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Induction of Xenobiotic metabolizing enzyme activities in primary culture of rainbow trout hepatocytes
Marine Em,ironmental Research 28 (1989} 113 116
Induction of Xenobiotic Metabolizing Enzyme Activities in Primary Culture of Rainbow Trout Hepatocytes Maija Pesonen," Anders Goksoyr t' & T o m m y Andersson" " Department of Zoophysiology, University of G6tcborg, Box 25059, S-40031 G6teborg, Sweden h Department of Biochemistry, University or Bergen, N-5009 Bergen, Norway
ABSTRACT
Rainhow trout hepatocytes in primarr monolayer culture maintained se~:eral important ('ell Junctions and enzyme activities int:olced in .\e'nohiotic metaholism. Furthermore. c vtochrome P-450-dependent 7-ethoxyresort{/in O-(teethvlase ( E R O D ) activity and the amount o/" the major polycyclic aromatic hydrocarhons (PAH)-inducihle / a r m o f cvtochrome P-450 (measured hv E L I S A using antihodr towards cod P-450(') #wreased a/ter treatment q f ('ells with fl-naphtholtal~one ( B N F ) or 2.3.7.8tetrachlorodihenzo-p-~#oxin ( T C D D ). Cultured trout hel)alocytes seems to he a promisinj~ in vitro model which ma)' serz'e as a .s'('reenin,g test.lor to,\-i( constiluents in hutustrial (Jfluents arm could sert'e as at, brier(sting s3"s'tem J+)r .s'tlidi('.s"oft regulation o[.venohiolic metaholLvtt h v (,xo~enous and endogenous liwtors.
Xenobiotic metabolizing enzymes biotransform various lipophilic, foreign compounds (xenobiotics) to water-soluble products. The transformation reactions can be oxidative, mediated by cytochrome P-450-dependent monooxygenases, and the product may be conjugated by, for example, glucuronic acid. An important characteristic of these enzymes is their inducibility by various xenobiotics. Fish liver cytochrome P-450 enzymes are induced by polycyclic aromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCBs). Environmental pollutants such as petroleum products and pulp bleach plant effluents are also potent inducers of fish cytochrome P-450. Induction ofcytochrome P-450 in fish has therefore been 113 .14arim, Enriron. Res. 0141-1136.90/$03.50 ~' 1990 Elsevier Science Publishers ktd, England. Printed in Great Britain
114
Ma!]a Pesonen, Anders Goksoyr, Tommy A ndersson
utilized as a sensitive biological response in monitoring of the aquatic environment.t -4 Freshly isolated and cultured liver cells have been used as a valuable model to study properties and cellular regulation of xenobiotic metabolizing enzymes in mammals. Thus, we have prepared primary liver cell culture from rainbow trout and characterized metabolic capabilities of several xenobiotic metabolizing enzymes in culture. The effects of PAH-type inducers (/3-naphthoflavone, BNF and 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and extracts from the pulpmill effluents on cytochrome P-450dependent 7-ethoxyresorufin O-deethylase activity (EROD) were also studied. Hepatocytes were isolated from immature rainbow trout (Oncorhynchus mykiss) by modification of the two-step collagenase perfusion technique of Seglen. 