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Plasma levels of vitellogenin and eggshell zona radiata proteins in 4-nonylphenol and o,p′-DDT treated juvenile Atlantic salmon (Salmo salar)
Marine Environmental 0 PII:
SOl41-1136(98)00002-6
Research, Vol. 46, No. 1-5, pp. 133-136, 1998 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-1136/98 $19.00+0.00
ELSEVlER
Plasma Levels of Vitellogenin and Eggshell Zona Radiata Proteins in 4-Nonylphenol and o,p’-DDT Treated Juvenile Atlantic Salmon (Salmo salar) A. Arukwe,* Department
T. Celius,
B. T. Walther
and
A. Gokseryr
of Molecular Biology, University of Bergen, HIB, N-5020 Bergen, Norway
ABSTRACT Induction of vitellogenin (Vtg) in males and juveniles of oviparous vertebrates has been used as a biomarker for xenoestrogens. Recently, we have demonstrated that synthesis of eggshell zona radiata proteins (Zrp) or zonagenesis is an integral aspect of$sh oogenesis (Oppen-Berntsen et al. (1994) Journal of Experimental Zoology 268,59-70)) and that Zrp synthesis is a sensitive biomarker for nonylphenol (Arukwe et al. (1997) Environmental Health Perspective 105, 418-422). This study compares the responses of Vtg and Zrp in plasma of juvenile Atlantic salmon (Salmo salar) treated with 4-nonylphenol (NP) and o,p’DDT (both at 25mg kg-‘, singly and in combination). Validated ELISA and immunoblot analysis show that Vtg and Zrp respond signtj$cantly stronger to NP treatment alone and in combination with o,p’-DDT compared to control and o,p’DDT treatment alone. However, a slight reduction in NP-induced Zrp levels was indicated when NP was injected in combination with o,p’-DDT. 0 1998 Elsevier Science Ltd. All rights reserved
Accumulating evidence indicates that some persistent chemicals released into the environment influence vital endocrine functions in wildlife and humans. These include natural chemicals, such as phytoestrogens and mycoestrogens, and man-made compounds, such as some organochlorine pesticides, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), polychlorinated dibenzodioxins (PCDDs), surfactants and plasticizers (Colborn and Clement, 1992). Induction of the female specific yolk protein precursor (vitellogenin; Vtg) in males and juveniles has been used as a biomarker for estrogenic contaminants in the environment (Sumpter and Jobling, 1995). Recently, we have demonstrated that the eggshell zona radiata proteins (Zrp) another female phenotypic marker, are a more sensitive xenoestrogen biomarker than Vtg (Arukwe et al., 1997). Both Vtg and Zrp are synthesized in the liver in response to estrogen stimulation. They are secreted and transported through the blood to the ovary (Oppen-Bemtsen et al., *To whom correspondence
