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Interstrain variation on the hepatic monooxygenase enzyme activities of rainbow trout: Induction and sample preparation
Abstracts PII:
127
SOl41-1136(98)00037-3
Initial Purification of CYPZ-Related Isoforms of Hepatic Cytochrome P450 from Channel Catfish (Zc~afurus punctatus). E. J. PERKINS AND D. SCHLENK. Department of
Pharmacology, School of Pharmacy, University of Mississippi, University, Mississippi 38677, USA. The purification and characterization of the cytochrome P450 monooxygenase system in fish has historically been limited to a few species. The isoform composition and functional characteristics of this system have not been well characterized in the channel catfish, a species which is gaining increasing favor as a model for toxicology studies. Immunochemical studies show that the channel catfish expresses at least four proteins that are crossreactive with anti-CYP2Kl polyclonal antibodies. All of these putative isoforms are similarly reactive with anti-CYP2M 1 antibodies. Isoform-specific variations in the expression of these proteins appear to have age-, sex- and chemical-dependent characteristics. The object of the present study was to purify the CYP2-related proteins from liver microsomes prepared from untreated mature catfish. Microsomes were prepared by standard methods and solubilized with 0.6% sodium cholate. Solubilized microsomes were applied initially to a phenyl-sepharose CL4B column (25x30 cm) and eluted with increasing detergent concentrations, resulting in three P450-containing peaks. Pooled fractions were subsequently added to a DEAE-sepharose CL-6B column and eluted with a linear gradient of sodium chloride (0.5~). Two proteins which are cross-reactive with anti-CYP2 antibodies (47 and 52 kDa) were co-isolated, with 17-22-fold purification as assessed by SDS-PAGE and western blotting. Further purification experiments are ongoing.
PII:
SOl41-1136(98)00038-S
Interstrain Variation on the Hepatic Monooxygenase Enzyme Activities of Rainbow Trout: Induction and Sample Preparation. 0. RITOLA,” K. KOPONEN,” V. VAN MIEGEMb
AND P. LINDSTROM-SEPPA.” aDepartment of Physiology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland; bDepartment of Biochemistry, University of Lille, Lille, France. Monooxygenase enzyme activities are used as biomarkers of exposure in fish. It is common practice when conducting laboratory experiments with fish to minimize the variationcausing factors by standardizing the study conditions. Equally important should be the minimization of variability caused by the selected fish breed. In this study, interstrain variation on the 7-ethoxyresorufin-0-deethylase (EROD) activity of two rainbow trout strains was determined. Simultaneously, the effect of sample processing temperature on the possible loss of EROD activity was studied. Juvenile rainbow trout of two different strains, Savon taimen and Nilakka, were exposed to 20mg/kg of /!I-naphthoflavone (BNF) by intraperitoneal injection. Control fish were given a pure olive oil injection. Seven days after the injection fish were killed and the microsome preparation carried out according to common procedures. In order to determine the effect of temperature change during the procedure on final enzyme activities, liver samples were divided into equal sections to undergo four differently cooled homogenizing treatments: (1) ice/ice (homogenization and
128
Abstracts
resuspension); (2) ice/no ice; (3) no ice/ice; (4) no ice/no ice. EROD enzyme activities in control fish showed no statistical differences either between strains or between treatments. In BNF-exposed fish, however, an interstrain difference in EROD induction levels was denoted: induction potential of Strain 1 seemed to be about 1.5-fold higher than that of Strain 2. Furthermore, the high temperature-invoked loss of activity was more prominent in Strain 2. Differences of induced EROD activities between treatments were also statistically significant. In exposure studies the effects of toxic compounds can easily be plotted behind the variability caused by the strain itself. This can be taken care of by using the same fish population, at least in consistent studies. These results also emphasise the importance of proper icing as well as careful handling of samples during hepatic microsome preparation.
PII:
SOl41-1136(98)00039-7
Cytochrome P4051A Expression and Localization
in Organs of the Minke Whale
(Balaenoptera acutorostrafu). J. J. STEGEMAN,” C. A. MILLER,” J. BEYER,b M. J. MOORE” AND A. GOKSIZIYR. b aBiology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA; bLaboratory of Marine Molecular Biology, Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway.
