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Aluminium and Cadmium Inhibit Vitellogenin and Its mRNA Induction by Estradiol-17 β in the Primary Culture of Hepatocytes in the Rainbow Trout Oncorhynchus mykiss
General and Comparative Endocrinology 119, 69 –76 (2000) doi:10.1006/gcen.2000.7494, available online at http://www.idealibrary.com on
Aluminium and Cadmium Inhibit Vitellogenin and Its mRNA Induction by Estradiol-17  in the Primary Culture of Hepatocytes in the Rainbow Trout Oncorhynchus mykiss Un-Gi Hwang, Nao Kagawa, and Yasuo Mugiya Laboratory of Comparative Physiology, Faculty of Fisheries, Hokkaido University, 3-1-1 Minato, Hakodate 041-8611, Japan Accepted March 23, 2000
such metals that impaired vitellogenin (VTG) production. VTG, an egg yolk precursor protein, is synthesized in the liver in response to estrogen and is incorporated into eggs to be processed as yolk proteins in maturing females. Impaired oogenesis due to poor accumulation of egg yolk (Lee and Gerking, 1980) and a reduction in egg number (Runn et al., 1977) have been reported in acidic waters, where Al reaches high concentrations of 3.7–34.8 M (Brumbaugh and Kane, 1985; Borg, 1986). Mugiya and Tanahashi (1998) showed directly that Al inhibited the induction of VTG synthesis in hepatocyte culture of rainbow trout. However, the intracellular mechanisms of inhibition are unknown. Cd is another metal that has been reported to inhibit the in vivo induction of VTG synthesis. Exposure to Cd decreased plasma VTG (alkaline-labile phosphate) concentrations in maturing winter flounder (Pereira et al., 1993). Coinjection of Cd with estrogen resulted in decreased plasma protein (mostly VTG) levels (Olsson et al., 1995) and it was suggested that Cd inhibited the transcription of VTG. Hepatocyte culture is an excellent system to confirm and analyze these in vivo results directly. The present study was undertaken to determine at which levels these metals interfere with the synthesis of VTG in rainbow trout. Hepatocytes were cultured with a combination of estradiol and Al or Cd, and
Effects of Al and Cd on vitellogenin (VTG) and VTG mRNA induction by estradiol-17  (E 2) were examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then E 2 (2 ⴛ 10 ⴚ6 M) and Al (10 ⴚ6–10 ⴚ4 M) or Cd (10 ⴚ9–10 ⴚ6 M) were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days. Media and hepatocytes were then analyzed by SDS-PAGE and Northern blotting for VTG and VTG mRNA, respectively. These metals had no appreciable effect on the viability of hepatocytes in culture. However, Al and Cd interfered with VTG production and VTG mRNA expression. Al reduced VTG production in a concentration-dependent way and a significant reduction occurred at Al concentrations greater than 5 ⴛ 10 ⴚ5 M. VTG mRNA expression also decreased with a negative correlation with Al concentrations (r ⴝ ⴚ0.98). The inhibition of VTG production by Cd was not concentration-dependent. This metal markedly inhibited VTG production and VTG mRNA expression at 10 ⴚ6 M. The Al-induced inhibition of VTG production was restored 7 days after Al removal, but the Cdinduced inhibition was not restored. These results suggest that Al and Cd inhibit VTG production at the transcriptional level to reduce VTG mRNA expression by different mechanisms. © 2000 Academic Press Metal contaminants in the aquatic environment may interfere with reproduction in fish. Al and Cd are two 0016-6480/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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VTG production and VTG mRNA expression were analyzed by electrophoresis and Northern blotting, respectively. Recovery experiments following removal of these metals were also conducted to clarify whether metal-induced inhibition is reversible.
MATERIALS AND METHODS Rainbow trout, Oncorhynchus mykiss, weighing 110 – 350 g were obtained from a commercial dealer and kept in outdoor ponds with running water at about 14°C. They were fed trout food pellets once a day but were starved on the last day before sampling to reduce the production of bile. Maturing females were not included.
Hwang, Kagawa, and Mugiya
ture, the media and hepatocytes were analyzed for VTG protein and VTG mRNA, respectively. The effects of Al and Cd on cell viability were also examined using crystal violet as described by Mugiya and Tanahashi (1998). Cell viability was taken as the number of living cells on Day 5 expressed as a percentage of the number of living cells just before metal addition. Treatment 2. To examine the effects of Al and Cd removal on the metal-induced inhibition of VTG production, hepatocytes were cultured with E 2 and Al (10 ⫺4 M) or Cd (10 ⫺6 M) for 3 days and then these metals were removed from the media (defined as Day 0). The cultures were continued for another 7 days. Media samples were taken for analysis on Days 0, 1, 2, 3, 4, 5, and 7.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) Hepatocyte Preparation and Incubation Hepatocytes were prepared following Hayashi and Ooshiro (1975) as described by Kwon et al. (1993). Cell yield and viability were determined by the trypan blue exclusion test. Cells were plated into a 60-mm plastic petri dish with a positive charge (Falcon) at a density of 0.5–1 ⫻ 10 6 cells/dish. William’s medium E (Life Technologies, Inc.) containing 0.2 M bovine insulin (Sigma, St. Louis, MO), streptomycin (100 g/ml), and penicillin (70 g/ml) was used for cell culture. All incubations were carried out in 3 ml of the medium at 15°C under 5% CO 2. Preculture was conducted for 2 days before each experiment. The whole medium was changed every day throughout the preculture and experimental periods.
Electrophoresis was done as described by Mugiya and Tanahashi (1998). Briefly, proteins were precipitated from the media by cold trichloroacetic acid, dissolved in sample buffer (0.175 M Tris-HCl, 8 M urea, 1% SDS, and 0.5% 2-mercaptoethanol, pH 7.4), and subjected to 5–20% gradient SDS-PAGE. The gels were stained with 0.25% Coomassie brilliant blue R-250 (CBB). It was difficult to apply a fixed amount of proteins to each lane, because the present experiment was intended to inhibit VTG (protein) synthesis. Standard proteins used for molecular weight (MW) determinations were carbonic anhydrase (MW 29,000), ovalbumin (45,000), bovine serum albumin (66,000), phosphorylase b (97,400), -galactosidase (116,000), and myosin (205,000).
