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Effect of dietary PAH on intestinal mixed-function oxygenase (MFO) activity in spot (Leiostomus xanthurus)
Abstracts
115
Oceanic and Atmospheric Administration, Great Lakes Environmental Research Laboratory, Ann Arbor, Michigan 48104, USA. Many species offish have been demonstrated to possess a cytochrome P-450 system which functions in metabolizing xenobiotics like polynuclear aromatic hydrocarbons (PAH). Since these compounds often induce enzyme activity, it may be possible to use cytochrome P-450 as an indicator of chemical exposure on fish populations. To test this hypothesis, brown bullhead (Ictalurus nebulosus) were collected from the Black River, Ohio, a site characterized by high levels of PAHs, and a reference site, Old Woman's Creek (OWC), Ohio. A radiometric assay using tritiated benzo(a)pyrene (BP) was initiated in the field to determine aryl hydrocarbon hydroxylase (AHH) activity. AHH activity was determined and the metabolites produced were examined in the laboratory. Fish from the Black River were found to have significantly higher levels of A H H activity than OWC fish. Sex did not influence A H H activity while age and time of collection did. In a laboratory study, OWC fish were collected and injected (i.p.) with either corn oil or BP in corn oil (30 mg/kg) for three days, and then were assayed periodically for the following week. AHH activity significantly increased in the OWC fish treated with BP as compared with the oil and non-treated controls. This demonstrates that A H H activity in bullhead can be induced by PAHs such as BP and is consistent with other studies, suggesting that the elevated levels of AHH activity seen in the field experiments could be linked to the presence of PAH contaminants in the environment.
Effect of Dietary PAH on Intestinal Mixed-function Oxygenase (MFO) Activity in Spot (Leiostomus xanthuras). P. A. VAN VELD,* J. J. S T E G E M A N , t J. S. PATTON:~ & R I C H A R D F. LEE.* *Skidaway Institute of Oceanography, Savannah, Georgia 31416, USA; t Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA & ~Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080, USA. Spot (Leiostomus xanthurus), a marine teleost fish, were administered polycyclic aromatic hydrocarbon (PAH)-contaminated diets to evaluate the response of the intestinal mixed-function oxygenase (MFO) system to dietary PAH exposure. Activities of ethoxyresorufin-O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) in intestinal microsomes from control-fed fish were very low relative to hepatic E R O D and A H H activities. However, following exposure to a 3-methylcholanthrene (3-MC)contaminated diet (10 mg 3-MC/kg food), the activity of intestinal EROD
116
Abstracts
and AHH increased 36- and 17-fold, respectively, such that intestinal MFO activities exceeded those of the liver. A significant increase in intestinal MFO activity occurred in fish receiving dietary benzo[a]pyrene (BP) at concentrations as low as 10/~g BP/kg food. A large (c. 5-fold) increase in intestinal EROD activity was observed within three hours after administration of dietary BP. A plateau in MFO activity occurred after approximately three days of PAH exposure. Activities decreased to control levels within three days after replacing the PAH diet with the control diet. Starvation resulted in disappearance of detectable MFO activity. MFO activities were highest in the pyloric caeca and in the proximal half of the intestine. Monoclonal antibody (MAb 1-12-3) against the major PAH-inducible cytochrome P-450 (P-450E) in the liver of the marine teleost (Stenotomus chrysops) [Park et al., Arch. Biochem. Biophys., 249, 339-50 (1986)] recognized a protein in intestinal microsomes from spot intestine having a molecular weight (c. 57 K) similar to that of PAH-inducible cytochromes P450 in hepatic microsomes of other species. The presence and activity of the PAH-inducible cytochrome P-450 of the spot intestine appeared to be entirely dependent upon dietary PAH exposure. The results of this study suggest that the fish intestinal MFO activity plays a major role in the metabolism of dietary PAH. Supported in part by USPHS grant ES-4220 to J.J.S.
