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A fish hepatocyte model for the investigation of the effects of environmental contaminants
Marine Environmental Research 24 (1988) 141-145
A Fish Hepatocyte Model for the Investigation of the Effects of Environmental Contaminants
Sandra M. Baksi & John M. Frazier The Johns Hopkins University, School of Hygiene and Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA
An isolated fish hepatocyte culture system was developed as a model system to investigate the mechanisms of action of environmental contaminants. Hepatocytes were isolated from striped bass (Morone saxatilis) by an adaptation of the two stage perfusion technique of Seglen. 1 The system was used to evaluate metal binding protein ( M B P ) induction in response to cadmium, a primary inducer of metallothionein ( MT) in rat hepatocytes. Striped bass hepatocytes appeared to be refractor), to the induction of MBP by cadmium, since there was no significant increase in the synthesis of MBP .for an)' of the doses at any of the time points investigated. However, when a similar experiment was performed using rat hepatocytes there was induction of MBP that was related to both dose and time. These comparative experiments indicate that although there are similarities between the hepatocytes of the two species in regard to 3sS incorporation into low molecular weight metal-binding proteins, there appear to be significant quantitative differences as well in regard to MBP kinetics. This in vitro model system could potentially be utilized to investigate the toxicological properties of other environmental contaminants.
A n isolated fish hepatocyte culture system was developed as a model system to investigate the mechanisms o f action of environmental contaminants. Hepatocytes were isolated from striped bass (Morone saxatilis) by a retrograde two-stage perfusion m e t h o d modified from Seglen.X A cannula w a s inserted below the heart into the sinus venosus and the hepatic portal veins were severed to allow clearance o f blood from the liver. The i n i t i a l perfusion media was a HEPES-buffered Hanks' calcium and magnesiumfree physiological salt solution (pH 7.4) containing E G T A (1.2mM). The 141 Marine Environ. Res. 0141-1136/88/$03"50 © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
142
Sandra M. Baksi, John M. Frazier
initial perfusion was performed at room temperature. After 15 min the liver was then perfused with a HEPES-buffered Hanks' solution containing collagenase (0"5 mg/ml) and calcium (5-1 mM) at 30°C. After perfusing for 15 min the liver was carefully excised and the hepatocytes dispersed in initial perfusion media (without EGTA). Under the conditions employed, approximately 300-600 × 106 cells per liver were obtained with 90-95% viability as assessed by trypan blue exclusion. Isolated hepatocytes were incubated at a density of 3 × 106 cells/ml in R P M I 1640 media supplemented with 1% bovine serum albumin. This culture system was used to evaluate metal-binding protein (MBP) induction in response to cadmium, a primary inducer of metallothionein (MT) in mammalian hepatocytes. Following a 30 min exposure to 0, 5, or 10 pM cadmium, striped bass hepatocytes were washed with cadmium-free media, then incubated for 1, 3 or 6 h in the presence of 35S-cysteine. Cells were harvested, lysed, and the cytosol chromatographed on an SG-75 column. An aliquot of each fraction was analyzed by liquid scintillation counting, and 35S incorporation into MBP was computed. There was no appreciable increase in the level of metal-binding protein for any of the doses at any of the time points. However, at the 5 pM dose at 6 h there was a slight elevation of MBP indicating minimal induction of MPB (Fig. 1). One possible explanation for the lack of response in striped bass hepatocytes was that the doses were inappropriate to induce metal-binding protein synthesis. To determine if the concentrations in the preliminary experiments were too low, an experiment was performed that involved exposure of striped bass hepatocytes to a series of concentrations of cadmium (0-150pM) and the determination of metal binding protein synthesis by the 35S-cysteine incorporation method. After a 6 h incubation there was no significant increase in 35S incorporation into metal-binding proteins at any of the doses, although .there was a dose-related decrease in cell viability. It had been established in previous studies that in rat hepatocytes exposed to cadmium there was an increase in metallothionein at the incubation times used in the fish experiment. 2 In order to verify that the lack of response observed in striped bass hepatocytes was genuine and not a problem with culture conditions or techniques, a similar experiment was performed with rat hepatocytes. There was a significant induction of MT in rat hepatocytes that was related to time. This is evidenced by the 5.6-fold increase in 35S incorporation into MT in hepatocytes exposed to 5/~M cadmium for 6 h (Fig. 2). This indicated that there was not a problem with experimental techniques, as the 3SS-cysteine incorporation method for assessing metalbinding protein levels gave good results when used with rat hepatocytes. These experiments indicated that fish hepatocytes are more refractory to MBP induction by cadmium than rat hepatocytes under the incubation
A fish model.]'or investigation o/: environmental contaminants
143
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Fig. 1. In vitro induction of metal-binding proteins in striped bass hepatocytes exposed to 0 or 5/~M cadmium for 30 min and incubated in cadmium-free media for (a) 3 h or (b) 6 h. Metalbinding proteins of interest are located in fractions 30-40.
