanced chamiluminescence's system according to the manufacture's protocol (Amersham, UK). The protein signal is quantified by scanning densitometry using a bio-image analysis system (C-DiGit Blot Scanner, with Image Studio software; Lincoln, NE, United States) and the results are expressed as relative integrated intensity compared to controls.
Fig. 1C shows the effects of PCB-126 on immunnohistochemical detection of Vtg after pretreatment with genistein in fishes. The immunohistochemistry results demonstrate an increase of immunoreactivity at 12 h in liver of treated fishes than the control. After 24 h this immunoreactivity is lower than 12 h and then slightly increased at 72 h. Regarding effects of only genistein on immunoistochemical detection of Vtg, its immunoreactivity progressively increases from 12 h to 72 h (Fig. 1D).
2.4. Statistical analysis
3.1.2. CYP1A1 immunoreactivity CYP1A1 progressively increases from 12 h to 72 h in all groups (Fig. 2A, B, C, D). This increase in immunoreactivity emerges more in the hepatic parenchyma of fish treated with PCB-126 instead it has a smaller increase when the fish is treated with genistein and PCB-126 together.
All data are expressed as mean values standard deviation (SD). Data are analyzed by analysis of variance (ANOVA) for multiple comparison of results. To compare group means, the Bonferroni's test is conducted. A P ≤ 0.05 is considered statistically significant. The data are analyzed using STATISTICA 7.5 software package (Stat Soft Inc.). 3. Results 3.1. Immunohistochemical investigation 3.1.1. Vitellogenin immunoreactivity Fig. 1A shows the control sample of Vtg at 12, 24 and 72 h. In Fig. 1B, where only PCB-126 is administered, a marked immunoreactivity of Vtg at 12 h than the control is observed. This immunoreactivity decrease only at 24 h and it increases slightly at 72 h than the control as reported in our previous work by Calò et al. (2012).
3.1.3. Western-blot analysis Immunohistochemical results confirm Western-blot analysis. Fig. 3 shows effect of time on immunoreactivity detection of Vtg and CYP1A1 after treatment with PCB-126 and/or genistein in zebrafish. In particularly, the control group of Vtg shows a statistical significance at 12, 24 and 72 h. In PCB-126 group is observed a marked immunoreactivity of Vtg at 12 h compared to control group. This immunoreactivity decrease only at 24 h and it increases slightly at 72 h respect to control as reported in previous work by Calò et al., 2012. The immunoreactivity of Vtg in Genistein group progressively increases from 12 h to 72 h. The fishes treated with PCB-126 and genistein (Group C) show a lower
VITELLOGENIN A
CONTROL
B
PCB126
C PCB126+GEN ..
D
GENISTEIN
12h CONTR. 12h Vtg
PCB 12h Vtg
PCB+G. 12h Vtg
G.12h Vtg
24h CONTR. 24h Vtg
PCB 24h Vtg
PCB+ G. 24h Vtg
G.24h Vtg
72h
CONTR. 72h Vtg
PCB 72h Vtg
PCB + G.72h Vtg
G. 72h Vtg
Fig. 1. Effects of PCB-126, genistein+PCB-126 and genistein on immunohistochemical detection of vitellogenin in fish. Representative images in control fishes (Group A); representative images of vitellogenin in fishes exposed to PCB-126 (Group B), to PCB-126 + genistein (Group D) and genistein (Group C) (original magnification, ×400) Scale Bars 50 μm.
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CYP1A1 A
12h
CONTROL
CONTR. CYP1A
B
PCB126
PCB12h-CYP1A
C
PCB126+GEN.
