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Induction of cytochrome P4501A in endothelium: the rete mirabile as a model for the study of endothelial CYP1A
Abstracts
305
Evaluation of the risk to these- animals requires an understanding of their capacity to respond to contaminant exposure, including the activity and regulation of enzymes involved in pollutant metabolism, such as the cytochromes P450. These studies focused on the presence and metabolic capabilities of P450 subfamilies 1A and 2B in livers of fur seals (Cullorhinus ursinus) from the Pribiloff Islands Alaska. A CYPlAl-specific monoclonal antibody (MAb l-12-3) recognized a single band on immunoblots of hepatic microsomal protein from adult seals and pups. This apparent seal CYPlA form showed variable expression, suggesting induction by environmental chemicals in some animals. Ethoxyresorufin 0-deethylase (EROD activity, specific for CYPlA forms in other vertebrates, was correlated with P450lA protein levels. Levels of the CYP2B-associated activity pentoxyresorufin 0-deethylase (PROD) were very low and were highly correlated with EROD activity, suggesting that there is a single catalyst (CYPlA). Other investigators have speculated that CYP2B function is reduced in pinmpeds, based on patterns of PCB congeners in blubber. Immunoblotting of fur seal hepatic microsomes with polyclonal antibodies to mammalian 2B forms showed recognition of seal proteins by anti-CYP2B4 (rabbit) and anti-CYP2Bll (dog) but not by antXYP2Bl (rat). The results indicate the possibility of one or more CYP2B-related forms in fur seals, but the functional significance of these forms is not yet known.
Induction of Cytochrome P45OlA in Endothelium: the Rete Mirabile as a Model for the Study of Endothelial CYPlA. J. S. JOY,” M. J. MOORE,” J. SCHELLb & J. J. STEGEMAN.” aBiology Department, Woocis Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA; bTerra, Inc., Tallahassee, Florida 32303, USA.
Previous studies have shown that endothelium is a major site of induction of cytochrome P450lA (CYPlA) in vertebrates, from fish to mammals (Mol. Pharmacof., 36, 723-729, 1989). CYPlA induction in endothelium could affect cardiovascular function, elicit toxicity and possibly act in removing compounds, thus contributing to the ‘barrier’ function of endothelium. We are investigating the removal of AhR agonists/CYPlA substrates from blood by examining the dose-dependent penetration of induction of endothelial cell into the counter-current capillary bed of the rete mirabile in the swim bladder of the American eel, Anguillu rostra&. Increasing doses of 3,3’,4,4’-tetrachlorobiphenyl (TCB; 0.1, 1.O, and 10 mg/kg) resulted in dose-dependent increases in CYPlA content and EROD activities in eel hepatic microsomes, although there was no significant increase in induction at 20 mg/kg TCB. Immunohistochemical analysis with MAb l-12-3 revealed increasing induction of CYPlA in the liver, heart, gill and kidney in the 1, 10 and 20 mg/kg TCB dose groups. Likewise, CYPlA in the endothelium of the rete mirabile was induced in all dose groups. In control eels and eels dosed at 0.1 mg/kg TCB, CYPlA staining was seen only in pre-rete arteries. Eels dosed at 1, 10, and 20 mg/kg TCB also showed CYPlA staining in the afferent arteriolar capillaries of the rete and a dose-dependent penetration of endothelial cell CYPlA staining into the capillary bed. Increasing doses of P-naphthoflavone (BNF; 0.1, 1, 5, 10, and 100 mg/kg) resulted in increases in EROD activity and CYPlA content in hepatic microsomes even at the higher doses. As with the TCB-treated eels, immunohistochemical analysis revealed induction in gill, kidney, heart and liver in those animals dosed with BNF. Eels which received BNF showed dose-dependent increases in the degree and the penetration of endothelial cell staining into the rete itself. Only one
306
Abstracts
of the eels dosed with 0.1 mg/kg BNF showed CYPlA staining in the heart pole endothelium; however, all of the eels dosed with 1.O mg/kg BNF were induced in the heart pole endothelium. Moreover, some animals given 5, 10 and lOmg/kg BNF showed induction of CYPlA throughout the rete, including the endothelium of efferent venular capillaries. We conclude that the penetration of endothelial cell CYPlA expression into this vascular bed reflects greater concentrations of inducer reaching the distal circulation. The results are consistent with the hypothesis that endothelial cell CYPlA may participate in removal of AhR agonist/CYPlA substrates from the circulation. Supported by the Air Force Office of Scientific Research and the Lyons Fellowship.
Cytochrome P4501A Expression in Teleost Chondroid Cells: a Possible Site of Endogenous Function of the Ah-Receptor-CYPlA Loop. J. J. STEGEMAN,’ P. LINDSTROM-SEPPA,b T. KNIPE,” S. SUTER,’ R. M. SMOLOWITZ” & E. HESTERMANN.” “Biology Department, Woods Hole Oceanographic Institution, Woo& Hole, Massachusetts 02543, USA; bDepartment of Physiology, University of Kuopio, 70211 Kuopio, Finland.