5 Each isolation gave 100-200 x 106 cells/g liver weight with a mean viability over 90% when tested by Trypan blue exclusion test. Hepatocytes were seeded on Petri dishes at a density of 106 viable cells/ml medium and were incubated at 10°C. The culture medium (medium 199) was changed one day after seeding and then every 2 days. After incubation cells were frozen in liquid nitrogen and stored at -80'~C until analysis. Inducing agents BN F and T C D D were dissolved in dimethyl sulfoxide (DMSO) and added in 10 Itl aliquots to 10 ml of culture medium. The same amount of DMSO was added to control hepatocytes. Fresh medium without inducers was changed every TABLE I Cytochromc P-450," Total Glutathione and Protein Contents and Several Xcnobiotic Metabolizing Enzyme Activities in Trout Primary Hepatocyte Cultureb During a Period of 120h
Cytochrome P-450 (pmol/mg protein) Total glutathione ( n m o l / m g proteint Proteins [mg/ml) AHH
0h
24 h
72 I1
120 h
1800
1100
700
5.00
10-26 -4. 1.5
943 _4 3'8
12.73 -4. 1"2
1.27+0.01 21.22 -4. 3.1
1"13_+0"05
1'13,4.0"2 25'55 -4. 3"7
19.27 + 0 7 1-24 -4. 0'08
(pmol/'min mg protein) EROD
(pmol,min nag proteinl UDP-glucuronosyltransferase" (pmol/min mg proteinl Glutathione S-transferase ~ (nmol/min mg protein)
19.07 + 1.0
20,50 -4. 1'1
111-12 4- 19,0 100.00 -4. 18.6 90'16 + 8 3
29,63 + 13
28.55 +_ 4'4
224,18 4-_ 35.0
259.03 + 35.7
90.01 -4. 3'8
79.71 -4. 7 6
"Values represent one experiment. h Values represent means ± SD of one two experiments each with three four replicates.
' Substratc was p-nitrophenol. J Substrate was 1-chloro-2,4-dinitrobenzene.
115
Xenobiotie metabolizing enzyme activities in trout
48 h when the duration of culture was longer than 2 days. Extracts from three different kraft mill effluents were added to the cell culture. One liter of the effluents or water from a clean area was extracted with dichlormethane, evaporated and dissolved in 1 ml acetone. Aliquots of the acetone extract or three dilutions were added to the culture dishes. EROD activity was measured in whole-cell homogenate according to Burke & Mayer. 6 Freshly isolated hepatocytes were usually individual, round and morphologically intact. Hepatocytes attached to the surface on dishes and flattened out after one day in culture. Survival of cells in culture conditions used was over 80% during 1-5 day culture period. Retention of functional capacities of cells was judged by measuring the level of intracellular lactate dehydrogenase (LDH) activity and its leakage to the culture medium as a function of time. The amount of LDH released into the culture medium was at highest 15% of the total LDH activity during first 24 h (total LDH activity represents combined intracellular and released activity at each time point} and decreased with time, being about 5% at day 5 in culture. The cytochrome P-450 content in hepatocytes decreased gradually from the value found in the freshly isolated cells and after 5 days in culture 29% of cytochrome P-450 content was left (Table 1). The loss of cytochrome P-450 content was not as high as usually reported for studies with mammalian cell cultures. ~ The cytochrome P-450 monooxygenases (aryl hydrocarbon hydroxylase, AHH, and 7-ethoxyresorufin O-deethylase, EROD activities)
EROD 80
40
20 Control
0
!
!
!
!
!
I
II
20
40
60
80
100
120
140
pmoI TCDD/I
Fig. I. Dose response curve of the induction of 7-ethoxyresorufin O-deethylase in trout hepatocytc primary culture treated with 2,3,7,8-tetrachlorodibenzo-p-dioxinat 24 h in culture and assayed for 7-ethoxyresorufin O-dethylase activity at 72h in culture. Values arc means ± SD of three four determinations.