should be addressed. 133
134
A. Arukwe et al.
1994). There, Vtg is sequestered to form the yolk proteins that serve as nutrient reserve (Mommsen and Walsh, 1988), while Zrp forms the eggshell that prevents polyspermy and provides mechanical protection for the developing embryo. The aim of this experiment was to study the possible interactions of combined nonylphenol and o,$-DDT treatment on plasma vitellogenin and eggshell z~na radiuta protein levels, using immunochemical assay methods. A homogeneous group of juvenile Atlantic salmon (75-100 g) belonging to the Norwegian salmon (NLA) strain was purchased from Savareid smolt producer in Fusa (near Bergen, Norway). They were acclimated and kept in sea water (salinity of 34%) at 10 (& 0.4)“C and natural photoperiod. Four subgroups of six fish each were injected intraperitoneally (i.p.) with NP and o,$-DDT (both at 25 mg kg-‘) singly and in combination. The chemicals were dissolved in acetone and Alkamuls (1: 1) as vehicle. The control group was injected with the vehicle, and all treated groups were kept in separate tanks. Blood samples were collected from all groups after 2 weeks and centrifuged immediately (5000 rpm for 5 min). Plasma levels of Vtg and Zrp were measured using validated ELISA (Arukwe et al., 1997) and immunoblot (Towbin et al., 1979) analysis with homologous rabbit polyclonal antisera against salmon Vtg and Zrp. In plasma of juvenile Atlantic salmon, a cross-reacting Vtg protein (170 kDa) was detected in the groups receiving NP alone and in combination with oq’-DDT using western blotting (result not shown). Similarly, Zrp (both (Y,/l and r) were also detected in these group. In accordance with the immunoblot analysis, the inducibility of these proteins was also demonstrated with ELISA. Significant differences (p < 0.001) in Vtg and Zrp levels were observed in the group treated with NP alone and in combination with oq’-DDT (Fig. 1) compared with the control group. It should be noted that the DDT-exposure used in this experiment is below DDT-doses shown to induce Zrp in Atlantic salmon (Celius and Walther, 1998). This experiment was designed to detect synergism or antagonism between submaximal xenoestrogen doses, and the results may indicate a slight, but unexpected, reduction in plasma Zrp-levels after combined NP and o,$-DDT treatment (Fig. 1). Many of the estrogenic chemicals identified to date, such as alkylphenol polyethoxylates (APES) and organochlorine pesticides, are lipophilic and may potentially accumulate in organisms that are constantly exposed (Ahel et al., 1993). In real life, organisms are less likely to be exposed to a single estrogenic chemical, than to cocktails of such chemicals. Therefore, interactive effects of xenobiotic estrogens are of ecological relevance. Cell cultures or other in vitro systems have been used extensively to study the estrogenic activity of these chemicals (White et al., 1994). Given that the metabolic processes typical of in vivo systems are lacking in vitro, results obtained in vitro do not always correlate with those obtained in vivo. Immunochemical analysis, using western blotting and indirect ELISA with homologous polyclonal antisera against Vtg and Zrp from Atlantic salmon, were used to study the responses of these proteins in plasma of control, NP and o,$-DDT treated juvenile salmon. The sensitivity of the antisera and methods has been demonstrated earlier (Oppen-Berntsen et al., 1994; Arukwe et al., 1997). Vtg and Zrp were detected in the group treated with NP alone and in combination with o,p’-DDT. The consequences of xenoestrogen-induced synthesis of Vtg and Zrp outside normal reproductive cycle is presently unknown, as are long-term effects of adult or juvenile fish exposure to xenoestrogens. However, estrogens are essential for female reproduction, and disruption of normal levels of circulating estrogens may likely have negative consequences. Herman and Kincaid (1988) have reported
Zona radiata proteins in Salmo
135
Treatment group analysis using indirect ELBA of vitellogenin (Vtg) and zona radiata proteins (Zrp) in plasma from control, 4-nonylphenol and o,p’-DDT (both at 25mg kg-’ administered intraperitoneally, singly and in combination) treated juvenile Atlantic salmon. Data are given as mean ELBA-absorbance values & standard error (SE; n = 6). *Significantly different from control (p < 0.001). %ignificantly different from oq’-DDT treated group alone (p < 0.001). Fig. 1. Immunochemical
that prolonged estrogen-induced synthesis of Vtg resulted in considerable metabolic stress with subsequent necrosis, kidney and liver damage, with possible mortal outcome for both
male and female rainbow trout. Furthermore, continued synthesis of Vtg diverts available energy resources (lipids, proteins). Loss of calcium from bones and also from the scales (Carragher and Sumpter, 1991) may increase the susceptibility of fish to disease. At present, research efforts are mostly directed towards screening environmental chemicals for estrogenic activity and identifying their occurrence in sewage recipient river and estuarine systems. Little progress has been made in determining the ecological significance of such findings at the fish population level. This important task is highly complex, since it is not feasible to empirically determine the sensitivity, susceptibility, or resistance of every species to each compound. In addition, sewage and industrial discharges contain mixtures of estrogenic compounds, and also compounds that may produce reproductive toxicity not mediated through hormonal pathways. Xenoestrogen-induced changes of vitellogenin and eggshell zona radiata protein levels appear to possess a potential for adverse ecological effects, but studies are needed of critical population parameters such as offspring
