- Email: [email protected]
Regulation of hepatic metallothionein in estradiol-treated rainbow trout
Marine Environmental Research 39 (1995) 121-129 0 1995 Elsevier Science Limited Printed in Great Britain. All rights resewed 0141-I 136/95/$09.50 0141-1136(94)00060-3
ELSEVIER
Regulation of Hepatic Metallothionein in Estradiol-Treated Rainbow Trout Per-Erik Olsson & Peter Kling Department
of Cellular and Developmental Biology, Umei University, S-90 187 Umel, Sweden
ABSTRACT Metallothionein has previously been shown to be regulated during the period of exogenous vitellogenesis (Olsson et al. (1987); Fish Physiol, Biochem., 3(l), 39-47). Following estradiol injection of rainbow trout it has been shown that hepatic metallothionein remains at basal levels until the vitellogenin mRNA levels begin to decline and Zn is released from highmolecular-weight proteins (Olsson et al. (1989); Biochem. J., 257,5X-9). In the present study the effects of estradiol treatment on the metal-inducibility of metallothionein have been investigated. Estradiol-treated fish that were injected with low doses of cadmium or zinc did not respond by induction of metallothionein.
The recent advances in the field of teleost metallothionein (MT) gene structure and regulation has greatly improved our understanding of the basic regulation of MT in fish. Teleost MT genes have so far been shown to contain multiple metal regulatory elements (MRE) (Zafarullah et al., 1988; Murphy et al., 1990; Olsson, 1994). Besides being induced by heavy metals the MT and MT mRNA levels increase in response after glucocorticoid treatment of teleost cell lines and primary cell cultures (Hyllner et al., 1989; George et al., 1992). On the other hand, in-vivo and in-vitro experiments have shown that MT is not directly inducible by estradiol in fish (Olsson et al., 1989). However, after estradiol treatment the hepatic zinc levels rise drastically, and after cessation of the period of exogenous vitellogenesis it appears to be the redistribution of Zn within the liver that triggers the induction of MT. The Zn that enters the liver during this period must be sequestered by non-MT proteins and enzymes, since the hepatic MT levels do not increase with the initial hepatic accumulation of Zn. To determine if the MT genes in rainbow trout liver are responsive to metal induction during this period, juvenile rainbow trout were injected with a combination of estradiol and cadmium or estradiol and zinc and followed the regulation of MT in the liver using differential pulse polarography. Juvenile rainbow trout were injected intraperitoneally with 10 mg estradiol/kg body weight alone or in combination with 0.2 mg (low dose) and 2.0 mg (high 127
128
P. -E. Olsson, P. Kling
dose) Cd/kg body weight or 0.5 mg Zn/kg body weight. Since the estradiol had to be finely dispersed in peanut oil, one group of fish (control) was injected with peanut oil alone. One additional group was injected with the low dose of Cd alone as a positive control. At sampling, after 1, 5, 9 and 15 days, the livers were collected and immediately frozen in liquid nitrogen. A 100 mg sample of each liver was homogenized in 1.0 ml ice-cold 10 mM Tris-HCl buffer, pH 7.6, heat denatured at 95°C and centrifuged at 10000 x g for 5 min at 4°C. The resulting supernatant was used to determine MT by differential pulse polarography as previously described (Olafson & Olsson, 1991). In the present study, the liver somatic index (LSI) was elevated in estradiol and estradiol/Zn injected fish five days post injection (Fig. 1). In both the estradioltreated and the estradiol/Zn-treated fish the LSI peaked after nine days and began to decline again after 15 days. Estradiol in combination with low dose Cdtreatment fish showed a delay in the elevation of LSI. The LSI levels in these fish increased significantly only after 15 days. The delay in elevation of LSI in the estradiol/low dose Cd group indicates that Cd has a definite effect on exogenous vitellogenesis. The delayed elevation of LSI indicates that Cd interferes with the production of vitellogenin in the liver. Reduced levels of vitellogenin has earlier been indicated in flounder in response to estradiol/Cd treatment (Faaborg Povlsen et al., 1990). Measurement of the hepatic MT levels five days after treatment revealed that neither injection of low dose Cd or Zn in combination with estradiol resulted in induction of MT (Fig. 2). However, injection of high dose Cd in combination with estradiol resulted in a close to three-fold induction of hepatic MT. This increase is within the same range as that found after the injection of 0.2 mg Cd/kg body weight alone. The present results thus indicate that Cd has an inhibitory effect on the vitellogenin production, as has also been observed by others (Faaborg Povlsen et al., 1990) and that this effect may be due to a low ability of Cd to induce MT during the period of vitellogenin production. Further experiments need to be performed to localize the injected Cd and to determine the factor(s) that reduce the Cd inducibility of MT after estradiol-induced induction of vitellogenesis.