conditions used in the experiments. This response pattern was also observed in striped bass exposed to cadmium in vivo. There was no increase in the level of metal-binding protein as measured by 35S-cysteine incorporation 24 h after a single i.m. injection of cadmium (2.2 mg/kg). However, 24 h after the last of three successive daily injections there was a significant induction of MBP in treated fish when compared to controls (1.6-fold) or sham (1.5-fold) injected fish. This suggests that the kinetics of MBP induction in fish in vivo is different from that observed in rats, i.e. requires longer time. This hypothesis
144
Sandra M. Baksi, John M. Frazier
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Fig. 2. In vitro induction of metal-binding proteins in rat hepatocytes exposed to 5 #M cadmium for 15 rain and incubated in cadmium-free media for 1 or 6 h. Metallothionein is located in fractions 29 39.
is consistent with the results obtained in the short-term experiments done in vitro.
There are several possible explanations for the observed differences in responses between rats and fish. There may be differences between isolated hepatocytes of the two species in the rate of uptake of metals from the culture media. This could affect the amount of metal that actually enters the cell during the pulse exposure to cadmium. Once the metal is inside the cell, there may be differences in intracellular distribution and the binding of the metal to intracellular proteins. The regulation of the synthesis of metalbinding proteins in aquatic species is not as well understood as it is in mammalian systems. There may be differences in the control of MBP synthesis at the m R N A level. The results in these studies could be due to differences in the rate of transcription of the m R N A for MBP, or in the rate of translation of the MBP m R N A . In addition, little is known about the effect of pre-existing MBP on the intracellular kinetics of metals in fish hepatocytes. In rat hepatocytes it has been observed that the existence of high levels of intracellular Z n - M T significantly supresses the induction of new MT synthesis by cadmium (Din and Frazier, unpublished results). Another possible explanation is that the process of MBP induction is sensitive to temperature, and the differences observed are related to the incubation temperature, which was 37°C for rat hepatocytes and 22°C for
A fish model for investigation of environmental contaminants
145
striped bass hepatocytes. The latter hypothesis is presently being investigated. In conclusion, striped bass hepatocytes can be successfully used to investigate many of the toxicological problems for which mammalian hepatocytes are presently being utilized, including metal regulation. Although there are similarities between the hepatocytes of the two species there appear to be differences as well. Fish hepatocytes appear to be much more refractory to the effects to cadmium than rat hepatocytes under similar culture conditions. Understanding the mechanisms behind these differences would provide information concerning the practicality of extrapolating toxicological data between teleost and mammalian species.
REFERENCES 1. Seglen, P. O. Methods Cell Biol., 13, 29 (1975). 2. Frazier, J. M. & Din, W. S. In Safety Evaluation and Regulation of Chemicals (F. Hamburger, ed.) Karger, New York, pp. 295-308, 1985.