Qualitative and Quantitative Analyses of VTG Treatment with Al and Cd Treatment 1. After a 2-day preculture, estradiol-17  (E 2, 2 ⫻ 10 ⫺6 M in 3 l of 95% ethanol) and Al (AlCl 3 in 3 l of redistilled water) or Cd (CdCl 2 in 3 l of redistilled water) were simultaneously added to the dishes. Final metal concentrations were 10 ⫺6, 10 ⫺5, 5 ⫻ 10 ⫺5, and 10 ⫺4 M for Al and 10 ⫺9, 10 ⫺8, 10 ⫺7, and 10 ⫺6 M for Cd. The effects of these metals on VTG production were examined after 5 days, during which time the media were changed daily. Control cultures received the equivalent amount of the solvents only. After cul-
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The identification of the VTG band was based on the results of a previous study (Kwon et al., 1993) in which isolated rainbow trout hepatocytes incubated with E 2 synthesized a protein with the same electrophoretic mobility (175 kDa) as in the present study. Kwon et al. identified this band as VTG (main band) by immunoblot analysis and immunoelectrophoresis. After SDS-PAGE, the integrated optical density (IOD) of the VTG band was measured by a Bio Image System (Millipore, Bedford) and was expressed as a percentage of the IOD of the total protein including
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Al and Cd Effects on in Vitro Vitellogenesis
VTG. Minor subunits of VTG were not considered as VTG, because the subunits constituted only a fairly small part of VTG and overlapped with other proteins (Kwon et al., 1993). An excellent correlation (r ⫽ 0.99) was reported between the amount (100 –1600 ng/ml) of VTG applied to the electrophoretic lanes and the IOD (Yeo and Mugiya, 1997).
RNA Extraction Total RNA was extracted from cultured hepatocytes using an RNA extraction kit, Isogen (Nippon Gene), according to the acid guanidinium thiocyanate phenol chloroform method (Chomczynski and Sacchi, 1987). After homogenization of the hepatocytes in Isogen, the homogenate was centrifuged at 13,000g for 30 min. Chloroform was added to the supernatant and the mixture was centrifuged at 13,000g for 30 min. Then, 2-propanol was added to the supernatant and the mixture was centrifuged at 13,000g for 30 min. The pellet was washed in 80% ethanol, lysed with water as a total RNA sample, and stored at ⫺75°C for Northern blot analyses.
RT-PCR for VTG cDNA Rainbow trout were injected ip with estradiol-17  (5 mg/kg body weight) in 95% ethanol three times at 5-day intervals and total RNA (including VTG mRNA) was extracted from the liver, as described above, 7 days after the last injection. The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using an AMV RNA PCR kit (TaKaRa) by a DNA thermal cycler (Perkin-Elmer). Primers were chosen so as to include partial sequences of the rainbow trout VTG cDNA (Le Guellec et al., 1988). The upstream primer was a 20-mer sense oligonucleotide (5⬘-AGCAAACACAAAGTCAATGC-3⬘) and the downstream primer was a 20-mer antisense oligonucleotide (5⬘-TCCACACTCTTCTCATAGAT3⬘). Total RNA (1 g) was reverse-transcribed at 96°C for 10 min and then PCR amplifications were made for 35 cycles (92°C for 30 s, 55°C for 30 s, and 72°C for 30 s). A 1196-bp RT-PCR product was analyzed by electrophoresis on a 2% agarose gel and confirmed to be identical to that reported for rainbow trout VTG cDNA (Le Guellec et al., 1988) using a DNA sequencer (Model-373A, Applied Biosystems).
Northern Blotting Total RNA was quantified by UV spectrophotometry at 260 nm. Ten micrograms of each RNA sample was mixed with 10% 3-morpholinopropane sulfonic acid (Mops) buffer (200 mM Mops, 50 mM sodium acetate, 10 mM ethylenediamine-N,N,N⬘,N⬘-tetraacetic acid (EDTA), pH 7.0), 16.25% formaldehyde, and 50% formamide, and then was denatured at 65°C for 5 min. After mixing with a 1/10 vol of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromphenol blue), samples were applied to a 1% agarose-formaldehyde gel. After 1-h electrophoresis at 40 mA, the gel was stained with ethidium bromide for 25 min and bleached for 25 min with 200 mM sodium acetate solution (pH 4.0). Then, RNA was transferred overnight to polyvinylidene difluoride membranes (Millipore). The transfer buffer was 20⫻ saline sodium citrate (SSC: 333 mM NaCl, 333 mM sodium citrate, pH 7.0). The completion of transfer was confirmed by ultraviolet transillumination. After being dried and heated at 80°C for 2 h, the membranes were prehybridized with 10⫻ SSC containing 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.1 mg/ml calf thymus DNA at 65°C for 2 h. They were hybridized overnight under the above conditions with a random-primed 32P-labeled VTG probe (100,000 cpm/ml). The membranes were washed in 1⫻ SSC containing 0.1% SDS for 30 min and submitted to the quantification of VTG mRNA expression by autoradiography using a Bio Image System (BAS2000; Fujix, Tokyo).
Statistical Analysis Data were analyzed by one-way ANOVA (Fisher PLSD test). Fisher’s r test was also used to examine the significance of correlation coefficients. Significance was accepted at P ⬍ 0.05. Percentage data were statistically analyzed after being arcsine transformed.