116
Mai/a Pesonen, Anders Goksoyr, Tommy Andersson
and conjugating enzymes (UDP~glucuronosyltransferase and glutathione Stransferase activities) stayed at their initial levels during the culture period (Table 1). Exposure of the cells to BNF (0.01 and 0.1 ktg BNF/ml, these doses were found to give maximal induction of E R O D activities), commencing one day after initiating the culture, led to decrease in E R O D activity during first 12 h of the exposure. Thereafter, a significant increase in the activities could be seen and maximal values were reached 48 h after addition of BNF. T C D D caused an increase in E R O D activity without the initial drop seen with BNF. The lowest dose which produced a significant increase in E R O D activity compared to the control values was 4 pmol TCDD/liter, and maximum induction (10-fold) was found with dose of 100 pmol TCDD/liter (Fig. 1}. Furthermore, the major PAH-inducible cytochrome P-450 form was measured by ELISA technique using anti-cod P-450c. 8 This cytochrome P-450 form seemed to be increased 1'5 h after addition of T C D D to the culture and reached 160% of the control value 48 h after treatment. Dose-dependent increase (l'7-4-fold) in EROD-activity was also seen when hepatocytes were incubated with extracts from pulpmill effluents. The potency of the fractions to induce E R O D activity in cells was dependent on the amount of chlorine used in the bleaching process.
ACKNOWLEDGEMENTS This study was supported by the Finnish Ministry of Agriculture and Forestry, Maj and Tot Nessling Foundation, Finland, and the Swedish Environmental Protection Board.
REFERENCES 1. Vodicnik, M., Elcombe, C. R. & Lech, J. J., Toxicol. Appl. Pharmacol., 59(19811 364 74. 2. Stegeman, J. J., Kloepper-Sams, P. J. & Farrington, J. W., Science, 231 t1986) 1287 9. 3. Andersson, T., F6rlin, L., Hfirdig, J. & Larsson,/~., Can. J. Fish. Aquat. Sci., 45 (1988) 1525-36. 4. Payne, J., Fancey, L., Kiceniuk, J., Williams, U., Osborne, J. & Rahimtula, A., Mar. Environ. Res., 17 (1985) 330 2. 5. Seglen, P., Exp. Cell Res., 82 (1973} 391 8. 6. Burke, M. D. & Mayer, R. T., Drug Metah. Di.sT)os., 2 (1974) 583 8. 7. Suolinna, E-M., Med. Biol., 60 (1982) 237 54. 8. Goksoyr, A., Andersson, T., Hansson, T., Klurgsoyr, J., Zhang, Y. & Farlin, L., Toxicol. Appl. Pharmacol., 89 (1987) 347 60.
Induction of Xenobiotic Metabolizing Enzyme Activities in Primary Culture of Rainbow Trout Hepatocytes Maija Pesonen," Anders Goksoyr t' & T o m m y Andersson" " Department of Zoophysiology, University of G6tcborg, Box 25059, S-40031 G6teborg, Sweden h Department of Biochemistry, University or Bergen, N-5009 Bergen, Norway
ABSTRACT
Rainhow trout hepatocytes in primarr monolayer culture maintained se~:eral important ('ell Junctions and enzyme activities int:olced in .\e'nohiotic metaholism. Furthermore. c vtochrome P-450-dependent 7-ethoxyresort{/in O-(teethvlase ( E R O D ) activity and the amount o/" the major polycyclic aromatic hydrocarhons (PAH)-inducihle / a r m o f cvtochrome P-450 (measured hv E L I S A using antihodr towards cod P-450(') #wreased a/ter treatment q f ('ells with fl-naphtholtal~one ( B N F ) or 2.3.7.8tetrachlorodihenzo-p-~#oxin ( T C D D ). Cultured trout hel)alocytes seems to he a promisinj~ in vitro model which ma)' serz'e as a .s'('reenin,g test.lor to,\-i( constiluents in hutustrial (Jfluents arm could sert'e as at, brier(sting s3"s'tem J+)r .s'tlidi('.s"oft regulation o[.venohiolic metaholLvtt h v (,xo~enous and endogenous liwtors.