Xenobiotic and Steroid Metabolism in Juvenile Rainbow Trout Kidney. MAIJA PESONEN,* LARS FORLIN,* TIIU HANSSONt & TOMMY ANDERSSON.* *Department of Zoophysiology, University of G6teborg, Box 25059, S-400 31 G6teborg, Sweden & ~fDepartment of Medical Nutrition, Karolinska Institution, Huddinge Hospital, S-149 86 Huddinge, Sweden. Fish kidney is an important excretory as well as endocrine organ. The trunk kidney contains mainly nephrons and tubules, whereas the head kidney also contains adrenal and hematopoietic tissue. Microsomal cytochrome P-450 content in trout kidney was approximately 5-fold lower than the content in the liver microsomes. The renal ethoxycoumarin- and ethoxyresorufin-Odeethylase activities calculated on a cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The results indicate different isoenzyme profiles and/or catalytic properties of renal and hepatic cytochrome P-450 enzymes in rainbow trout. Marked differences in cytochrome P-450-mediated activities between the trunk and head kidney were found. Ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were mainly localized in the trunk kidney, whereas the activities in the head kidney were barely detectable. The same distribution pattern, although not
115
Oceanic and Atmospheric Administration, Great Lakes Environmental Research Laboratory, Ann Arbor, Michigan 48104, USA. Many species offish have been demonstrated to possess a cytochrome P-450 system which functions in metabolizing xenobiotics like polynuclear aromatic hydrocarbons (PAH). Since these compounds often induce enzyme activity, it may be possible to use cytochrome P-450 as an indicator of chemical exposure on fish populations. To test this hypothesis, brown bullhead (Ictalurus nebulosus) were collected from the Black River, Ohio, a site characterized by high levels of PAHs, and a reference site, Old Woman's Creek (OWC), Ohio. A radiometric assay using tritiated benzo(a)pyrene (BP) was initiated in the field to determine aryl hydrocarbon hydroxylase (AHH) activity. AHH activity was determined and the metabolites produced were examined in the laboratory. Fish from the Black River were found to have significantly higher levels of A H H activity than OWC fish. Sex did not influence A H H activity while age and time of collection did. In a laboratory study, OWC fish were collected and injected (i.p.) with either corn oil or BP in corn oil (30 mg/kg) for three days, and then were assayed periodically for the following week. AHH activity significantly increased in the OWC fish treated with BP as compared with the oil and non-treated controls. This demonstrates that A H H activity in bullhead can be induced by PAHs such as BP and is consistent with other studies, suggesting that the elevated levels of AHH activity seen in the field experiments could be linked to the presence of PAH contaminants in the environment.
Effect of Dietary PAH on Intestinal Mixed-function Oxygenase (MFO) Activity in Spot (Leiostomus xanthuras). P. A. VAN VELD,* J. J. S T E G E M A N , t J. S. PATTON:~ & R I C H A R D F. LEE.* *Skidaway Institute of Oceanography, Savannah, Georgia 31416, USA; t Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA & ~Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080, USA. Spot (Leiostomus xanthurus), a marine teleost fish, were administered polycyclic aromatic hydrocarbon (PAH)-contaminated diets to evaluate the response of the intestinal mixed-function oxygenase (MFO) system to dietary PAH exposure. Activities of ethoxyresorufin-O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) in intestinal microsomes from control-fed fish were very low relative to hepatic E R O D and A H H activities. However, following exposure to a 3-methylcholanthrene (3-MC)contaminated diet (10 mg 3-MC/kg food), the activity of intestinal EROD
116
Abstracts
and AHH increased 36- and 17-fold, respectively, such that intestinal MFO activities exceeded those of the liver. A significant increase in intestinal MFO activity occurred in fish receiving dietary benzo[a]pyrene (BP) at concentrations as low as 10/~g BP/kg food. A large (c. 5-fold) increase in intestinal EROD activity was observed within three hours after administration of dietary BP. A plateau in MFO activity occurred after approximately three days of PAH exposure. Activities decreased to control levels within three days after replacing the PAH diet with the control diet. Starvation resulted in disappearance of detectable MFO activity. MFO activities were highest in the pyloric caeca and in the proximal half of the intestine. Monoclonal antibody (MAb 1-12-3) against the major PAH-inducible cytochrome P-450 (P-450E) in the liver of the marine teleost (Stenotomus chrysops) [Park et al., Arch. Biochem. Biophys., 249, 339-50 (1986)] recognized a protein in intestinal microsomes from spot intestine having a molecular weight (c. 57 K) similar to that of PAH-inducible cytochromes P450 in hepatic microsomes of other species. The presence and activity of the PAH-inducible cytochrome P-450 of the spot intestine appeared to be entirely dependent upon dietary PAH exposure. The results of this study suggest that the fish intestinal MFO activity plays a major role in the metabolism of dietary PAH. Supported in part by USPHS grant ES-4220 to J.J.S.
Xenobiotic and Steroid Metabolism in Juvenile Rainbow Trout Kidney. MAIJA PESONEN,* LARS FORLIN,* TIIU HANSSONt & TOMMY ANDERSSON.* *Department of Zoophysiology, University of G6teborg, Box 25059, S-400 31 G6teborg, Sweden & ~fDepartment of Medical Nutrition, Karolinska Institution, Huddinge Hospital, S-149 86 Huddinge, Sweden. Fish kidney is an important excretory as well as endocrine organ. The trunk kidney contains mainly nephrons and tubules, whereas the head kidney also contains adrenal and hematopoietic tissue. Microsomal cytochrome P-450 content in trout kidney was approximately 5-fold lower than the content in the liver microsomes. The renal ethoxycoumarin- and ethoxyresorufin-Odeethylase activities calculated on a cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The results indicate different isoenzyme profiles and/or catalytic properties of renal and hepatic cytochrome P-450 enzymes in rainbow trout. Marked differences in cytochrome P-450-mediated activities between the trunk and head kidney were found. Ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were mainly localized in the trunk kidney, whereas the activities in the head kidney were barely detectable. The same distribution pattern, although not