RESULTS The collagenase perfusion method yielded about 3 ⫻ 10 8 hepatocytes per fish. Cell viability was estimated to be over 90% by trypan blue staining. After being transferred to a positively charged dish, hepa-
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Hwang, Kagawa, and Mugiya
the control culture, but this value decreased with increasing Al concentrations (Fig. 2). Significant inhibition was confirmed at Al concentrations of 5 ⫻ 10 ⫺5 and 10 ⫺4, in which the VTG production was reduced to 48% (P ⬍ 0.05) and 20% (P ⬍ 0.01) of the control, respectively. Cd had no effect on VTG production at concentrations of 10 ⫺9–10 ⫺7 M. However, it markedly inhibited the production at 10 ⫺6 M. Figure 3 shows the quantitative results. The inhibitory effect of Cd on VTG production was not concentration-dependent. VTG production was reduced to 34% of the control at 10 ⫺6 M (P ⬍ 0.01). FIG. 1. SDS-PAGE showing the inhibition of VTG (arrowhead) production by Al in hepatocyte cultures with E 2 in rainbow trout. Spent media were analyzed on Day 5 after Al addition. m, molecular weight (MW) marker. CBB stain.
tocytes firmly attached to the dish in the serum-free medium and started to spread within 2 days of preculture. Although hepatocytes maintained good viability for at least 15 days, they spread rather slowly and formed a few aggregations. When expressed as a percentage of the number of living cells on Day 5 after addition of the metal to the number of living cells just before addition, Al and Cd had no appreciable toxic effect on cell viability showing survival rates of 78 – 91% regardless of the concentrations used.
Effects of Al and Cd on VTG mRNA Expression After a 5-day culture with E 2 and Al or Cd, hepatocytes were analyzed by Northern blotting. VTG mRNA bands were detected at about 6.6 kb in cultures with E 2, while the culture without E 2 did not induce an equivalent band (Fig. 4). Al reduced the intensity of the VTG mRNA bands in a concentration-dependent way (Fig. 4). The level of VTG mRNA expression was quantified by autoradiography (Fig. 5). VTG mRNA expression decreased with increasing Al concentration (r ⫽ ⫺0.98, P ⬍ 0.01).
Effects of Al and Cd on VTG Production After a 2-day preculture, hepatocytes were incubated in the serum-free medium with E 2 and Al or Cd for 5 days and the spent medium was analyzed by SDS-PAGE. A newly synthesized protein band was detected at a molecular weight position of 175 kDa (Fig. 1). This band was identified as a main VTG band by Kwon et al. (1993) on the basis of immunoblotting and immunoelectrophoresis. The control culture without E 2 did not produce an equivalent protein. The addition of Al to the incubation medium reduced the intensity of CBB staining for the VTG band in a concentration-dependent fashion (Fig. 1). Protein bands other than VTG were less affected by Al. The relative amount of VTG was determined by IOD. VTG accounted for 10.3% of the total proteins in
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FIG. 2. Concentration-dependent inhibition of VTG production by Al in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG production was estimated as a percentage of VTG to total proteins after SDS-PAGE on Day 5 after Al addition. Vertical bars represent the SE of the mean for three individuals. *P ⬍ 0.05 and **P ⬍ 0.01 for control (E 2 ⫹ 0 M Al).
Al and Cd Effects on in Vitro Vitellogenesis
FIG. 3. Concentration-independent inhibition of VTG production by Cd in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG production was estimated as a percentage of VTG to total proteins after SDS-PAGE on Day 5 after Cd addition. Vertical bars represent the SE of the mean for three individuals. *P ⬍ 0.01 for control (E 2 ⫹ 0 M Cd).
Addition of Cd to the incubation medium did not change the intensity of VTG mRNA bands at concentrations of 10 ⫺9–10 ⫺7 M. However, the band intensity
FIG. 4. Northern blotting of VTG mRNA. (A) The inhibition of VTG mRNA (arrowhead) expression by Al in hepatocyte cultures with E 2 in rainbow trout. Hepatocytes were extracted for total RNA on Day 5 after Al addition. (B) Ribosome bands with 28 S (open arrowhead) and 18 S (arrowhead).
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FIG. 5. Concentration-dependent inhibition of VTG mRNA expression by Al in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG mRNA expression was estimated on Day 5 after Al addition by autoradiography of Northern blotting. The regression was significant (P ⬍ 0.05).
was markedly weakened at 10 ⫺6 M. Figure 6 shows these profiles quantitatively. Cd reduced VTG mRNA expression to 35% of the control at 10 ⫺6 M. However, a linear regression did not fit the data, showing no concentration dependency between Cd concentrations and VTG mRNA expression (r ⫽ ⫺0.82, P ⬎ 0.05).
FIG. 6. Concentration-independent inhibition of VTG mRNA expression by Cd in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG mRNA expression was estimated on Day 5 after Cd addition by autoradiography of Northern blotting. A linear regression did not fit the data and the correlation coefficient was not significant.
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FIG. 7. Recovery profiles of Al and Cd-induced inhibition of VTG production after removal of the metals. Rainbow trout hepatocytes were incubated with E 2 and Al (10 ⫺4 M) or Cd (10 ⫺6 M) for 3 days and then these metals were removed (Day 0). Incubation continued for another 7 days. The activity of VTG production was estimated as a percentage of VTG to total proteins after SDS-PAGE. Values are the means ⫾ SE of three fish.
Effects of Al and Cd Removal on VTG Production After a 3-day incubation with E 2 and Al (10 ⫺4 M) or Cd (10 ⫺6 M), these metals were removed and hepatocytes were further incubated with E 2 for 7 more days. VTG production in the control culture increased with time (Fig. 7). However, the Al-induced inhibition of VTG production remained unchanged until Day 4. Then it was restored slightly on Day 5 and reached 78% of the control on Day 7 after Al removal. This level was statistically equal to the level of the control. In contrast, VTG production was not restored by removal of Cd even on Day 7 (Fig. 7). VTG mRNA expression showed a recovery profile similar to that of the VTG production in both metals (data not shown).