Fig. 1. Liver somatic index of rainbow trout in response to different treatments. Control fish were injected with peanut oil only (filled circles). The different treatments were 10 mg estradiol/kg body weight (filled square), 10 mg estradiol + 0.2 mg Cd/kg body weight (open circles) and 10 mg estradiol + 0.5 mg &/kg body weight (open squares).
Regulation of hepatic MT in estradiol-treated
rainbow trout
129
nmoles/g
2
3 4 Inducer
I
5
6
Fig. 2. MT levels in livers of fish five days after treatment. 1, control; 2, 1Omg estradiol/ kg; 3, 1Omg estradiol + 0.2 mg Cd/kg; 4, 10 mg estradiol + 0.5 mg Zn/kg; 5, 10 mg estradiol + 2.0mg Cd/kg; 6,0.2 mg Cd/kg. All results are presented as mean f S.D., n = 4.
ACKNOWLEDGEMENT This study Council.
was made
possible
by a grant
from
the Swedish
Natural
Research
REFERENCES Faaborg Povlsen, A., Korsgaard, B. & Bjerregaard, P. (1990). Aquat. Toxicol., 17, 25342. George, S., Burgess, D., Leaver, M. & Frerichs, N. (1992). Fish Physiol. Biochem., 10, 43-54. Hyllner, S.J., Andersson, T., Haux, C. & Olsson, P.-E. (1989). J. Cell. Physiol., 139, 248. Murphy, M.F., Collier, J., Koutz, P. & Howard, B. (1990). Nucl. Acids Res. 18( 15), 4622. Olafson, R.W. & Olsson, P.-E. (1991). Meth. Enzymol., 205, 205-l 3. Olsson, P.-E. (1994). The biochemistry and molecular biology of fishes. In: Molecular Biology of$shes, Vo1.2. P.W. Hochachka & T.P. Mommsen (eds). pp. 259-78. Olsson, P.-E., Zafarullah, M. & Gedamu, L. (1989). Biochem. J., 257, 555-9. Zafarullah, M., Bonham, K. & Gedamu, L. (1988). Mol. Cell. Biol., 8(10), 4469-76.
ELSEVIER
Regulation of Hepatic Metallothionein in Estradiol-Treated Rainbow Trout Per-Erik Olsson & Peter Kling Department
of Cellular and Developmental Biology, Umei University, S-90 187 Umel, Sweden
ABSTRACT Metallothionein has previously been shown to be regulated during the period of exogenous vitellogenesis (Olsson et al. (1987); Fish Physiol, Biochem., 3(l), 39-47). Following estradiol injection of rainbow trout it has been shown that hepatic metallothionein remains at basal levels until the vitellogenin mRNA levels begin to decline and Zn is released from highmolecular-weight proteins (Olsson et al. (1989); Biochem. J., 257,5X-9). In the present study the effects of estradiol treatment on the metal-inducibility of metallothionein have been investigated. Estradiol-treated fish that were injected with low doses of cadmium or zinc did not respond by induction of metallothionein.
