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Effects of tributyltin chloride in vitro on hepatic microsomal cytochrome P450 and associated enzyme activities in the marine fish Stenotomus chrysops
21o
Abstracts
University of G6teborg, Box 25059, S-40031 G6teborg, Sweden & ~Department of Biochemistry, University of Bergen, Jlrstadvein 19, N-5009 Bergen, Norway. Primary cultures of rainbow trout hepatocytes were used to evaluate the expression of cytochrome P4501A 1 protein, m RNA, and catalytic activities during 96 h after exposure of cells to/~-naphthoflavone (BNF) and 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes were isolated from immature rainbow trout by a two-step perfusion method and incubated at 10°C. The cells were exposed to the inducers for 48 h, starting after a 24-h preculture. Exposure of hepatocytes to BNF increased cytochrome P4501A1 activity (EROD) over the control levels at 12 h after addition of the inducers. The activities peaked at 48 h and then declined to control levels at 72 h. In cells exposed to TCDD, a significant increase in E R O D activity could be seen at 12 h, but, in contrast to BNF exposure, activities continued to increase and reached maximal values at 96h. Cytochrome P4501A1 protein, measured by the ELISA technique, paralleled the changes in E R O D activities. Cytochrome P4501A1 m R N A levels, determined by Northern and slot-blots analysis, were hardly detectable in control cells. In BNF- and TCDD-treated cells, the m R N A content increased before the protein and catalytic activities. The elevated level of cytochrome P4501A1 m R N A was more sustained by T C D D than by BNF exposure. The results indicate differences in induction of cytochrome P4501A1 mRNA, its protein product, and catalytic activity between BNF and T C D D exposure.
Effects of Tributyltin Chloride In Vitro on Hepatic MicrosomM Cytochrome P450 and Associated Enzyme Activities in the Marine Fish Stenotomus chrysops. K A R L VENT* & JOHN J. STEGEMAN.~ *Swiss Federal Institute for Water Resources and Water Pollution Control (EA WAG), CH604 7 Kastanienbaum, Switzerland & ~ Woods Hole Oceanographic Institution, Biology' Department, Woods Hole, Massachusetts 02543, USA. Tributyltin (TBT) is introduced into the aquatic environment principally by leaching from antifouling paints but also via wastewaters. TBT has been shown to be highly toxic to a number of aquatic species, but the mode(s) of action on a biochemical level remain(s) to be elucidated. In this study, we investigated the interaction in vitro of TBT with hepatic microsomal cytochrome P450 and associated enzyme activities in scup (Stenotomus chrysops). Hepatic microsomes from/%naphthoflavone (/~NF)-induced scup were incubated in vitro with TBT and various components analyzed. TBT led to a time- and dose-dependent decrease in total (spectrophotometrically
Abstracts
211
measured) microsomal P450 content. This decrease in total P450 was accompanied by the formation of cytochrome P420. A complete loss of P450 occurred with 1 mM TBT after a 30-min incubation. Ethoxyresorufin-Odeethylase (EROD) activity was strongly inhibited by TBT in vitro in a dosedependent manner, with a complete inhibition at 0.3 mM TBT after a 15-min incubation. Other components of the microsomal electron transport system were also investigated. Neither cytochrome b 5 content nor N A D P H cytochrome c reductase activity was affected by TBT at concentrations up to 0-5 mM TBT. N A D H cytochrome c reductase activity, however, showed a dose-dependent increase, with more than a doubling of activity at 0"5 mM TBT. This indicates that TBT has different effects on these reductases. In conclusion, TBT can strongly interact with microsomal P450, leading to destruction of native enzyme and inhibition of enzyme activity. Further investigations are under way on the mechanism by which TBT acts on cytochrome P450, whether similar mechanisms might operate in vivo, and to identify any selectivity in the action of TBT on P450 forms. Supported by the EAWAG and NIH ES04220.