DISCUSSION ELISA is a sensitive method for VTG determination and has been widely used for this purpose. In the present study, however, SDS-PAGE was used to separate VTG, before optical quantification. VTG produc-
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Hwang, Kagawa, and Mugiya
tion in the presence and absence of metals was expressed as a percentage of total proteins. This type of expression has the benefit of excluding the effects of variation in the number of cultured cells and in the amount of proteins applied to the gel. It also has the advantage of checking the effects of metals on the production of proteins other than VTG. A significant decrease in the percentage means that the synthesis of VTG is more susceptible to metals than are other hepatocyte-derived proteins. Al and Cd are nonessential biological elements and therefore fish may have no regulatory mechanisms for them. Cd predominantly exists as a free aquo ion (Cd 2⫹) in solution in the pH range 4 –7 (Campbell and Stokes, 1985), while the chemical form of Al depends on the pH. At neutral pH, it occurs as an Al(OH) 3 precipitate and/or an Al(OH) 4⫺-soluble form (Verbost et al., 1992). Because no precipitates were found during the present incubation, Al must exert its effect on vitellogenesis as an ion of Al(OH) 4⫺. Al and Cd are known to be toxic to fish, interfering with ion regulation (Verbost et al., 1987; Booth et al., 1988) and respiratory function (Karlsson-Norrgren et al., 1985; Wood et al., 1988) in the gill. Fish reproduction was also impaired by these metals (Haux et al., 1988; Rask et al., 1990). Al reaches high levels in acidic waters and aggravated unfavorable effects of low pH on reproduction. For example, the spawning of perch was delayed in acidic lakes with high Al levels (Rask et al., 1990). Further discussion about these aspects is available in Mugiya and Tanahashi (1998). In vivo exposure to Cd decreased both estrogen production and vitellogenesis (Singh et al., 1989; Pereira et al., 1993). Victor et al. (1986) reported that Cd caused ovarian lesions and suggested that the primary disruption was at the ovarian or pituitary level rather than on hepatic synthesis. Kim (1998) also stated that decreased plasma VTG due to Cd exposure does not necessarily demonstrate a disruption of hepatic function and is more likely to result from low ovarian estrogen secretion. The present in vitro study, however, demonstrated that this metal inhibits VTG mRNA expression in hepatocytes of rainbow trout. The present study showed that Al and Cd inhibited VTG production and VTG mRNA expression. The inhibition by Al was reversible, while that by Cd was not. Yao et al. (1996) found that Zn and Cu reversibly inhibited VTG production in goldfish, while the ef-
Al and Cd Effects on in Vitro Vitellogenesis
fects of Hg and Cd were irreversible. It was suggested that Zn and Cu acted at the receptor level to inhibit VTG gene activation by E 2, while Cd and Hg acted at the transcriptional level to reduce VTG mRNA production. Another difference in action of Al and Cd in the present study is that Al inhibited VTG production in a concentration-dependent manner, while Cd inhibited it discontinuously. Cd at concentrations of 10 ⫺9–10 ⫺7 M had no effect on VTG production, while it markedly inhibited VTG production at 10 ⫺6 M. Treatment of hepatocytes with Cd induced the synthesis of metallothioneins, which bind to and detoxify Cd (Roch and McCarter, 1984). Therefore, Cd might exert an inhibitory effect on VTG production when the amount of Cd added exceeded the hepatocytes’ ability to synthesize metallothioneins (McCarter et al., 1982). In conclusion, Al and Cd inhibited VTG production reversibly and irreversibly, respectively, at the transcriptional level.
ACKNOWLEDGMENT This study was financially supported in part by Grant 10460078 from the Ministry of Education of Japan.
REFERENCES Booth, C. E., McDonald, D. G., Simons, B. P., and Wood, C. M. (1988). Effects of aluminium and low pH on net ion fluxes and ion balance in the brook trout, Salvelinus fontinalis. Can. J. Fish. Aquat. Sci. 45, 1563–1574. Borg, H. (1986). Metal speciation in acidified mountain streams in central Sweden. Water Air Soil Pollut. 30, 1007–1014. Brumbaugh, W. G., and Kane, D. A. (1985). Variability of aluminium concentrations in organs and whole bodies of smallmouth bass (Micropterus dolomieui). Environ. Sci. Technol. 19, 828 – 831. Campbell, P. G. C., and Stokes, P. M. (1985). Acidification and toxicity of metals to aquatic biota. Can. J. Fish. Aquat. Sci. 42, 2034 –2049. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156 –159. Haux, C., Bjo¨rnsson, B. Th., Fo¨rlin, L., Larsson, Å., and Deftos, L. J. (1988). Influence of cadmium exposure on plasma calcium, vitel-
75 logenin and calcitonin in vitellogenic rainbow trout. Mar. Environ. Res. 24, 199 –202. Hayashi, S., and Ooshiro, Z. (1975). Gluconeogenesis and glycolysis in isolated perfused liver of the eel. Bull. Jpn. Soc. Sci. Fish. 41, 201–208. Karlsson-Norrgren, L., Runn, P., Haux, C., and Forlin, L. (1985). Cadmium-induced changes in gill morphology of zebrafish, Brachydanio rerio (Hamilton-Buchanan), and rainbow trout, Salmo gairdneri Richardson. J. Fish. Biol. 27, 81–95. Kim, D. E. (1998). “Endocrine Disruption in Fish.” Kluwer Academic, Boston. Kwon, H. C., Hayashi, S., and Mugiya, Y. (1993). Vitellogenin induction by estradiol-17  in primary hepatocyte culture in the rainbow trout, Oncorhynchus mykiss. Comp. Biochem. Physiol. B 104, 381–386. Le Guellec, K., Lawless, K., Valotaire, Y., Kress, M., and Tenniswood, M. (1988). Vitellogenin gene expression in male rainbow trout (Salmo gairdnerii). Gen. Comp. Endocrinol. 71, 359 –371. Lee, R. M., and Gerking, S. D. (1980). Survival and reproductive performance of the desert pupfish, Cyprinodon n. nevadensis (Eigenmann and Eigenmann), in acid water. J. Fish Biol. 17, 507– 515. McCarter, J. A., Matheson, A. T., Roch, M., Olatson, R. W., and Buckley, J. T. (1982). Chronic exposure of coho salmon to sublethal concentrations of copper. II. Distribution of copper between high- and low-molecular-weight proteins in liver cytosol and the possible role of metallothionein in detoxification. Comp. Biochem. Physiol. C 72, 21–26. Mugiya, Y., and Tanahashi, A. (1998). Inhibitory effects of aluminium on vitellogenin induction by estradiol-17  in the primary culture of hepatocytes in the rainbow trout Oncorhynchus mykiss. Gen. Comp. Endocrinol. 109, 37– 43. Olsson, P.-E., Kling, P., Petterson, C., and Silversand, C. (1995). Interaction of cadmium and oestradiol-17  on metallothionein and vitellogenin synthesis in rainbow trout (Oncorhynchus mykiss). Biochem. J. 307, 197–203. Pereira, J. J., Mercaldo-Allen, R., Kuropat, C., Luedke, D., and Sennefelder, G. (1993). Effect of cadmium accumulation on serum vitellogenin levels and hepatosomatic and gonadosomatic indices of winter flounder (Pleuronectes americanus). Arch. Environ. Contam. Toxicol. 24, 427– 431. Rask, M., Vuorinen, P. J., and Vuorinen, M. (1990). Delayed spawning of perch, Perca fluviatilis L., in acidified lakes. J. Fish. Biol. 36, 317–325. Roch, M., and McCarter, J. A. (1984). Hepatic metallothionein production and resistance to heavy metals by rainbow trout (Salmo gairdneri). I. Exposed to an artificial mixture of zinc, copper and cadmium. Comp. Biochem. Physiol. C 77, 71–75. Runn, P., Johansson, N., and Milbrink, G. (1977). Some effects of low pH on the hatchability of eggs of perch, Perca fluviatilis L. Zoon 5, 115–125. Singh, T. P., Yadav, A. K., Lal, B., and Singh, S. (1989). Impact of pesticide on fish reproduction: A review. Sci. Res. J. 1, 9 –20. Verbost, P. M., Flick, G., Lock, R. A. C., and Wendelaar Bonga, S. E. (1987). Cadmium inhibition of Ca 2⫹ uptake in rainbow trout gills. Am. J. Physiol. 253, R216 –R221.