The recent advances in the field of teleost metallothionein (MT) gene structure and regulation has greatly improved our understanding of the basic regulation of MT in fish. Teleost MT genes have so far been shown to contain multiple metal regulatory elements (MRE) (Zafarullah et al., 1988; Murphy et al., 1990; Olsson, 1994). Besides being induced by heavy metals the MT and MT mRNA levels increase in response after glucocorticoid treatment of teleost cell lines and primary cell cultures (Hyllner et al., 1989; George et al., 1992). On the other hand, in-vivo and in-vitro experiments have shown that MT is not directly inducible by estradiol in fish (Olsson et al., 1989). However, after estradiol treatment the hepatic zinc levels rise drastically, and after cessation of the period of exogenous vitellogenesis it appears to be the redistribution of Zn within the liver that triggers the induction of MT. The Zn that enters the liver during this period must be sequestered by non-MT proteins and enzymes, since the hepatic MT levels do not increase with the initial hepatic accumulation of Zn. To determine if the MT genes in rainbow trout liver are responsive to metal induction during this period, juvenile rainbow trout were injected with a combination of estradiol and cadmium or estradiol and zinc and followed the regulation of MT in the liver using differential pulse polarography. Juvenile rainbow trout were injected intraperitoneally with 10 mg estradiol/kg body weight alone or in combination with 0.2 mg (low dose) and 2.0 mg (high 127
128
P. -E. Olsson, P. Kling
dose) Cd/kg body weight or 0.5 mg Zn/kg body weight. Since the estradiol had to be finely dispersed in peanut oil, one group of fish (control) was injected with peanut oil alone. One additional group was injected with the low dose of Cd alone as a positive control. At sampling, after 1, 5, 9 and 15 days, the livers were collected and immediately frozen in liquid nitrogen. A 100 mg sample of each liver was homogenized in 1.0 ml ice-cold 10 mM Tris-HCl buffer, pH 7.6, heat denatured at 95°C and centrifuged at 10000 x g for 5 min at 4°C. The resulting supernatant was used to determine MT by differential pulse polarography as previously described (Olafson & Olsson, 1991). In the present study, the liver somatic index (LSI) was elevated in estradiol and estradiol/Zn injected fish five days post injection (Fig. 1). In both the estradioltreated and the estradiol/Zn-treated fish the LSI peaked after nine days and began to decline again after 15 days. Estradiol in combination with low dose Cdtreatment fish showed a delay in the elevation of LSI. The LSI levels in these fish increased significantly only after 15 days. The delay in elevation of LSI in the estradiol/low dose Cd group indicates that Cd has a definite effect on exogenous vitellogenesis. The delayed elevation of LSI indicates that Cd interferes with the production of vitellogenin in the liver. Reduced levels of vitellogenin has earlier been indicated in flounder in response to estradiol/Cd treatment (Faaborg Povlsen et al., 1990). Measurement of the hepatic MT levels five days after treatment revealed that neither injection of low dose Cd or Zn in combination with estradiol resulted in induction of MT (Fig. 2). However, injection of high dose Cd in combination with estradiol resulted in a close to three-fold induction of hepatic MT. This increase is within the same range as that found after the injection of 0.2 mg Cd/kg body weight alone. The present results thus indicate that Cd has an inhibitory effect on the vitellogenin production, as has also been observed by others (Faaborg Povlsen et al., 1990) and that this effect may be due to a low ability of Cd to induce MT during the period of vitellogenin production. Further experiments need to be performed to localize the injected Cd and to determine the factor(s) that reduce the Cd inducibility of MT after estradiol-induced induction of vitellogenesis.
Fig. 1. Liver somatic index of rainbow trout in response to different treatments. Control fish were injected with peanut oil only (filled circles). The different treatments were 10 mg estradiol/kg body weight (filled square), 10 mg estradiol + 0.2 mg Cd/kg body weight (open circles) and 10 mg estradiol + 0.5 mg &/kg body weight (open squares).
Regulation of hepatic MT in estradiol-treated
rainbow trout
129
nmoles/g
2
3 4 Inducer
I
5
6
Fig. 2. MT levels in livers of fish five days after treatment. 1, control; 2, 1Omg estradiol/ kg; 3, 1Omg estradiol + 0.2 mg Cd/kg; 4, 10 mg estradiol + 0.5 mg Zn/kg; 5, 10 mg estradiol + 2.0mg Cd/kg; 6,0.2 mg Cd/kg. All results are presented as mean f S.D., n = 4.
ACKNOWLEDGEMENT This study Council.
was made
possible
by a grant
from
the Swedish
Natural
Research
REFERENCES Faaborg Povlsen, A., Korsgaard, B. & Bjerregaard, P. (1990). Aquat. Toxicol., 17, 25342. George, S., Burgess, D., Leaver, M. & Frerichs, N. (1992). Fish Physiol. Biochem., 10, 43-54. Hyllner, S.J., Andersson, T., Haux, C. & Olsson, P.-E. (1989). J. Cell. Physiol., 139, 248. Murphy, M.F., Collier, J., Koutz, P. & Howard, B. (1990). Nucl. Acids Res. 18( 15), 4622. Olafson, R.W. & Olsson, P.-E. (1991). Meth. Enzymol., 205, 205-l 3. Olsson, P.-E. (1994). The biochemistry and molecular biology of fishes. In: Molecular Biology of$shes, Vo1.2. P.W. Hochachka & T.P. Mommsen (eds). pp. 259-78. Olsson, P.-E., Zafarullah, M. & Gedamu, L. (1989). Biochem. J., 257, 555-9. Zafarullah, M., Bonham, K. & Gedamu, L. (1988). Mol. Cell. Biol., 8(10), 4469-76.