Xenobiotic metabolizing enzymes biotransform various lipophilic, foreign compounds (xenobiotics) to water-soluble products. The transformation reactions can be oxidative, mediated by cytochrome P-450-dependent monooxygenases, and the product may be conjugated by, for example, glucuronic acid. An important characteristic of these enzymes is their inducibility by various xenobiotics. Fish liver cytochrome P-450 enzymes are induced by polycyclic aromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCBs). Environmental pollutants such as petroleum products and pulp bleach plant effluents are also potent inducers of fish cytochrome P-450. Induction ofcytochrome P-450 in fish has therefore been 113 .14arim, Enriron. Res. 0141-1136.90/$03.50 ~' 1990 Elsevier Science Publishers ktd, England. Printed in Great Britain
114
Ma!]a Pesonen, Anders Goksoyr, Tommy A ndersson
utilized as a sensitive biological response in monitoring of the aquatic environment.t -4 Freshly isolated and cultured liver cells have been used as a valuable model to study properties and cellular regulation of xenobiotic metabolizing enzymes in mammals. Thus, we have prepared primary liver cell culture from rainbow trout and characterized metabolic capabilities of several xenobiotic metabolizing enzymes in culture. The effects of PAH-type inducers (/3-naphthoflavone, BNF and 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and extracts from the pulpmill effluents on cytochrome P-450dependent 7-ethoxyresorufin O-deethylase activity (EROD) were also studied. Hepatocytes were isolated from immature rainbow trout (Oncorhynchus mykiss) by modification of the two-step collagenase perfusion technique of Seglen. 5 Each isolation gave 100-200 x 106 cells/g liver weight with a mean viability over 90% when tested by Trypan blue exclusion test. Hepatocytes were seeded on Petri dishes at a density of 106 viable cells/ml medium and were incubated at 10°C. The culture medium (medium 199) was changed one day after seeding and then every 2 days. After incubation cells were frozen in liquid nitrogen and stored at -80'~C until analysis. Inducing agents BN F and T C D D were dissolved in dimethyl sulfoxide (DMSO) and added in 10 Itl aliquots to 10 ml of culture medium. The same amount of DMSO was added to control hepatocytes. Fresh medium without inducers was changed every TABLE I Cytochromc P-450," Total Glutathione and Protein Contents and Several Xcnobiotic Metabolizing Enzyme Activities in Trout Primary Hepatocyte Cultureb During a Period of 120h
Cytochrome P-450 (pmol/mg protein) Total glutathione ( n m o l / m g proteint Proteins [mg/ml) AHH
0h
24 h
72 I1
120 h
1800
1100
700
5.00
10-26 -4. 1.5
943 _4 3'8
12.73 -4. 1"2
1.27+0.01 21.22 -4. 3.1
1"13_+0"05
1'13,4.0"2 25'55 -4. 3"7
19.27 + 0 7 1-24 -4. 0'08
(pmol/'min mg protein) EROD
(pmol,min nag proteinl UDP-glucuronosyltransferase" (pmol/min mg proteinl Glutathione S-transferase ~ (nmol/min mg protein)
19.07 + 1.0
20,50 -4. 1'1
111-12 4- 19,0 100.00 -4. 18.6 90'16 + 8 3
29,63 + 13
28.55 +_ 4'4
224,18 4-_ 35.0
259.03 + 35.7
90.01 -4. 3'8
79.71 -4. 7 6
"Values represent one experiment. h Values represent means ± SD of one two experiments each with three four replicates.
' Substratc was p-nitrophenol. J Substrate was 1-chloro-2,4-dinitrobenzene.