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76 Verbost, P. M., Lafeber, F. P. J. G., Spanings, F. A. T., Aarden, E. M., and Wendelaar Bonga, S. E. (1992). Inhibition of Ca 2⫹ uptake in freshwater carp, Cyprinus carpio, during short-term exposure to aluminum. J. Exp. Zool. 262, 247–254. Victor, B., Mahalingam, S., and Sarojini, R. (1986). Toxicity of mercury and cadmium on oocyte differentiation and vitellogenesis of the teleost, Lepidocephalichthys thermalis (Bleeker). J. Environ. Biol. 7, 209 –214. Wood, C. M., Playle, R. C., Simons, B. P., Goss, G. G., and McDonald, D. G. (1988). Blood gases, acid-base status, ions, and
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Hwang, Kagawa, and Mugiya
hematology in adult brook trout, Salvelinus fontinalis, under acid/ aluminum exposure. Can. J. Fish. Aquat. Sci. 45, 1575–1586. Yao, Z., Van der Kraak, G. J., and Squires, E. J. (1996). Effect of heavy metals on in vitro vitellogenin production from cultured hepatocytes of goldfish, Proc. 3rd Int. Symp. Fish Endocrinol. 27–31 May, 1996, Hakodate, p. 172. Yeo, I. Y., and Mugiya, Y. (1997). Effects of extracellular calcium concentrations and calcium antagonists on vitellogenin induction by estradiol-17  in primary hepatocyte culture in the rainbow trout Oncorhynchus mykiss. Gen. Comp. Endocrinol. 105, 294 –301.
Aluminium and Cadmium Inhibit Vitellogenin and Its mRNA Induction by Estradiol-17  in the Primary Culture of Hepatocytes in the Rainbow Trout Oncorhynchus mykiss Un-Gi Hwang, Nao Kagawa, and Yasuo Mugiya Laboratory of Comparative Physiology, Faculty of Fisheries, Hokkaido University, 3-1-1 Minato, Hakodate 041-8611, Japan Accepted March 23, 2000
such metals that impaired vitellogenin (VTG) production. VTG, an egg yolk precursor protein, is synthesized in the liver in response to estrogen and is incorporated into eggs to be processed as yolk proteins in maturing females. Impaired oogenesis due to poor accumulation of egg yolk (Lee and Gerking, 1980) and a reduction in egg number (Runn et al., 1977) have been reported in acidic waters, where Al reaches high concentrations of 3.7–34.8 M (Brumbaugh and Kane, 1985; Borg, 1986). Mugiya and Tanahashi (1998) showed directly that Al inhibited the induction of VTG synthesis in hepatocyte culture of rainbow trout. However, the intracellular mechanisms of inhibition are unknown. Cd is another metal that has been reported to inhibit the in vivo induction of VTG synthesis. Exposure to Cd decreased plasma VTG (alkaline-labile phosphate) concentrations in maturing winter flounder (Pereira et al., 1993). Coinjection of Cd with estrogen resulted in decreased plasma protein (mostly VTG) levels (Olsson et al., 1995) and it was suggested that Cd inhibited the transcription of VTG. Hepatocyte culture is an excellent system to confirm and analyze these in vivo results directly. The present study was undertaken to determine at which levels these metals interfere with the synthesis of VTG in rainbow trout. Hepatocytes were cultured with a combination of estradiol and Al or Cd, and
Effects of Al and Cd on vitellogenin (VTG) and VTG mRNA induction by estradiol-17  (E 2) were examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then E 2 (2 ⴛ 10 ⴚ6 M) and Al (10 ⴚ6–10 ⴚ4 M) or Cd (10 ⴚ9–10 ⴚ6 M) were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days. Media and hepatocytes were then analyzed by SDS-PAGE and Northern blotting for VTG and VTG mRNA, respectively. These metals had no appreciable effect on the viability of hepatocytes in culture. However, Al and Cd interfered with VTG production and VTG mRNA expression. Al reduced VTG production in a concentration-dependent way and a significant reduction occurred at Al concentrations greater than 5 ⴛ 10 ⴚ5 M. VTG mRNA expression also decreased with a negative correlation with Al concentrations (r ⴝ ⴚ0.98). The inhibition of VTG production by Cd was not concentration-dependent. This metal markedly inhibited VTG production and VTG mRNA expression at 10 ⴚ6 M. The Al-induced inhibition of VTG production was restored 7 days after Al removal, but the Cdinduced inhibition was not restored. These results suggest that Al and Cd inhibit VTG production at the transcriptional level to reduce VTG mRNA expression by different mechanisms. © 2000 Academic Press Metal contaminants in the aquatic environment may interfere with reproduction in fish. Al and Cd are two 0016-6480/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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VTG production and VTG mRNA expression were analyzed by electrophoresis and Northern blotting, respectively. Recovery experiments following removal of these metals were also conducted to clarify whether metal-induced inhibition is reversible.