136
A. Arukwe et al.
survival and recruitment. This hypothesis may be tested through long-term exposure of fish to low levels of xenoestrogen. We are continuing the exploration of possible synergistic or antagonistic interactions between xenobiotics on zonagenesis and vitellogenesis in juvenile salmon (Arukwe et al., submitted).
ACKNOWLEDGEMENT Supported by the Norwegian Research Council (NFR; Programme for Ecotoxicology).
REFERENCES Ahel, M., McEvoy, J. and Giger, W. (1993) Environmental Pollution 79, 243-248. Arukwe, A., Knudsen, F. R. and Goksoyr, A. (1997) Environmental Health
Perspective
105,
418422.
Carragher, J. F. and Sumpter, J. P. (1991) Comparative Biochemistry and Physiology A 99, 169-172. Colborn, T. and Clement, C. (eds) (1992) Advances in Modern Environmental Toxicology, Vol. 21. Princeton Scientific Publishing, Princeton, New Jersey. Herman, R. L. and Kincaid, H. L. (1988) Aquaculture 72, 165-172. Mommsen, P. T. and Walsh, P. J. (1988) In Fish Physiology, eds W. S. Hoar and D. J. Randall, pp. 347406. Academic Press, New York. Oppen-Berntsen, D. O., Olsen, S. O., Rong, C. J., Taranger, G. L., Swanson, P. and Walther, B. T. (1994) Journal of Experimental Zoology 268, 59-70. Sumpter, J. P. and Jobling, S. (1995) Environmentaf Health Perspective 103 (Suppl. 7), 173-178. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proceedings of the National Academy of Science, USA 76,435&4354.
White, R., Jobling, S., Hoare, S. A., Sumpter, J. P. and Parker, M. G. (1994) Endocrinology 175-182.
135,
SOl41-1136(98)00002-6
Research, Vol. 46, No. 1-5, pp. 133-136, 1998 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-1136/98 $19.00+0.00
ELSEVlER
Plasma Levels of Vitellogenin and Eggshell Zona Radiata Proteins in 4-Nonylphenol and o,p’-DDT Treated Juvenile Atlantic Salmon (Salmo salar) A. Arukwe,* Department
T. Celius,
B. T. Walther
and
A. Gokseryr
of Molecular Biology, University of Bergen, HIB, N-5020 Bergen, Norway
ABSTRACT Induction of vitellogenin (Vtg) in males and juveniles of oviparous vertebrates has been used as a biomarker for xenoestrogens. Recently, we have demonstrated that synthesis of eggshell zona radiata proteins (Zrp) or zonagenesis is an integral aspect of$sh oogenesis (Oppen-Berntsen et al. (1994) Journal of Experimental Zoology 268,59-70)) and that Zrp synthesis is a sensitive biomarker for nonylphenol (Arukwe et al. (1997) Environmental Health Perspective 105, 418-422). This study compares the responses of Vtg and Zrp in plasma of juvenile Atlantic salmon (Salmo salar) treated with 4-nonylphenol (NP) and o,p’DDT (both at 25mg kg-‘, singly and in combination). Validated ELISA and immunoblot analysis show that Vtg and Zrp respond signtj$cantly stronger to NP treatment alone and in combination with o,p’-DDT compared to control and o,p’DDT treatment alone. However, a slight reduction in NP-induced Zrp levels was indicated when NP was injected in combination with o,p’-DDT. 0 1998 Elsevier Science Ltd. All rights reserved
Accumulating evidence indicates that some persistent chemicals released into the environment influence vital endocrine functions in wildlife and humans. These include natural chemicals, such as phytoestrogens and mycoestrogens, and man-made compounds, such as some organochlorine pesticides, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), polychlorinated dibenzodioxins (PCDDs), surfactants and plasticizers (Colborn and Clement, 1992). Induction of the female specific yolk protein precursor (vitellogenin; Vtg) in males and juveniles has been used as a biomarker for estrogenic contaminants in the environment (Sumpter and Jobling, 1995). Recently, we have demonstrated that the eggshell zona radiata proteins (Zrp) another female phenotypic marker, are a more sensitive xenoestrogen biomarker than Vtg (Arukwe et al., 1997). Both Vtg and Zrp are synthesized in the liver in response to estrogen stimulation. They are secreted and transported through the blood to the ovary (Oppen-Bemtsen et al., *To whom correspondence