A Fish Hepatocyte Model for the Investigation of the Effects of Environmental Contaminants
Sandra M. Baksi & John M. Frazier The Johns Hopkins University, School of Hygiene and Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA
An isolated fish hepatocyte culture system was developed as a model system to investigate the mechanisms of action of environmental contaminants. Hepatocytes were isolated from striped bass (Morone saxatilis) by an adaptation of the two stage perfusion technique of Seglen. 1 The system was used to evaluate metal binding protein ( M B P ) induction in response to cadmium, a primary inducer of metallothionein ( MT) in rat hepatocytes. Striped bass hepatocytes appeared to be refractor), to the induction of MBP by cadmium, since there was no significant increase in the synthesis of MBP .for an)' of the doses at any of the time points investigated. However, when a similar experiment was performed using rat hepatocytes there was induction of MBP that was related to both dose and time. These comparative experiments indicate that although there are similarities between the hepatocytes of the two species in regard to 3sS incorporation into low molecular weight metal-binding proteins, there appear to be significant quantitative differences as well in regard to MBP kinetics. This in vitro model system could potentially be utilized to investigate the toxicological properties of other environmental contaminants.
A n isolated fish hepatocyte culture system was developed as a model system to investigate the mechanisms o f action of environmental contaminants. Hepatocytes were isolated from striped bass (Morone saxatilis) by a retrograde two-stage perfusion m e t h o d modified from Seglen.X A cannula w a s inserted below the heart into the sinus venosus and the hepatic portal veins were severed to allow clearance o f blood from the liver. The i n i t i a l perfusion media was a HEPES-buffered Hanks' calcium and magnesiumfree physiological salt solution (pH 7.4) containing E G T A (1.2mM). The 141 Marine Environ. Res. 0141-1136/88/$03"50 © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
142
Sandra M. Baksi, John M. Frazier
initial perfusion was performed at room temperature. After 15 min the liver was then perfused with a HEPES-buffered Hanks' solution containing collagenase (0"5 mg/ml) and calcium (5-1 mM) at 30°C. After perfusing for 15 min the liver was carefully excised and the hepatocytes dispersed in initial perfusion media (without EGTA). Under the conditions employed, approximately 300-600 × 106 cells per liver were obtained with 90-95% viability as assessed by trypan blue exclusion. Isolated hepatocytes were incubated at a density of 3 × 106 cells/ml in R P M I 1640 media supplemented with 1% bovine serum albumin. This culture system was used to evaluate metal-binding protein (MBP) induction in response to cadmium, a primary inducer of metallothionein (MT) in mammalian hepatocytes. Following a 30 min exposure to 0, 5, or 10 pM cadmium, striped bass hepatocytes were washed with cadmium-free media, then incubated for 1, 3 or 6 h in the presence of 35S-cysteine. Cells were harvested, lysed, and the cytosol chromatographed on an SG-75 column. An aliquot of each fraction was analyzed by liquid scintillation counting, and 35S incorporation into MBP was computed. There was no appreciable increase in the level of metal-binding protein for any of the doses at any of the time points. However, at the 5 pM dose at 6 h there was a slight elevation of MBP indicating minimal induction of MPB (Fig. 1). One possible explanation for the lack of response in striped bass hepatocytes was that the doses were inappropriate to induce metal-binding protein synthesis. To determine if the concentrations in the preliminary experiments were too low, an experiment was performed that involved exposure of striped bass hepatocytes to a series of concentrations of cadmium (0-150pM) and the determination of metal binding protein synthesis by the 35S-cysteine incorporation method. After a 6 h incubation there was no significant increase in 35S incorporation into metal-binding proteins at any of the doses, although .there was a dose-related decrease in cell viability. It had been established in previous studies that in rat hepatocytes exposed to cadmium there was an increase in metallothionein at the incubation times used in the fish experiment. 2 In order to verify that the lack of response observed in striped bass hepatocytes was genuine and not a problem with culture conditions or techniques, a similar experiment was performed with rat hepatocytes. There was a significant induction of MT in rat hepatocytes that was related to time. This is evidenced by the 5.6-fold increase in 35S incorporation into MT in hepatocytes exposed to 5/~M cadmium for 6 h (Fig. 2). This indicated that there was not a problem with experimental techniques, as the 3SS-cysteine incorporation method for assessing metalbinding protein levels gave good results when used with rat hepatocytes. These experiments indicated that fish hepatocytes are more refractory to MBP induction by cadmium than rat hepatocytes under the incubation
A fish model.]'or investigation o/: environmental contaminants
143
76 24 2.2 2
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0.4 0.2 £,
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CONTROL -
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FRACTION NUM~I~R + S" ~M CADMIUM - 3 H
3 ZP~ 25 24 2~ 0 I
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2 ~S I $
O. 0
I 4
1 Z 1 0~. 06 0.4 02 0
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40
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CONTROL -- 614
f'RACTION NUMBER + S ~M CADMIUM -
$ H
Fig. 1. In vitro induction of metal-binding proteins in striped bass hepatocytes exposed to 0 or 5/~M cadmium for 30 min and incubated in cadmium-free media for (a) 3 h or (b) 6 h. Metalbinding proteins of interest are located in fractions 30-40.