115
Xenobiotie metabolizing enzyme activities in trout
48 h when the duration of culture was longer than 2 days. Extracts from three different kraft mill effluents were added to the cell culture. One liter of the effluents or water from a clean area was extracted with dichlormethane, evaporated and dissolved in 1 ml acetone. Aliquots of the acetone extract or three dilutions were added to the culture dishes. EROD activity was measured in whole-cell homogenate according to Burke & Mayer. 6 Freshly isolated hepatocytes were usually individual, round and morphologically intact. Hepatocytes attached to the surface on dishes and flattened out after one day in culture. Survival of cells in culture conditions used was over 80% during 1-5 day culture period. Retention of functional capacities of cells was judged by measuring the level of intracellular lactate dehydrogenase (LDH) activity and its leakage to the culture medium as a function of time. The amount of LDH released into the culture medium was at highest 15% of the total LDH activity during first 24 h (total LDH activity represents combined intracellular and released activity at each time point} and decreased with time, being about 5% at day 5 in culture. The cytochrome P-450 content in hepatocytes decreased gradually from the value found in the freshly isolated cells and after 5 days in culture 29% of cytochrome P-450 content was left (Table 1). The loss of cytochrome P-450 content was not as high as usually reported for studies with mammalian cell cultures. ~ The cytochrome P-450 monooxygenases (aryl hydrocarbon hydroxylase, AHH, and 7-ethoxyresorufin O-deethylase, EROD activities)
EROD 80
40
20 Control
0
!
!
!
!
!
I
II
20
40
60
80
100
120
140
pmoI TCDD/I
Fig. I. Dose response curve of the induction of 7-ethoxyresorufin O-deethylase in trout hepatocytc primary culture treated with 2,3,7,8-tetrachlorodibenzo-p-dioxinat 24 h in culture and assayed for 7-ethoxyresorufin O-dethylase activity at 72h in culture. Values arc means ± SD of three four determinations.
116
Mai/a Pesonen, Anders Goksoyr, Tommy Andersson
and conjugating enzymes (UDP~glucuronosyltransferase and glutathione Stransferase activities) stayed at their initial levels during the culture period (Table 1). Exposure of the cells to BNF (0.01 and 0.1 ktg BNF/ml, these doses were found to give maximal induction of E R O D activities), commencing one day after initiating the culture, led to decrease in E R O D activity during first 12 h of the exposure. Thereafter, a significant increase in the activities could be seen and maximal values were reached 48 h after addition of BNF. T C D D caused an increase in E R O D activity without the initial drop seen with BNF. The lowest dose which produced a significant increase in E R O D activity compared to the control values was 4 pmol TCDD/liter, and maximum induction (10-fold) was found with dose of 100 pmol TCDD/liter (Fig. 1}. Furthermore, the major PAH-inducible cytochrome P-450 form was measured by ELISA technique using anti-cod P-450c. 8 This cytochrome P-450 form seemed to be increased 1'5 h after addition of T C D D to the culture and reached 160% of the control value 48 h after treatment. Dose-dependent increase (l'7-4-fold) in EROD-activity was also seen when hepatocytes were incubated with extracts from pulpmill effluents. The potency of the fractions to induce E R O D activity in cells was dependent on the amount of chlorine used in the bleaching process.
ACKNOWLEDGEMENTS This study was supported by the Finnish Ministry of Agriculture and Forestry, Maj and Tot Nessling Foundation, Finland, and the Swedish Environmental Protection Board.
REFERENCES 1. Vodicnik, M., Elcombe, C. R. & Lech, J. J., Toxicol. Appl. Pharmacol., 59(19811 364 74. 2. Stegeman, J. J., Kloepper-Sams, P. J. & Farrington, J. W., Science, 231 t1986) 1287 9. 3. Andersson, T., F6rlin, L., Hfirdig, J. & Larsson,/~., Can. J. Fish. Aquat. Sci., 45 (1988) 1525-36. 4. Payne, J., Fancey, L., Kiceniuk, J., Williams, U., Osborne, J. & Rahimtula, A., Mar. Environ. Res., 17 (1985) 330 2. 5. Seglen, P., Exp. Cell Res., 82 (1973} 391 8. 6. Burke, M. D. & Mayer, R. T., Drug Metah. Di.sT)os., 2 (1974) 583 8. 7. Suolinna, E-M., Med. Biol., 60 (1982) 237 54. 8. Goksoyr, A., Andersson, T., Hansson, T., Klurgsoyr, J., Zhang, Y. & Farlin, L., Toxicol. Appl. Pharmacol., 89 (1987) 347 60.