should be addressed. 133
134
A. Arukwe et al.
1994). There, Vtg is sequestered to form the yolk proteins that serve as nutrient reserve (Mommsen and Walsh, 1988), while Zrp forms the eggshell that prevents polyspermy and provides mechanical protection for the developing embryo. The aim of this experiment was to study the possible interactions of combined nonylphenol and o,$-DDT treatment on plasma vitellogenin and eggshell z~na radiuta protein levels, using immunochemical assay methods. A homogeneous group of juvenile Atlantic salmon (75-100 g) belonging to the Norwegian salmon (NLA) strain was purchased from Savareid smolt producer in Fusa (near Bergen, Norway). They were acclimated and kept in sea water (salinity of 34%) at 10 (& 0.4)“C and natural photoperiod. Four subgroups of six fish each were injected intraperitoneally (i.p.) with NP and o,$-DDT (both at 25 mg kg-‘) singly and in combination. The chemicals were dissolved in acetone and Alkamuls (1: 1) as vehicle. The control group was injected with the vehicle, and all treated groups were kept in separate tanks. Blood samples were collected from all groups after 2 weeks and centrifuged immediately (5000 rpm for 5 min). Plasma levels of Vtg and Zrp were measured using validated ELISA (Arukwe et al., 1997) and immunoblot (Towbin et al., 1979) analysis with homologous rabbit polyclonal antisera against salmon Vtg and Zrp. In plasma of juvenile Atlantic salmon, a cross-reacting Vtg protein (170 kDa) was detected in the groups receiving NP alone and in combination with oq’-DDT using western blotting (result not shown). Similarly, Zrp (both (Y,/l and r) were also detected in these group. In accordance with the immunoblot analysis, the inducibility of these proteins was also demonstrated with ELISA. Significant differences (p < 0.001) in Vtg and Zrp levels were observed in the group treated with NP alone and in combination with oq’-DDT (Fig. 1) compared with the control group. It should be noted that the DDT-exposure used in this experiment is below DDT-doses shown to induce Zrp in Atlantic salmon (Celius and Walther, 1998). This experiment was designed to detect synergism or antagonism between submaximal xenoestrogen doses, and the results may indicate a slight, but unexpected, reduction in plasma Zrp-levels after combined NP and o,$-DDT treatment (Fig. 1). Many of the estrogenic chemicals identified to date, such as alkylphenol polyethoxylates (APES) and organochlorine pesticides, are lipophilic and may potentially accumulate in organisms that are constantly exposed (Ahel et al., 1993). In real life, organisms are less likely to be exposed to a single estrogenic chemical, than to cocktails of such chemicals. Therefore, interactive effects of xenobiotic estrogens are of ecological relevance. Cell cultures or other in vitro systems have been used extensively to study the estrogenic activity of these chemicals (White et al., 1994). Given that the metabolic processes typical of in vivo systems are lacking in vitro, results obtained in vitro do not always correlate with those obtained in vivo. Immunochemical analysis, using western blotting and indirect ELISA with homologous polyclonal antisera against Vtg and Zrp from Atlantic salmon, were used to study the responses of these proteins in plasma of control, NP and o,$-DDT treated juvenile salmon. The sensitivity of the antisera and methods has been demonstrated earlier (Oppen-Berntsen et al., 1994; Arukwe et al., 1997). Vtg and Zrp were detected in the group treated with NP alone and in combination with o,p’-DDT. The consequences of xenoestrogen-induced synthesis of Vtg and Zrp outside normal reproductive cycle is presently unknown, as are long-term effects of adult or juvenile fish exposure to xenoestrogens. However, estrogens are essential for female reproduction, and disruption of normal levels of circulating estrogens may likely have negative consequences. Herman and Kincaid (1988) have reported
