- Email: [email protected]
Metabolic activation of carcinogens by three marine invertebrates
Abstracts
213
ethoxycoumarin-O-deethylase (ECOD) activities over 6pmol/mg protein per min. The pituitary gland from both cod and rainbow trout showed activities of 2-4 pmol/mg protein per min towards 7-ethoxycoumarin and 7ethoxyresorufin. The P450 system in cod and rainbow trout brain was found to be inducible to various extents. Treatment of rainbow trout with flnaphthoflavone (BNF) increased the 7-ethoxyresorufin-O-deethylase (EROD) activity from four- to eight-fold in the various brain parts whereas in the cod a significant induction (eight-fold) could be seen only in the olfactory bulbs. The ECOD activity was induced (from four- to five-fold) in the rainbow trout hypothalamus and the optic lobe, whereas no significant changes were seen in the cod brain by BNF treatment. The present study shows that the cod and rainbow trout brains contain an active cytochrome P450 system, which, at least in some parts of the tissue, could be induced. The toxicological consequences and physiological function of the brain P450 in fishes should be further investigated.
Metabolic Activation of Carcinogens by Three Marine Invertebrates. JANE W. MARSH,* JAMES K. C H I P M A N * & DAVID R. LIVINGSTONE.:~ * School of Biochemistry, University of Birmingham, Edgbaston, Birmingham, UK, BI5 2TT & ~Plymouth Marine Laboratory, Citadel Hill, Plymouth, UK, PL1 2PB. The ability of mussels (Mytilus edulis), shore crabs (Carcinus maenus), and common starfish (Asterias rubens) to convert 2-acetylaminofluorene (2AAF), 2-aminofluorene (2AF), N-hydroxy-2AAF, and benzo(a)pyrene (BP) to reactive and mutagenic products was compared. In a Salmonella typhimurium pre-incubation assay, with strain TA98, post-mitochondrial supernatants ($9) of crab hepatopancreas and mussel digestive gland converted 2AAF (1-100 #g/plate), 2AF (1-10/~g/plate), and N-hydroxy2AAF (0.1-1#g/plate) to products that were mutagenic. The mussel appeared to possess a soluble deacetylase (paraoxon inhibited mutagenicity by 96%) capable of converting 2AAF to 2AF, and an enzyme, similar to mammalian microsomal flavin-containing monooxygenase, which oxidized 2AF (methimazole inhibited activation by 69% but cytochrome P450 inhibitors, DPEA, ~-naphthoflavone, and metyrapone had no effect). Activation of 2AAF by the crab was also inhibited by paraoxon (88%) but the oxidative activation step was not inhibited by either methimazole or the above cytochrome P450 inhibitors. The starfish pyloric caeca $9 also activated 2AF and N-hydroxy-2AAF at the doses shown above but surprisingly did not convert 2AAF to mutagens (dose up to 500/~g/plate). The deacetylation and/or oxidation systems of the starfish, although
214
Abstracts
activating N-hydroxy-2AAF and 2AF, respectively, therefore appear not to metabolize 2AAF. In smilar assays, no mutagenicity was seen with BP (up to 200 #g/plate), although covalent binding to protein (2-3-5"9 fmol bound/mg protein) and to D N A (4.1-5.5 fmol bound/mg DNA) was seen in all three invertebrates after administration of [3H]BE The results show the ability of marine invertebrates to activate certain mammalian carcinogens to reactive and mutagenic products and illustrate differences in the enzyme systems involved. The authors acknowledge the support of the NERC.
Fractionation of Planar and Non-planar Polychlorinated Biphenyis for Determination in Environmental Samples. DONALD E. TILLITT, TED R. SCHWARTZ, KEVIN P. FELTZ & JON LEBO. US Fish and Wildlife Service, National Fisheries Contaminant Research Center, Rt 2, 4200 New Haven Rd, Columbia, Missouri 65201, USA. Concern about the occurrence of PCB congeners in the environment centers on their persistent nature, propensity for bioaccumulation, and toxicity to fish and wildlife. A subset of the PCBs, mono- or non-ortho-C1 substituted planar congeners, elicit dioxin-like activity and are extremely toxic to fish and wildlife. Analytic methods for the determination of the planar PCBs have improved in recent years, but these methods remain time-consuming and costly, and are almost exclusively limited to GC/MS quantitation. We report the development and application of an automated HPLC-based method for fractionation and enrichment of the non-o,o'-C1 substituted PCB congeners (IUPAC Nos 77, 126, 169, and 81), the mono-o,o'-C1 substituted PCB congeners (IUPAC Nos 81, 105, 114, 118, 123, 156, 157, 167, and 189), and the di-o,o'-C1 PCB congeners (IUPAC Nos 128, 158, and 166) with activated carbon by using commercially available instrumentation. Environmental samples (fish tissue, fish eggs, bird eggs, and sediments from fresh and estuary waters) were selected for the validation of this method. Quantitation of these analyses was successful by GC/ECD with dualcolumn confirmation to a limit of approximately 100 ppt (pg/g). Recoveries were nearly quantitative in this range and there were no interferences encountered in the samples tested as confirmed by GC/MS. In conclusion, the method described allows simple, repeatable separation and quantitation of planar PCBs from environmental samples. Additionally, this method should allow more laboratories access to the capabilities of planar PCB analysis, which will in turn help with their assessment in cases of environmental pollution by PCBs.