MATERIALS AND METHODS Rainbow trout, Oncorhynchus mykiss, weighing 110 – 350 g were obtained from a commercial dealer and kept in outdoor ponds with running water at about 14°C. They were fed trout food pellets once a day but were starved on the last day before sampling to reduce the production of bile. Maturing females were not included.
Hwang, Kagawa, and Mugiya
ture, the media and hepatocytes were analyzed for VTG protein and VTG mRNA, respectively. The effects of Al and Cd on cell viability were also examined using crystal violet as described by Mugiya and Tanahashi (1998). Cell viability was taken as the number of living cells on Day 5 expressed as a percentage of the number of living cells just before metal addition. Treatment 2. To examine the effects of Al and Cd removal on the metal-induced inhibition of VTG production, hepatocytes were cultured with E 2 and Al (10 ⫺4 M) or Cd (10 ⫺6 M) for 3 days and then these metals were removed from the media (defined as Day 0). The cultures were continued for another 7 days. Media samples were taken for analysis on Days 0, 1, 2, 3, 4, 5, and 7.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) Hepatocyte Preparation and Incubation Hepatocytes were prepared following Hayashi and Ooshiro (1975) as described by Kwon et al. (1993). Cell yield and viability were determined by the trypan blue exclusion test. Cells were plated into a 60-mm plastic petri dish with a positive charge (Falcon) at a density of 0.5–1 ⫻ 10 6 cells/dish. William’s medium E (Life Technologies, Inc.) containing 0.2 M bovine insulin (Sigma, St. Louis, MO), streptomycin (100 g/ml), and penicillin (70 g/ml) was used for cell culture. All incubations were carried out in 3 ml of the medium at 15°C under 5% CO 2. Preculture was conducted for 2 days before each experiment. The whole medium was changed every day throughout the preculture and experimental periods.
Electrophoresis was done as described by Mugiya and Tanahashi (1998). Briefly, proteins were precipitated from the media by cold trichloroacetic acid, dissolved in sample buffer (0.175 M Tris-HCl, 8 M urea, 1% SDS, and 0.5% 2-mercaptoethanol, pH 7.4), and subjected to 5–20% gradient SDS-PAGE. The gels were stained with 0.25% Coomassie brilliant blue R-250 (CBB). It was difficult to apply a fixed amount of proteins to each lane, because the present experiment was intended to inhibit VTG (protein) synthesis. Standard proteins used for molecular weight (MW) determinations were carbonic anhydrase (MW 29,000), ovalbumin (45,000), bovine serum albumin (66,000), phosphorylase b (97,400), -galactosidase (116,000), and myosin (205,000).
Qualitative and Quantitative Analyses of VTG Treatment with Al and Cd Treatment 1. After a 2-day preculture, estradiol-17  (E 2, 2 ⫻ 10 ⫺6 M in 3 l of 95% ethanol) and Al (AlCl 3 in 3 l of redistilled water) or Cd (CdCl 2 in 3 l of redistilled water) were simultaneously added to the dishes. Final metal concentrations were 10 ⫺6, 10 ⫺5, 5 ⫻ 10 ⫺5, and 10 ⫺4 M for Al and 10 ⫺9, 10 ⫺8, 10 ⫺7, and 10 ⫺6 M for Cd. The effects of these metals on VTG production were examined after 5 days, during which time the media were changed daily. Control cultures received the equivalent amount of the solvents only. After cul-
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The identification of the VTG band was based on the results of a previous study (Kwon et al., 1993) in which isolated rainbow trout hepatocytes incubated with E 2 synthesized a protein with the same electrophoretic mobility (175 kDa) as in the present study. Kwon et al. identified this band as VTG (main band) by immunoblot analysis and immunoelectrophoresis. After SDS-PAGE, the integrated optical density (IOD) of the VTG band was measured by a Bio Image System (Millipore, Bedford) and was expressed as a percentage of the IOD of the total protein including
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Al and Cd Effects on in Vitro Vitellogenesis
VTG. Minor subunits of VTG were not considered as VTG, because the subunits constituted only a fairly small part of VTG and overlapped with other proteins (Kwon et al., 1993). An excellent correlation (r ⫽ 0.99) was reported between the amount (100 –1600 ng/ml) of VTG applied to the electrophoretic lanes and the IOD (Yeo and Mugiya, 1997).
RNA Extraction Total RNA was extracted from cultured hepatocytes using an RNA extraction kit, Isogen (Nippon Gene), according to the acid guanidinium thiocyanate phenol chloroform method (Chomczynski and Sacchi, 1987). After homogenization of the hepatocytes in Isogen, the homogenate was centrifuged at 13,000g for 30 min. Chloroform was added to the supernatant and the mixture was centrifuged at 13,000g for 30 min. Then, 2-propanol was added to the supernatant and the mixture was centrifuged at 13,000g for 30 min. The pellet was washed in 80% ethanol, lysed with water as a total RNA sample, and stored at ⫺75°C for Northern blot analyses.
RT-PCR for VTG cDNA Rainbow trout were injected ip with estradiol-17  (5 mg/kg body weight) in 95% ethanol three times at 5-day intervals and total RNA (including VTG mRNA) was extracted from the liver, as described above, 7 days after the last injection. The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using an AMV RNA PCR kit (TaKaRa) by a DNA thermal cycler (Perkin-Elmer). Primers were chosen so as to include partial sequences of the rainbow trout VTG cDNA (Le Guellec et al., 1988). The upstream primer was a 20-mer sense oligonucleotide (5⬘-AGCAAACACAAAGTCAATGC-3⬘) and the downstream primer was a 20-mer antisense oligonucleotide (5⬘-TCCACACTCTTCTCATAGAT3⬘). Total RNA (1 g) was reverse-transcribed at 96°C for 10 min and then PCR amplifications were made for 35 cycles (92°C for 30 s, 55°C for 30 s, and 72°C for 30 s). A 1196-bp RT-PCR product was analyzed by electrophoresis on a 2% agarose gel and confirmed to be identical to that reported for rainbow trout VTG cDNA (Le Guellec et al., 1988) using a DNA sequencer (Model-373A, Applied Biosystems).