There are speculations that the Ah receptor functions in regulating cell growth or differentiation (Ann. Rev. Pharmacol. Toxicof., 22, 517, 1982), and that an endogenous ligand might be metabolized by CYPl A, (Mol. Endocrinof., 5, 1202, 1991) constituting a ‘cell regulation loop’. An endogenous regulatory function has not been defined, partly because a tissue has not been found where AhR-regulated gene(s) are distinctly and highly expressed, without exogenous or endogenous toxic ligands possibly having evoked the response. We have identified a cell type in fish in which CYPlA is very highly expressed, in the absence of exogenous ligand for the AhR, and in the absence of high level CYPlA expression in any other cells in the body. Untreated fathead minnow (Pimephales prome/as) were fixed, decalcified, embedded, and sectioned whole (5 pm sections). Immunohistochemical analysis of untreated animals with the CY PI Al -specific monoclonal antibody 1-12-3 produced intense staining in novel, structural mesenchymal cells (termed chondroid cells) located in regions resembling growth plates and at articular surfaces. An identical pattern of staining was seen with monoclonal antibody 1-71-3, that recognizes a distinct fish CYPlA epitope. Northern blot analysis of mRNA from tissue carved from a chondroid-rich region showed a band of appropriate size hybridizing to a fish CYPl Al cDNA. In situ reverse-transcriptase-PCR using teleost CYPlAl specific primers also indicated expression of CYPlA mRNA in chondroid cells. In untreated fish CYPlA was not detectable by immunohistochemistry in any cell types other than chondroid. Treatment with the AhR agonists 3,3’,4,4’-tetrachlorobiphenyl or fl-naphthoflavone elicited dose-dependent induction of CYPlA in liver and extrahepatic organs, detected by immunoblot and by IHC with MAb 1-12-3 and 1-71-3. Subsequent IHC studies have identified ‘endogenous’ expression or induction of CYPlA in similar cells (chondroid structures) in at least 8 species, including in salmonids and zebrafish, confirming that chondroid CYPlA expression is widespread. A strong, endogenous expression of CYPlA in cells associated with the vertebrate condition is consistent with our observations (Arch. Biochem. Biophys., 310, 218, 1994) that a recognizable AhR appears in phylogeny soon after vertebrate emergence. The results suggest that the Ah-receptor and CYPlA may have endogenous functions involving growth or differentiation of bone or associated
305
Evaluation of the risk to these- animals requires an understanding of their capacity to respond to contaminant exposure, including the activity and regulation of enzymes involved in pollutant metabolism, such as the cytochromes P450. These studies focused on the presence and metabolic capabilities of P450 subfamilies 1A and 2B in livers of fur seals (Cullorhinus ursinus) from the Pribiloff Islands Alaska. A CYPlAl-specific monoclonal antibody (MAb l-12-3) recognized a single band on immunoblots of hepatic microsomal protein from adult seals and pups. This apparent seal CYPlA form showed variable expression, suggesting induction by environmental chemicals in some animals. Ethoxyresorufin 0-deethylase (EROD activity, specific for CYPlA forms in other vertebrates, was correlated with P450lA protein levels. Levels of the CYP2B-associated activity pentoxyresorufin 0-deethylase (PROD) were very low and were highly correlated with EROD activity, suggesting that there is a single catalyst (CYPlA). Other investigators have speculated that CYP2B function is reduced in pinmpeds, based on patterns of PCB congeners in blubber. Immunoblotting of fur seal hepatic microsomes with polyclonal antibodies to mammalian 2B forms showed recognition of seal proteins by anti-CYP2B4 (rabbit) and anti-CYP2Bll (dog) but not by antXYP2Bl (rat). The results indicate the possibility of one or more CYP2B-related forms in fur seals, but the functional significance of these forms is not yet known.
Induction of Cytochrome P45OlA in Endothelium: the Rete Mirabile as a Model for the Study of Endothelial CYPlA. J. S. JOY,” M. J. MOORE,” J. SCHELLb & J. J. STEGEMAN.” aBiology Department, Woocis Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA; bTerra, Inc., Tallahassee, Florida 32303, USA.