Hepatic CYPlA expression has been used as a marker of exposure of animals to aryl hydrocarbon receptor agonists in most vertebrate groups, including marine mammals (White et al. (1994) Toxicology and Applied Pharmacology 126, 45-57). Analysis of extrahepatic organs can show whether expression occurs elsewhere in the body. We examined the levels of CYPlA activity (EROD) in liver microsomes of minke whale, comparing these to rates of PROD activity, and to the content of polychlorinated biphenyls (PCBs) and other contaminant residues in blubber. Then multiple organs were examined by immunohistochemistry for expression of CYPlA. Animals were sampled in the summer of 1992 from near 6769”N and 12-16”E. Hepatic microsomes were prepared from liver tissue. In seven animals examined the rates of EROD ranged from 352 to 740 pmol/min/mg. PROD rates ranged from 10 to 26 pmol/min/mg, and did not correlate with EROD rates. Neither EROD nor PROD activity correlated with the content of PCBs, dichloro-diphenyl-trichloroethane (DDT), chlordanes or hexachlorocyclohexanes. Organs examined by immunohistochemistry included liver, lung, kidney, heart, spleen and brain. Sections of formalin-fixed, paraffin-embedded tissue were stained with monoclonal antibody 1-12-3 to scup CYPlA, which preferentially recognizes CYPlAl forms in mammals. The degree of staining varied among the seven animals examined. In liver, the staining of hepatocytes ranged from mild to very strong; endothelial cells in the liver stained less frequently and less strongly than did hepatocytes. In kidney, there was staining of endothelial cells in most vessels, and occasional staining of urinary epithelium. In heart and lung there was common and occasionally strong endothelial cell staining. In spleen there was rare capillary endothelial staining. In brain (obtained from one individual only) there was rare but moderate endothelial staining. The staining of CYPlA in liver and in endothelium in multiple organs of minke whales indicates that Ah receptor agonists are acting throughout the body of these animals. Similar results have been seen in odontocetes fromthewesternNorthAtlantic.Thecausativeagent(s)remainto beidentified.(Supportfrom NOAA-Sea Grants NA46RG0470-R/P61 and R/P-53, and RARGOM.)
127
SOl41-1136(98)00037-3
Initial Purification of CYPZ-Related Isoforms of Hepatic Cytochrome P450 from Channel Catfish (Zc~afurus punctatus). E. J. PERKINS AND D. SCHLENK. Department of
Pharmacology, School of Pharmacy, University of Mississippi, University, Mississippi 38677, USA. The purification and characterization of the cytochrome P450 monooxygenase system in fish has historically been limited to a few species. The isoform composition and functional characteristics of this system have not been well characterized in the channel catfish, a species which is gaining increasing favor as a model for toxicology studies. Immunochemical studies show that the channel catfish expresses at least four proteins that are crossreactive with anti-CYP2Kl polyclonal antibodies. All of these putative isoforms are similarly reactive with anti-CYP2M 1 antibodies. Isoform-specific variations in the expression of these proteins appear to have age-, sex- and chemical-dependent characteristics. The object of the present study was to purify the CYP2-related proteins from liver microsomes prepared from untreated mature catfish. Microsomes were prepared by standard methods and solubilized with 0.6% sodium cholate. Solubilized microsomes were applied initially to a phenyl-sepharose CL4B column (25x30 cm) and eluted with increasing detergent concentrations, resulting in three P450-containing peaks. Pooled fractions were subsequently added to a DEAE-sepharose CL-6B column and eluted with a linear gradient of sodium chloride (0.5~). Two proteins which are cross-reactive with anti-CYP2 antibodies (47 and 52 kDa) were co-isolated, with 17-22-fold purification as assessed by SDS-PAGE and western blotting. Further purification experiments are ongoing.