conditions used in the experiments. This response pattern was also observed in striped bass exposed to cadmium in vivo. There was no increase in the level of metal-binding protein as measured by 35S-cysteine incorporation 24 h after a single i.m. injection of cadmium (2.2 mg/kg). However, 24 h after the last of three successive daily injections there was a significant induction of MBP in treated fish when compared to controls (1.6-fold) or sham (1.5-fold) injected fish. This suggests that the kinetics of MBP induction in fish in vivo is different from that observed in rats, i.e. requires longer time. This hypothesis
144
Sandra M. Baksi, John M. Frazier
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Fig. 2. In vitro induction of metal-binding proteins in rat hepatocytes exposed to 5 #M cadmium for 15 rain and incubated in cadmium-free media for 1 or 6 h. Metallothionein is located in fractions 29 39.
is consistent with the results obtained in the short-term experiments done in vitro.
There are several possible explanations for the observed differences in responses between rats and fish. There may be differences between isolated hepatocytes of the two species in the rate of uptake of metals from the culture media. This could affect the amount of metal that actually enters the cell during the pulse exposure to cadmium. Once the metal is inside the cell, there may be differences in intracellular distribution and the binding of the metal to intracellular proteins. The regulation of the synthesis of metalbinding proteins in aquatic species is not as well understood as it is in mammalian systems. There may be differences in the control of MBP synthesis at the m R N A level. The results in these studies could be due to differences in the rate of transcription of the m R N A for MBP, or in the rate of translation of the MBP m R N A . In addition, little is known about the effect of pre-existing MBP on the intracellular kinetics of metals in fish hepatocytes. In rat hepatocytes it has been observed that the existence of high levels of intracellular Z n - M T significantly supresses the induction of new MT synthesis by cadmium (Din and Frazier, unpublished results). Another possible explanation is that the process of MBP induction is sensitive to temperature, and the differences observed are related to the incubation temperature, which was 37°C for rat hepatocytes and 22°C for
A fish model for investigation of environmental contaminants
145
striped bass hepatocytes. The latter hypothesis is presently being investigated. In conclusion, striped bass hepatocytes can be successfully used to investigate many of the toxicological problems for which mammalian hepatocytes are presently being utilized, including metal regulation. Although there are similarities between the hepatocytes of the two species there appear to be differences as well. Fish hepatocytes appear to be much more refractory to the effects to cadmium than rat hepatocytes under similar culture conditions. Understanding the mechanisms behind these differences would provide information concerning the practicality of extrapolating toxicological data between teleost and mammalian species.
REFERENCES 1. Seglen, P. O. Methods Cell Biol., 13, 29 (1975). 2. Frazier, J. M. & Din, W. S. In Safety Evaluation and Regulation of Chemicals (F. Hamburger, ed.) Karger, New York, pp. 295-308, 1985.