Northern Blotting Total RNA was quantified by UV spectrophotometry at 260 nm. Ten micrograms of each RNA sample was mixed with 10% 3-morpholinopropane sulfonic acid (Mops) buffer (200 mM Mops, 50 mM sodium acetate, 10 mM ethylenediamine-N,N,N⬘,N⬘-tetraacetic acid (EDTA), pH 7.0), 16.25% formaldehyde, and 50% formamide, and then was denatured at 65°C for 5 min. After mixing with a 1/10 vol of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromphenol blue), samples were applied to a 1% agarose-formaldehyde gel. After 1-h electrophoresis at 40 mA, the gel was stained with ethidium bromide for 25 min and bleached for 25 min with 200 mM sodium acetate solution (pH 4.0). Then, RNA was transferred overnight to polyvinylidene difluoride membranes (Millipore). The transfer buffer was 20⫻ saline sodium citrate (SSC: 333 mM NaCl, 333 mM sodium citrate, pH 7.0). The completion of transfer was confirmed by ultraviolet transillumination. After being dried and heated at 80°C for 2 h, the membranes were prehybridized with 10⫻ SSC containing 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.1 mg/ml calf thymus DNA at 65°C for 2 h. They were hybridized overnight under the above conditions with a random-primed 32P-labeled VTG probe (100,000 cpm/ml). The membranes were washed in 1⫻ SSC containing 0.1% SDS for 30 min and submitted to the quantification of VTG mRNA expression by autoradiography using a Bio Image System (BAS2000; Fujix, Tokyo).
Statistical Analysis Data were analyzed by one-way ANOVA (Fisher PLSD test). Fisher’s r test was also used to examine the significance of correlation coefficients. Significance was accepted at P ⬍ 0.05. Percentage data were statistically analyzed after being arcsine transformed.
RESULTS The collagenase perfusion method yielded about 3 ⫻ 10 8 hepatocytes per fish. Cell viability was estimated to be over 90% by trypan blue staining. After being transferred to a positively charged dish, hepa-
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72
Hwang, Kagawa, and Mugiya
the control culture, but this value decreased with increasing Al concentrations (Fig. 2). Significant inhibition was confirmed at Al concentrations of 5 ⫻ 10 ⫺5 and 10 ⫺4, in which the VTG production was reduced to 48% (P ⬍ 0.05) and 20% (P ⬍ 0.01) of the control, respectively. Cd had no effect on VTG production at concentrations of 10 ⫺9–10 ⫺7 M. However, it markedly inhibited the production at 10 ⫺6 M. Figure 3 shows the quantitative results. The inhibitory effect of Cd on VTG production was not concentration-dependent. VTG production was reduced to 34% of the control at 10 ⫺6 M (P ⬍ 0.01). FIG. 1. SDS-PAGE showing the inhibition of VTG (arrowhead) production by Al in hepatocyte cultures with E 2 in rainbow trout. Spent media were analyzed on Day 5 after Al addition. m, molecular weight (MW) marker. CBB stain.
tocytes firmly attached to the dish in the serum-free medium and started to spread within 2 days of preculture. Although hepatocytes maintained good viability for at least 15 days, they spread rather slowly and formed a few aggregations. When expressed as a percentage of the number of living cells on Day 5 after addition of the metal to the number of living cells just before addition, Al and Cd had no appreciable toxic effect on cell viability showing survival rates of 78 – 91% regardless of the concentrations used.
Effects of Al and Cd on VTG mRNA Expression After a 5-day culture with E 2 and Al or Cd, hepatocytes were analyzed by Northern blotting. VTG mRNA bands were detected at about 6.6 kb in cultures with E 2, while the culture without E 2 did not induce an equivalent band (Fig. 4). Al reduced the intensity of the VTG mRNA bands in a concentration-dependent way (Fig. 4). The level of VTG mRNA expression was quantified by autoradiography (Fig. 5). VTG mRNA expression decreased with increasing Al concentration (r ⫽ ⫺0.98, P ⬍ 0.01).
Effects of Al and Cd on VTG Production After a 2-day preculture, hepatocytes were incubated in the serum-free medium with E 2 and Al or Cd for 5 days and the spent medium was analyzed by SDS-PAGE. A newly synthesized protein band was detected at a molecular weight position of 175 kDa (Fig. 1). This band was identified as a main VTG band by Kwon et al. (1993) on the basis of immunoblotting and immunoelectrophoresis. The control culture without E 2 did not produce an equivalent protein. The addition of Al to the incubation medium reduced the intensity of CBB staining for the VTG band in a concentration-dependent fashion (Fig. 1). Protein bands other than VTG were less affected by Al. The relative amount of VTG was determined by IOD. VTG accounted for 10.3% of the total proteins in
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FIG. 2. Concentration-dependent inhibition of VTG production by Al in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG production was estimated as a percentage of VTG to total proteins after SDS-PAGE on Day 5 after Al addition. Vertical bars represent the SE of the mean for three individuals. *P ⬍ 0.05 and **P ⬍ 0.01 for control (E 2 ⫹ 0 M Al).
Al and Cd Effects on in Vitro Vitellogenesis
FIG. 3. Concentration-independent inhibition of VTG production by Cd in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG production was estimated as a percentage of VTG to total proteins after SDS-PAGE on Day 5 after Cd addition. Vertical bars represent the SE of the mean for three individuals. *P ⬍ 0.01 for control (E 2 ⫹ 0 M Cd).
Addition of Cd to the incubation medium did not change the intensity of VTG mRNA bands at concentrations of 10 ⫺9–10 ⫺7 M. However, the band intensity
FIG. 4. Northern blotting of VTG mRNA. (A) The inhibition of VTG mRNA (arrowhead) expression by Al in hepatocyte cultures with E 2 in rainbow trout. Hepatocytes were extracted for total RNA on Day 5 after Al addition. (B) Ribosome bands with 28 S (open arrowhead) and 18 S (arrowhead).