D
GENISTEIN
PCB +G.12h-CYP1A
G12h-CYP1A
PCB+G24h-CYP1A
G24h-CYP1A
24h CONTR. CYP1A
PCB24h-CYP1A
72h CONTR. CYP1A
PCB72h-CYP1A
PCB+G72h-CYP1A
G72h-CYP1A
Fig. 2. Effects of PCB-126, genistein+PCB-126 and genistein on immunohistochemical detection of P-450 CYP1A1 in fish. Representative images in control fishes (Group A); representative images of vitellogenin in fishes exposed to PCB-126 (Group B), to PCB-126 + genistein (Group D) and genistein (Group C) (original magnification, ×400) Scale Bars 50 μm.
immunoreactivity at 12 h compared to PCB-126 (Group B). After 24 h this immunoreactivity is lower than 12 h with a slightly increase at 72 h. CYP1A1 progressively increases from 12 h to 72 h in all groups. The increase in immunoreactivity emerges more in the hepatic parenchyma of fish treated with PCB-126 instead it has a smaller increase when the fish is treated with PCB-126 and genistein. 4. Discussion PCBs as ubiquitous and persistent in the environment, they can give many toxic effects on the reproduction, nervous, endocrine system development and immunological function of animals. The toxicity of PCBs are largely based on the similarity in chemical structure and toxic mode of action with dioxin. Many steps of their effects are known to be ER-mediated by acting as estrogen mimics or as estrogen antagonists (Calò et al., 2010). Phytoestrogens such as genistein and resveratrol, contrary to chemically synthesized substances such as EDC, showed chemopreventive effects on specific cancers by contending with ER binding and regulating normal ER action in target structure of zebrafish. In this study adult zebrafish (Danio rerio) are used to show a significant increase of vitellogenin at 12 h in all groups treated than the control group (Lecomte et al., 2017). We show a reduction in the estrogenic effect when the fishes are treated with PCB-126 and genistein together at 12 h than the group treated with only PCB-126. Infact the most important result is the reduction of the estrogenic effect of PCB-126 when genistein is added antagonizing the effects. Moreover, we also demonstrate that low
concentrations of genistein decrease the marked P450 expression induced by PCB-126 as demonstrated in a work by Calò et al. (2009). This shows that genistein decreases the expression of P450 target genes mediated by AhR at 24 h. Also Piasecka-Srader et al. (2016) established the TCDD and genistein administered together decreased ERβ (48 h) and AhR (24 h) protein expression in the cells in contrast to the individual treatments. Messina and Wood (2008) showed that soyfoods are essentially a unique dietary source of isoflavones, compounds which bind to estrogen receptors and exhibit weak estrogen-like effects under certain experimental conditions. This work also demonstrates that higher levels of the isoflavone genistein inhibits the development of tumor cells and then breast cancer. Genistein has a biphasic effect in MCF-7 cells in vitro, cells proliferate when a low concentration of genistein is given, on the contrary an inhibition occurs when a high concentration is administered. In ovariectomized athymic mice implanted with MCF-7 cells, both genistein and soy protein stimulate tumor growth in a dose-dependent manner. In intact mice fed with estrogens and genistein, the latter inhibits tumor growth compared to mice fed only with estrogens. (Messina and Loprinzi, 2001). However, other research is necessary in the long term at lower doses and mixtures of phytoestrogens are required to demonstrate that dietary supplements do have beneficial rather than adverse effects on cancer. Moreover, the many targets in cancer cells and their ability to modulate epigenetic events associated with cancer and prevention may lead to new, non-toxic therapeutic approaches with the study of highly specific and long-acting analogues of phytoestrogens (Bilal et al., 2014).
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Fig. 3. Effect of time on immunoreactivity detection of Vtg and CYP1A1 after treatment with PCB-126 and/or genistein.
5. Conclusions These our studies indicate that a diet with low concentrations of phytoestrogens can have protective effects on estrogen-related diseases. Phytoestrogens have received recently great attention because of their beneficial effects, which include moreover the prevention of atherosclerosis, obesity, bone density loss, cancer and cardiovascular disease. Rietjens et al., 2017 established that the phytoestrogens are also considered endocrine disruptors, indicating that they have the potential to cause adverse health effects. Exposure to these compounds is excellent, but inappropriate or excessive exposure may be detrimental. Many natural substances can be potent and can have both good and bad health affects because if they are in excess or too little can alter hormone-dependent cellular and tissue functions. Phytoestrogens should be used in moderation to avoid any involuntary health repercussions (Patisaul and Jefferson, 2010). With the increase in industrial activity, the number of EDCs has also enhanced considerably in our environment, affecting nature and above all human. The potential risks of EDCs in endocrine disruption and carcinogenesis are important issues and needed to be better verified.
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