John J. Stegeman

Cytochrome P-450 monooxygenase systems in aquatic species: carcinogen metabolism and biomarkers for carcinogen and pollutant exposure.

High levels of polynuclear aromatic hydrocarbon (PAH) carcinogens commonly occur in aquatic systems where neoplasms arise in fish and other animals. Enzymes that transform PAHs can act in initiating these diseases and can indicate the contamination of fish by carcinogens and other pollutants. Cytochrome P-450 has similar roles in activating PAH carcinogens in fish and mammalian species. PAHs and many chlorinated hydrocarbons, e.g., polychlorinated biphenyls (PCBs) induce a form of cytochrome P-450 in fish that is the primary catalyst of PAH metabolism. The induction of this P-450 in fish can accelerate the disposition of hydrocarbons, but can also enhance the formation of carcinogenic derivatives of PAHs. Invertebrates have lower rates of PAH metabolism than fish. These rates are not obviously inducible by exposure to PAHs or PCBs. The lower rates of foreign compound metabolism contribute to higher pollutant residue levels in bivalve mollusks (clams, mussels, etc.) than in fish and may limit the involvement of some procarcinogens (requiring activation) in disease processes in invertebrates. The induction of P-450 forms can indicate the exposure of fish to PAHs, PCBs, and other toxic compounds. This is not restricted to carcinogens. Environmental induction has been detected in fish from contaminated areas by use of catalytic assay, antibodies to fish P-450, and cDNA probes that hybridize with P-450 messenger RNA. Application of these methods can provide sensitive biological monitoring tools that can detect environmental contamination of fish by some carcinogens and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

An alternative 7-ethoxyresorufin O-deethylase activity assay: a continuous visible spectrophotometric method for measurement of cytochrome P-450 monooxygenase activity

A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The two assays yielded identical results with both beta-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (epsilon 572 = 73 mM-1 cm-1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the K1 for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.

Cytochrome P4501A induction and inhibition by 3,3′,4,4′-tetrachlorobiphenyl in an Ah receptor-containing fish hepatoma cell line (PLHC-1)

The induction of cytochrome P4501A1 (CYP1A1) in rat hepatoma cells has been used by some investigators to determine ‘dioxin equivalents’ in environmental samples, including extracts of fish tissues. However, the relative potency of inducing compounds may vary between species, suggesting the need for taxon-specific model systems. In this paper we present an initial characterization of CYP1A induction in one such system, a teleost liver cell line (PLHC-1) derived from a hepatocellular carcinoma of Poeciliopsis lucida (Hightower, L.E. and Renfro, J.L., 1988. J. Exp. Zool. 248, 290). Specific binding of the photoaffinity ligand 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin([125I]N3Br2DD) to proteins in PLHC-1 cytosol indicated the presence of the Ah receptor, which is known to control CYP1A induction in mammals. 3,3′,4,4′-Tetrachlorobiphenyl (TCB) induced a microsomal protein in PLHC-1 cells that was recognized by monoclonal antibody (MAb) 1-12-3 to scup CYP1A1 (P450E) on immunoblots. Immunohistochemical staining of whole cells with MAb 1-12-3 showed specific recognition of CYP1A induced by TCB. No staining was seen in untreated or vehicle-treated cells. There was an excellent quantitative correlation between amounts of CYP1A protein detected immunohistochemically and in immunoblots of cell homogenates. In a dose response experiment, maximal induction of ethoxyresorufin O-deethylase (EROD) activity occurred at 0.1 μM TCB; at higher concentrations (1 and 10 μM), EROD activity was reduced as compared to the activity at 0.1 μM TCB. In contrast, immunoreactive CYP1A protein increased with increasing TCB concentration up to 10 μM. The loss of EROD activity at high concentrations of TCB did not result from changes in cell number or viability. The apparent inhibition or inactivation of CYP1A catalytic activity by the higher concentrations of halogenated biphenyls has been seen, but not generally recognized, both in vivo and in cultured cells from diverse vertebrate species. PLHC-1 cells may be a good model system for studying Ah receptor-mediated regulation of gene expression, for determining the fish-specific toxic or inducing potency of halogenated aromatic hydrocarbon congeners, and for investigating the mechanism of CYP1A inhibition or inactivation by environmental contaminants such as TCB.