PII:
SOl41-1136(98)00038-S
Interstrain Variation on the Hepatic Monooxygenase Enzyme Activities of Rainbow Trout: Induction and Sample Preparation. 0. RITOLA,” K. KOPONEN,” V. VAN MIEGEMb
AND P. LINDSTROM-SEPPA.” aDepartment of Physiology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland; bDepartment of Biochemistry, University of Lille, Lille, France. Monooxygenase enzyme activities are used as biomarkers of exposure in fish. It is common practice when conducting laboratory experiments with fish to minimize the variationcausing factors by standardizing the study conditions. Equally important should be the minimization of variability caused by the selected fish breed. In this study, interstrain variation on the 7-ethoxyresorufin-0-deethylase (EROD) activity of two rainbow trout strains was determined. Simultaneously, the effect of sample processing temperature on the possible loss of EROD activity was studied. Juvenile rainbow trout of two different strains, Savon taimen and Nilakka, were exposed to 20mg/kg of /!I-naphthoflavone (BNF) by intraperitoneal injection. Control fish were given a pure olive oil injection. Seven days after the injection fish were killed and the microsome preparation carried out according to common procedures. In order to determine the effect of temperature change during the procedure on final enzyme activities, liver samples were divided into equal sections to undergo four differently cooled homogenizing treatments: (1) ice/ice (homogenization and
128
Abstracts
resuspension); (2) ice/no ice; (3) no ice/ice; (4) no ice/no ice. EROD enzyme activities in control fish showed no statistical differences either between strains or between treatments. In BNF-exposed fish, however, an interstrain difference in EROD induction levels was denoted: induction potential of Strain 1 seemed to be about 1.5-fold higher than that of Strain 2. Furthermore, the high temperature-invoked loss of activity was more prominent in Strain 2. Differences of induced EROD activities between treatments were also statistically significant. In exposure studies the effects of toxic compounds can easily be plotted behind the variability caused by the strain itself. This can be taken care of by using the same fish population, at least in consistent studies. These results also emphasise the importance of proper icing as well as careful handling of samples during hepatic microsome preparation.
PII:
SOl41-1136(98)00039-7
Cytochrome P4051A Expression and Localization
in Organs of the Minke Whale
(Balaenoptera acutorostrafu). J. J. STEGEMAN,” C. A. MILLER,” J. BEYER,b M. J. MOORE” AND A. GOKSIZIYR. b aBiology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA; bLaboratory of Marine Molecular Biology, Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway.
Hepatic CYPlA expression has been used as a marker of exposure of animals to aryl hydrocarbon receptor agonists in most vertebrate groups, including marine mammals (White et al. (1994) Toxicology and Applied Pharmacology 126, 45-57). Analysis of extrahepatic organs can show whether expression occurs elsewhere in the body. We examined the levels of CYPlA activity (EROD) in liver microsomes of minke whale, comparing these to rates of PROD activity, and to the content of polychlorinated biphenyls (PCBs) and other contaminant residues in blubber. Then multiple organs were examined by immunohistochemistry for expression of CYPlA. Animals were sampled in the summer of 1992 from near 6769”N and 12-16”E. Hepatic microsomes were prepared from liver tissue. In seven animals examined the rates of EROD ranged from 352 to 740 pmol/min/mg. PROD rates ranged from 10 to 26 pmol/min/mg, and did not correlate with EROD rates. Neither EROD nor PROD activity correlated with the content of PCBs, dichloro-diphenyl-trichloroethane (DDT), chlordanes or hexachlorocyclohexanes. Organs examined by immunohistochemistry included liver, lung, kidney, heart, spleen and brain. Sections of formalin-fixed, paraffin-embedded tissue were stained with monoclonal antibody 1-12-3 to scup CYPlA, which preferentially recognizes CYPlAl forms in mammals. The degree of staining varied among the seven animals examined. In liver, the staining of hepatocytes ranged from mild to very strong; endothelial cells in the liver stained less frequently and less strongly than did hepatocytes. In kidney, there was staining of endothelial cells in most vessels, and occasional staining of urinary epithelium. In heart and lung there was common and occasionally strong endothelial cell staining. In spleen there was rare capillary endothelial staining. In brain (obtained from one individual only) there was rare but moderate endothelial staining. The staining of CYPlA in liver and in endothelium in multiple organs of minke whales indicates that Ah receptor agonists are acting throughout the body of these animals. Similar results have been seen in odontocetes fromthewesternNorthAtlantic.Thecausativeagent(s)remainto beidentified.(Supportfrom NOAA-Sea Grants NA46RG0470-R/P61 and R/P-53, and RARGOM.)