S E C T I O N 3: B I O T R A N S F O R M A T I O N , AND OXYGEN RADICALS
TOXICOKINETICS,
Comparison of In Vitro Benzo[a]pyrene Metabolism and Mutagenesis in Mussel, Sea Bass, and Rat. X. MICHEL, D. RIBERA, S. DECOUDU & J. F. N A R B O N N E . Laboratoire de Toxicologie Alimentaire, Institut des Sciences et Techniques des Aliments de Bordeaux, Universitb de Bordeaux, Av. des Facultbs, 33405 Talence Cedex, France. In-vitro metabolism of benzo(a)pyrene (B(a)P) was studied in both microsomal and post-mitochondrial ($9) fractions from the digestive gland of the mussel (Mytilus galloprovineialis) and from the liver of SpragueDawley rats and sea bass (Dicentrachus labrax). Moreover, the relative role of the different metabolic pathways and the production of mutagenic derivatives was investigated by using specific inhibitors. The overall conversion of B(a)P was very low for mussel (6pmol B(a)P/mn/mg MP) compared with rat and fish (about 2 0 0 p m o l B ( a ) P < / m n / m g M P ) . The metabolite profiles were similar with $9 and microsomes except for mussel, which showed a lack of diols and an increase in phenols in the $9 profile. Comparison of individual metabolites revealed that quinones were the major metabolites for mussel (80%), whereas they represent only 20% in rat and less than 10% in fish. Greater proportion of B(a)P was converted to diols in fish fractions (27%) than in rat or mussel (4%). B(a)P phenols remained
Abstracts
University of G6teborg, Box 25059, S-40031 G6teborg, Sweden & ~Department of Biochemistry, University of Bergen, Jlrstadvein 19, N-5009 Bergen, Norway. Primary cultures of rainbow trout hepatocytes were used to evaluate the expression of cytochrome P4501A 1 protein, m RNA, and catalytic activities during 96 h after exposure of cells to/~-naphthoflavone (BNF) and 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes were isolated from immature rainbow trout by a two-step perfusion method and incubated at 10°C. The cells were exposed to the inducers for 48 h, starting after a 24-h preculture. Exposure of hepatocytes to BNF increased cytochrome P4501A1 activity (EROD) over the control levels at 12 h after addition of the inducers. The activities peaked at 48 h and then declined to control levels at 72 h. In cells exposed to TCDD, a significant increase in E R O D activity could be seen at 12 h, but, in contrast to BNF exposure, activities continued to increase and reached maximal values at 96h. Cytochrome P4501A1 protein, measured by the ELISA technique, paralleled the changes in E R O D activities. Cytochrome P4501A1 m R N A levels, determined by Northern and slot-blots analysis, were hardly detectable in control cells. In BNF- and TCDD-treated cells, the m R N A content increased before the protein and catalytic activities. The elevated level of cytochrome P4501A1 m R N A was more sustained by T C D D than by BNF exposure. The results indicate differences in induction of cytochrome P4501A1 mRNA, its protein product, and catalytic activity between BNF and T C D D exposure.
Effects of Tributyltin Chloride In Vitro on Hepatic MicrosomM Cytochrome P450 and Associated Enzyme Activities in the Marine Fish Stenotomus chrysops. K A R L VENT* & JOHN J. STEGEMAN.~ *Swiss Federal Institute for Water Resources and Water Pollution Control (EA WAG), CH604 7 Kastanienbaum, Switzerland & ~ Woods Hole Oceanographic Institution, Biology' Department, Woods Hole, Massachusetts 02543, USA. Tributyltin (TBT) is introduced into the aquatic environment principally by leaching from antifouling paints but also via wastewaters. TBT has been shown to be highly toxic to a number of aquatic species, but the mode(s) of action on a biochemical level remain(s) to be elucidated. In this study, we investigated the interaction in vitro of TBT with hepatic microsomal cytochrome P450 and associated enzyme activities in scup (Stenotomus chrysops). Hepatic microsomes from/%naphthoflavone (/~NF)-induced scup were incubated in vitro with TBT and various components analyzed. TBT led to a time- and dose-dependent decrease in total (spectrophotometrically
Abstracts
211
measured) microsomal P450 content. This decrease in total P450 was accompanied by the formation of cytochrome P420. A complete loss of P450 occurred with 1 mM TBT after a 30-min incubation. Ethoxyresorufin-Odeethylase (EROD) activity was strongly inhibited by TBT in vitro in a dosedependent manner, with a complete inhibition at 0.3 mM TBT after a 15-min incubation. Other components of the microsomal electron transport system were also investigated. Neither cytochrome b 5 content nor N A D P H cytochrome c reductase activity was affected by TBT at concentrations up to 0-5 mM TBT. N A D H cytochrome c reductase activity, however, showed a dose-dependent increase, with more than a doubling of activity at 0"5 mM TBT. This indicates that TBT has different effects on these reductases. In conclusion, TBT can strongly interact with microsomal P450, leading to destruction of native enzyme and inhibition of enzyme activity. Further investigations are under way on the mechanism by which TBT acts on cytochrome P450, whether similar mechanisms might operate in vivo, and to identify any selectivity in the action of TBT on P450 forms. Supported by the EAWAG and NIH ES04220.
S E C T I O N 3: B I O T R A N S F O R M A T I O N , AND OXYGEN RADICALS
TOXICOKINETICS,
Comparison of In Vitro Benzo[a]pyrene Metabolism and Mutagenesis in Mussel, Sea Bass, and Rat. X. MICHEL, D. RIBERA, S. DECOUDU & J. F. N A R B O N N E . Laboratoire de Toxicologie Alimentaire, Institut des Sciences et Techniques des Aliments de Bordeaux, Universitb de Bordeaux, Av. des Facultbs, 33405 Talence Cedex, France. In-vitro metabolism of benzo(a)pyrene (B(a)P) was studied in both microsomal and post-mitochondrial ($9) fractions from the digestive gland of the mussel (Mytilus galloprovineialis) and from the liver of SpragueDawley rats and sea bass (Dicentrachus labrax). Moreover, the relative role of the different metabolic pathways and the production of mutagenic derivatives was investigated by using specific inhibitors. The overall conversion of B(a)P was very low for mussel (6pmol B(a)P/mn/mg MP) compared with rat and fish (about 2 0 0 p m o l B ( a ) P < / m n / m g M P ) . The metabolite profiles were similar with $9 and microsomes except for mussel, which showed a lack of diols and an increase in phenols in the $9 profile. Comparison of individual metabolites revealed that quinones were the major metabolites for mussel (80%), whereas they represent only 20% in rat and less than 10% in fish. Greater proportion of B(a)P was converted to diols in fish fractions (27%) than in rat or mussel (4%). B(a)P phenols remained