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

BackgroundIncreasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.ResultsZebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.ConclusionsTranscript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.

Cytochrome P450 forms in fish: Catalytic, immunological and sequence similarities

1. Hepatic microsomal enzymes from teleost and elasmobranch fishes catalyse a diversity of monooxygenase reactions, consistent with the presence of multiple, distinct P450 forms. Protein purification and immunological studies have confirmed that multiple microsomal P450s occur in teleosts. 2. A member of the aromatic hydrocarbon-inducible P450 IA family is present in all fish species examined to date. This protein appears to be most closely related to P450 IA1. Certain of the immunological probes for a teleost P450 IA1 (scup P450E) appear to be reagent antibodies, recognizing the homologous protein in members of all vertebrate groups examined. The nature of the epitope recognized by such antibodies is not known. 3. Based on immunological and amino acid sequence comparisons, teleost P450 IA1 appears to be orthologous to both P450 IA1 and P450 IA2 in mammals. Multiple P450 IA genes may appear in teleosts, but divergence on separate lines from that involving mammalian P450 IA2 could include additional, new members (P450 IA3?) of the P450 IA family. 4. There are greater similarities in the N-terminal amino acid sequences of different teleost (scup and trout) P450 IA1 forms, than seen in the N-terminal sequence relationships found in P450 IA1 of mammalian species. Whether this similarity extends to the rest of these teleost proteins is unknown. 5. The induction of P450 IA1 in teleosts involves transcriptional and translational events. However, the temporal patterns involved in induction of mRNA or protein are different from those in mammalian species, indicating additional aspects of the regulation in teleosts. 6. Relationships between other teleost and mammalian P450 forms, or between other P450 forms isolated from different teleosts, remain to be conclusively established. However, certain relationships are suggested, based on catalytic and other comparisons.

Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): Cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450☆

Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors.

Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish.

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.

An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish Stenotomus chrysops.

Hepatic microsomal cytochrome P-450 from the untreated coastal marine fish scup, Stenotomus chrysops, was solubilized and resolved into five fractions by ion-exchange chromatography. The major fraction, cytochrome P-450E (Mr = 54,300), was further purified to a specific content of 11.7 nmol heme/mg protein and contained a chromophore absorbing at 447 nm in the CO-ligated, reduced difference spectrum. NH2-terminal sequence analysis of cytochrome P-450E by Edman degradation revealed no homology with any known cytochrome P-450 isozyme in the first nine residues. S. chrysops liver NADPH-cytochrome P-450 reductase, purified 225-fold (Mr = 82,600), had a specific activity of 45-60 U/mg with cytochrome c, contained both FAD and FMN, and was isolated as the one-electron reduced semiquinone. Purified cytochrome P-450E metabolized several substrates including 7-ethoxycoumarin, acetanilide, and benzo[a]pyrene when reconstituted with lipid and hepatic NADPH-cytochrome P-450 reductase from either S. chrysops or rat. The purified, reconstituted monooxygenase system was sensitive to inhibition by 100 microM 7,8-benzoflavone, and analysis of products in reconstitutions with purified rat epoxide hydrolase indicated a preference for oxidation on the benzo-ring of benzo[a]pyrene consistent with the primary features of benzo[a]pyrene metabolism in microsomes. Cytochrome P-450E is identical to the major microsomal aromatic hydrocarbon-inducible cytochrome P-450 by the criteria of molecular weight, optical properties, and catalytic profile. It is suggested that substantial quantities of this aromatic hydrocarbon-inducible isozyme exist in the hepatic microsomes of some untreated S. chrysops. The characterization of this aryl hydrocarbon hydroxylase extends our understanding of the metabolism patterns observed in hepatic microsomes isolated from untreated fish.

Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish stenotomus versicolor

NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup (Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg2+. The kinetic properties of benzo[a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured Km values for benzo[a]pyrene between 4 × 10−7 M and 4 × 10−5 M. The Km for aminopyrine was 7 × 10−4 M, and NADPH-cytochrome c reductase had Km values of 2.1 × 10−5 M and 1.3 × 10−5 M for cytochrome c and NADPH. respectively. NADH supported benzo[a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.