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FIG. 5. Concentration-dependent inhibition of VTG mRNA expression by Al in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG mRNA expression was estimated on Day 5 after Al addition by autoradiography of Northern blotting. The regression was significant (P ⬍ 0.05).
was markedly weakened at 10 ⫺6 M. Figure 6 shows these profiles quantitatively. Cd reduced VTG mRNA expression to 35% of the control at 10 ⫺6 M. However, a linear regression did not fit the data, showing no concentration dependency between Cd concentrations and VTG mRNA expression (r ⫽ ⫺0.82, P ⬎ 0.05).
FIG. 6. Concentration-independent inhibition of VTG mRNA expression by Cd in hepatocyte cultures with E 2 in rainbow trout. The activity of VTG mRNA expression was estimated on Day 5 after Cd addition by autoradiography of Northern blotting. A linear regression did not fit the data and the correlation coefficient was not significant.
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74
FIG. 7. Recovery profiles of Al and Cd-induced inhibition of VTG production after removal of the metals. Rainbow trout hepatocytes were incubated with E 2 and Al (10 ⫺4 M) or Cd (10 ⫺6 M) for 3 days and then these metals were removed (Day 0). Incubation continued for another 7 days. The activity of VTG production was estimated as a percentage of VTG to total proteins after SDS-PAGE. Values are the means ⫾ SE of three fish.
Effects of Al and Cd Removal on VTG Production After a 3-day incubation with E 2 and Al (10 ⫺4 M) or Cd (10 ⫺6 M), these metals were removed and hepatocytes were further incubated with E 2 for 7 more days. VTG production in the control culture increased with time (Fig. 7). However, the Al-induced inhibition of VTG production remained unchanged until Day 4. Then it was restored slightly on Day 5 and reached 78% of the control on Day 7 after Al removal. This level was statistically equal to the level of the control. In contrast, VTG production was not restored by removal of Cd even on Day 7 (Fig. 7). VTG mRNA expression showed a recovery profile similar to that of the VTG production in both metals (data not shown).
DISCUSSION ELISA is a sensitive method for VTG determination and has been widely used for this purpose. In the present study, however, SDS-PAGE was used to separate VTG, before optical quantification. VTG produc-
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Hwang, Kagawa, and Mugiya
tion in the presence and absence of metals was expressed as a percentage of total proteins. This type of expression has the benefit of excluding the effects of variation in the number of cultured cells and in the amount of proteins applied to the gel. It also has the advantage of checking the effects of metals on the production of proteins other than VTG. A significant decrease in the percentage means that the synthesis of VTG is more susceptible to metals than are other hepatocyte-derived proteins. Al and Cd are nonessential biological elements and therefore fish may have no regulatory mechanisms for them. Cd predominantly exists as a free aquo ion (Cd 2⫹) in solution in the pH range 4 –7 (Campbell and Stokes, 1985), while the chemical form of Al depends on the pH. At neutral pH, it occurs as an Al(OH) 3 precipitate and/or an Al(OH) 4⫺-soluble form (Verbost et al., 1992). Because no precipitates were found during the present incubation, Al must exert its effect on vitellogenesis as an ion of Al(OH) 4⫺. Al and Cd are known to be toxic to fish, interfering with ion regulation (Verbost et al., 1987; Booth et al., 1988) and respiratory function (Karlsson-Norrgren et al., 1985; Wood et al., 1988) in the gill. Fish reproduction was also impaired by these metals (Haux et al., 1988; Rask et al., 1990). Al reaches high levels in acidic waters and aggravated unfavorable effects of low pH on reproduction. For example, the spawning of perch was delayed in acidic lakes with high Al levels (Rask et al., 1990). Further discussion about these aspects is available in Mugiya and Tanahashi (1998). In vivo exposure to Cd decreased both estrogen production and vitellogenesis (Singh et al., 1989; Pereira et al., 1993). Victor et al. (1986) reported that Cd caused ovarian lesions and suggested that the primary disruption was at the ovarian or pituitary level rather than on hepatic synthesis. Kim (1998) also stated that decreased plasma VTG due to Cd exposure does not necessarily demonstrate a disruption of hepatic function and is more likely to result from low ovarian estrogen secretion. The present in vitro study, however, demonstrated that this metal inhibits VTG mRNA expression in hepatocytes of rainbow trout. The present study showed that Al and Cd inhibited VTG production and VTG mRNA expression. The inhibition by Al was reversible, while that by Cd was not. Yao et al. (1996) found that Zn and Cu reversibly inhibited VTG production in goldfish, while the ef-
Al and Cd Effects on in Vitro Vitellogenesis
fects of Hg and Cd were irreversible. It was suggested that Zn and Cu acted at the receptor level to inhibit VTG gene activation by E 2, while Cd and Hg acted at the transcriptional level to reduce VTG mRNA production. Another difference in action of Al and Cd in the present study is that Al inhibited VTG production in a concentration-dependent manner, while Cd inhibited it discontinuously. Cd at concentrations of 10 ⫺9–10 ⫺7 M had no effect on VTG production, while it markedly inhibited VTG production at 10 ⫺6 M. Treatment of hepatocytes with Cd induced the synthesis of metallothioneins, which bind to and detoxify Cd (Roch and McCarter, 1984). Therefore, Cd might exert an inhibitory effect on VTG production when the amount of Cd added exceeded the hepatocytes’ ability to synthesize metallothioneins (McCarter et al., 1982). In conclusion, Al and Cd inhibited VTG production reversibly and irreversibly, respectively, at the transcriptional level.
ACKNOWLEDGMENT This study was financially supported in part by Grant 10460078 from the Ministry of Education of Japan.
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76 Verbost, P. M., Lafeber, F. P. J. G., Spanings, F. A. T., Aarden, E. M., and Wendelaar Bonga, S. E. (1992). Inhibition of Ca 2⫹ uptake in freshwater carp, Cyprinus carpio, during short-term exposure to aluminum. J. Exp. Zool. 262, 247–254. Victor, B., Mahalingam, S., and Sarojini, R. (1986). Toxicity of mercury and cadmium on oocyte differentiation and vitellogenesis of the teleost, Lepidocephalichthys thermalis (Bleeker). J. Environ. Biol. 7, 209 –214. Wood, C. M., Playle, R. C., Simons, B. P., Goss, G. G., and McDonald, D. G. (1988). Blood gases, acid-base status, ions, and
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