Zona radiata proteins in Salmo
135
Treatment group analysis using indirect ELBA of vitellogenin (Vtg) and zona radiata proteins (Zrp) in plasma from control, 4-nonylphenol and o,p’-DDT (both at 25mg kg-’ administered intraperitoneally, singly and in combination) treated juvenile Atlantic salmon. Data are given as mean ELBA-absorbance values & standard error (SE; n = 6). *Significantly different from control (p < 0.001). %ignificantly different from oq’-DDT treated group alone (p < 0.001). Fig. 1. Immunochemical
that prolonged estrogen-induced synthesis of Vtg resulted in considerable metabolic stress with subsequent necrosis, kidney and liver damage, with possible mortal outcome for both
male and female rainbow trout. Furthermore, continued synthesis of Vtg diverts available energy resources (lipids, proteins). Loss of calcium from bones and also from the scales (Carragher and Sumpter, 1991) may increase the susceptibility of fish to disease. At present, research efforts are mostly directed towards screening environmental chemicals for estrogenic activity and identifying their occurrence in sewage recipient river and estuarine systems. Little progress has been made in determining the ecological significance of such findings at the fish population level. This important task is highly complex, since it is not feasible to empirically determine the sensitivity, susceptibility, or resistance of every species to each compound. In addition, sewage and industrial discharges contain mixtures of estrogenic compounds, and also compounds that may produce reproductive toxicity not mediated through hormonal pathways. Xenoestrogen-induced changes of vitellogenin and eggshell zona radiata protein levels appear to possess a potential for adverse ecological effects, but studies are needed of critical population parameters such as offspring
136
A. Arukwe et al.
survival and recruitment. This hypothesis may be tested through long-term exposure of fish to low levels of xenoestrogen. We are continuing the exploration of possible synergistic or antagonistic interactions between xenobiotics on zonagenesis and vitellogenesis in juvenile salmon (Arukwe et al., submitted).
ACKNOWLEDGEMENT Supported by the Norwegian Research Council (NFR; Programme for Ecotoxicology).
REFERENCES Ahel, M., McEvoy, J. and Giger, W. (1993) Environmental Pollution 79, 243-248. Arukwe, A., Knudsen, F. R. and Goksoyr, A. (1997) Environmental Health
Perspective
105,
418422.
Carragher, J. F. and Sumpter, J. P. (1991) Comparative Biochemistry and Physiology A 99, 169-172. Colborn, T. and Clement, C. (eds) (1992) Advances in Modern Environmental Toxicology, Vol. 21. Princeton Scientific Publishing, Princeton, New Jersey. Herman, R. L. and Kincaid, H. L. (1988) Aquaculture 72, 165-172. Mommsen, P. T. and Walsh, P. J. (1988) In Fish Physiology, eds W. S. Hoar and D. J. Randall, pp. 347406. Academic Press, New York. Oppen-Berntsen, D. O., Olsen, S. O., Rong, C. J., Taranger, G. L., Swanson, P. and Walther, B. T. (1994) Journal of Experimental Zoology 268, 59-70. Sumpter, J. P. and Jobling, S. (1995) Environmentaf Health Perspective 103 (Suppl. 7), 173-178. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proceedings of the National Academy of Science, USA 76,435&4354.
White, R., Jobling, S., Hoare, S. A., Sumpter, J. P. and Parker, M. G. (1994) Endocrinology 175-182.
135,