Previous studies have shown that endothelium is a major site of induction of cytochrome P450lA (CYPlA) in vertebrates, from fish to mammals (Mol. Pharmacof., 36, 723-729, 1989). CYPlA induction in endothelium could affect cardiovascular function, elicit toxicity and possibly act in removing compounds, thus contributing to the ‘barrier’ function of endothelium. We are investigating the removal of AhR agonists/CYPlA substrates from blood by examining the dose-dependent penetration of induction of endothelial cell into the counter-current capillary bed of the rete mirabile in the swim bladder of the American eel, Anguillu rostra&. Increasing doses of 3,3’,4,4’-tetrachlorobiphenyl (TCB; 0.1, 1.O, and 10 mg/kg) resulted in dose-dependent increases in CYPlA content and EROD activities in eel hepatic microsomes, although there was no significant increase in induction at 20 mg/kg TCB. Immunohistochemical analysis with MAb l-12-3 revealed increasing induction of CYPlA in the liver, heart, gill and kidney in the 1, 10 and 20 mg/kg TCB dose groups. Likewise, CYPlA in the endothelium of the rete mirabile was induced in all dose groups. In control eels and eels dosed at 0.1 mg/kg TCB, CYPlA staining was seen only in pre-rete arteries. Eels dosed at 1, 10, and 20 mg/kg TCB also showed CYPlA staining in the afferent arteriolar capillaries of the rete and a dose-dependent penetration of endothelial cell CYPlA staining into the capillary bed. Increasing doses of P-naphthoflavone (BNF; 0.1, 1, 5, 10, and 100 mg/kg) resulted in increases in EROD activity and CYPlA content in hepatic microsomes even at the higher doses. As with the TCB-treated eels, immunohistochemical analysis revealed induction in gill, kidney, heart and liver in those animals dosed with BNF. Eels which received BNF showed dose-dependent increases in the degree and the penetration of endothelial cell staining into the rete itself. Only one
306
Abstracts
of the eels dosed with 0.1 mg/kg BNF showed CYPlA staining in the heart pole endothelium; however, all of the eels dosed with 1.O mg/kg BNF were induced in the heart pole endothelium. Moreover, some animals given 5, 10 and lOmg/kg BNF showed induction of CYPlA throughout the rete, including the endothelium of efferent venular capillaries. We conclude that the penetration of endothelial cell CYPlA expression into this vascular bed reflects greater concentrations of inducer reaching the distal circulation. The results are consistent with the hypothesis that endothelial cell CYPlA may participate in removal of AhR agonist/CYPlA substrates from the circulation. Supported by the Air Force Office of Scientific Research and the Lyons Fellowship.
Cytochrome P4501A Expression in Teleost Chondroid Cells: a Possible Site of Endogenous Function of the Ah-Receptor-CYPlA Loop. J. J. STEGEMAN,’ P. LINDSTROM-SEPPA,b T. KNIPE,” S. SUTER,’ R. M. SMOLOWITZ” & E. HESTERMANN.” “Biology Department, Woods Hole Oceanographic Institution, Woo& Hole, Massachusetts 02543, USA; bDepartment of Physiology, University of Kuopio, 70211 Kuopio, Finland.
There are speculations that the Ah receptor functions in regulating cell growth or differentiation (Ann. Rev. Pharmacol. Toxicof., 22, 517, 1982), and that an endogenous ligand might be metabolized by CYPl A, (Mol. Endocrinof., 5, 1202, 1991) constituting a ‘cell regulation loop’. An endogenous regulatory function has not been defined, partly because a tissue has not been found where AhR-regulated gene(s) are distinctly and highly expressed, without exogenous or endogenous toxic ligands possibly having evoked the response. We have identified a cell type in fish in which CYPlA is very highly expressed, in the absence of exogenous ligand for the AhR, and in the absence of high level CYPlA expression in any other cells in the body. Untreated fathead minnow (Pimephales prome/as) were fixed, decalcified, embedded, and sectioned whole (5 pm sections). Immunohistochemical analysis of untreated animals with the CY PI Al -specific monoclonal antibody 1-12-3 produced intense staining in novel, structural mesenchymal cells (termed chondroid cells) located in regions resembling growth plates and at articular surfaces. An identical pattern of staining was seen with monoclonal antibody 1-71-3, that recognizes a distinct fish CYPlA epitope. Northern blot analysis of mRNA from tissue carved from a chondroid-rich region showed a band of appropriate size hybridizing to a fish CYPl Al cDNA. In situ reverse-transcriptase-PCR using teleost CYPlAl specific primers also indicated expression of CYPlA mRNA in chondroid cells. In untreated fish CYPlA was not detectable by immunohistochemistry in any cell types other than chondroid. Treatment with the AhR agonists 3,3’,4,4’-tetrachlorobiphenyl or fl-naphthoflavone elicited dose-dependent induction of CYPlA in liver and extrahepatic organs, detected by immunoblot and by IHC with MAb 1-12-3 and 1-71-3. Subsequent IHC studies have identified ‘endogenous’ expression or induction of CYPlA in similar cells (chondroid structures) in at least 8 species, including in salmonids and zebrafish, confirming that chondroid CYPlA expression is widespread. A strong, endogenous expression of CYPlA in cells associated with the vertebrate condition is consistent with our observations (Arch. Biochem. Biophys., 310, 218, 1994) that a recognizable AhR appears in phylogeny soon after vertebrate emergence. The results suggest that the Ah-receptor and CYPlA may have endogenous functions involving growth or differentiation of bone or associated