213
ethoxycoumarin-O-deethylase (ECOD) activities over 6pmol/mg protein per min. The pituitary gland from both cod and rainbow trout showed activities of 2-4 pmol/mg protein per min towards 7-ethoxycoumarin and 7ethoxyresorufin. The P450 system in cod and rainbow trout brain was found to be inducible to various extents. Treatment of rainbow trout with flnaphthoflavone (BNF) increased the 7-ethoxyresorufin-O-deethylase (EROD) activity from four- to eight-fold in the various brain parts whereas in the cod a significant induction (eight-fold) could be seen only in the olfactory bulbs. The ECOD activity was induced (from four- to five-fold) in the rainbow trout hypothalamus and the optic lobe, whereas no significant changes were seen in the cod brain by BNF treatment. The present study shows that the cod and rainbow trout brains contain an active cytochrome P450 system, which, at least in some parts of the tissue, could be induced. The toxicological consequences and physiological function of the brain P450 in fishes should be further investigated.
Metabolic Activation of Carcinogens by Three Marine Invertebrates. JANE W. MARSH,* JAMES K. C H I P M A N * & DAVID R. LIVINGSTONE.:~ * School of Biochemistry, University of Birmingham, Edgbaston, Birmingham, UK, BI5 2TT & ~Plymouth Marine Laboratory, Citadel Hill, Plymouth, UK, PL1 2PB. The ability of mussels (Mytilus edulis), shore crabs (Carcinus maenus), and common starfish (Asterias rubens) to convert 2-acetylaminofluorene (2AAF), 2-aminofluorene (2AF), N-hydroxy-2AAF, and benzo(a)pyrene (BP) to reactive and mutagenic products was compared. In a Salmonella typhimurium pre-incubation assay, with strain TA98, post-mitochondrial supernatants ($9) of crab hepatopancreas and mussel digestive gland converted 2AAF (1-100 #g/plate), 2AF (1-10/~g/plate), and N-hydroxy2AAF (0.1-1#g/plate) to products that were mutagenic. The mussel appeared to possess a soluble deacetylase (paraoxon inhibited mutagenicity by 96%) capable of converting 2AAF to 2AF, and an enzyme, similar to mammalian microsomal flavin-containing monooxygenase, which oxidized 2AF (methimazole inhibited activation by 69% but cytochrome P450 inhibitors, DPEA, ~-naphthoflavone, and metyrapone had no effect). Activation of 2AAF by the crab was also inhibited by paraoxon (88%) but the oxidative activation step was not inhibited by either methimazole or the above cytochrome P450 inhibitors. The starfish pyloric caeca $9 also activated 2AF and N-hydroxy-2AAF at the doses shown above but surprisingly did not convert 2AAF to mutagens (dose up to 500/~g/plate). The deacetylation and/or oxidation systems of the starfish, although
214
Abstracts
activating N-hydroxy-2AAF and 2AF, respectively, therefore appear not to metabolize 2AAF. In smilar assays, no mutagenicity was seen with BP (up to 200 #g/plate), although covalent binding to protein (2-3-5"9 fmol bound/mg protein) and to D N A (4.1-5.5 fmol bound/mg DNA) was seen in all three invertebrates after administration of [3H]BE The results show the ability of marine invertebrates to activate certain mammalian carcinogens to reactive and mutagenic products and illustrate differences in the enzyme systems involved. The authors acknowledge the support of the NERC.
Fractionation of Planar and Non-planar Polychlorinated Biphenyis for Determination in Environmental Samples. DONALD E. TILLITT, TED R. SCHWARTZ, KEVIN P. FELTZ & JON LEBO. US Fish and Wildlife Service, National Fisheries Contaminant Research Center, Rt 2, 4200 New Haven Rd, Columbia, Missouri 65201, USA. Concern about the occurrence of PCB congeners in the environment centers on their persistent nature, propensity for bioaccumulation, and toxicity to fish and wildlife. A subset of the PCBs, mono- or non-ortho-C1 substituted planar congeners, elicit dioxin-like activity and are extremely toxic to fish and wildlife. Analytic methods for the determination of the planar PCBs have improved in recent years, but these methods remain time-consuming and costly, and are almost exclusively limited to GC/MS quantitation. We report the development and application of an automated HPLC-based method for fractionation and enrichment of the non-o,o'-C1 substituted PCB congeners (IUPAC Nos 77, 126, 169, and 81), the mono-o,o'-C1 substituted PCB congeners (IUPAC Nos 81, 105, 114, 118, 123, 156, 157, 167, and 189), and the di-o,o'-C1 PCB congeners (IUPAC Nos 128, 158, and 166) with activated carbon by using commercially available instrumentation. Environmental samples (fish tissue, fish eggs, bird eggs, and sediments from fresh and estuary waters) were selected for the validation of this method. Quantitation of these analyses was successful by GC/ECD with dualcolumn confirmation to a limit of approximately 100 ppt (pg/g). Recoveries were nearly quantitative in this range and there were no interferences encountered in the samples tested as confirmed by GC/MS. In conclusion, the method described allows simple, repeatable separation and quantitation of planar PCBs from environmental samples. Additionally, this method should allow more laboratories access to the capabilities of planar PCB analysis, which will in turn help with their assessment in cases of environmental pollution by PCBs.