Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, Mar 1, 2000
Previously we showed that the polychlorinated biphenyl 3,3%,4,4%-tetrachlorobiphenyl (TCB) caused... more Previously we showed that the polychlorinated biphenyl 3,3%,4,4%-tetrachlorobiphenyl (TCB) caused a release of reactive oxygen species (ROS) from cytochrome P450 1A (CYP1A) of the fish scup (Stenotomus chrysops), and from rat and human CYP1A1. This was linked to a TCB-and NADPH-dependent oxidative inactivation of the enzyme, which in scup and rat was inversely related to the rates of TCB oxidation. We examined the relationship between rates of TCB oxidation, CYP1A inactivation and ROS production in liver microsomes from additional vertebrate species, including skate (Raja erinacea), eel (Anguilla rostrata), killifish (Fundulus heteroclitus), winter flounder (Pleuronectes americanus), chicken (Gallus domesticus), cormorant (Phalacrocorax auritus), gull (Larus argentatus), and turtle (Chrysemys picta picta). TCB oxidation rates were induced in all fish and birds treated with aryl hydrocarbon receptor agonists. Induced rates of TCB oxidation were B1 pmol/min/mg microsomal protein in all fish, and 6-14 pmol/min/mg in the birds. In all species but one, TCB oxidation rates correlated positively with EROD rates, indicating likely involvement of CYP1A in TCB oxidation. Incubation of liver microsomes of most species with TCB + NADPH resulted in an immediate (TCB-dependent) inhibition of EROD, and a progressive loss of EROD capacity, indicating an oxidative inactivation of CYP1A like that in scup. NADPH stimulated production of ROS (H 2 O 2 and/or O 2 −) by liver microsomes, slightly in some species (eel) and greatly in others (chicken, turtle). Among the birds and the fish, NADPH-stimulated ROS production correlated positively with EROD activity. TCB caused a significant stimulation of ROS production by liver microsomes of flounder, killifish, cormorant and gull, as well as scup. The stimulation of CYP1A inactivation and ROS generation indicates an uncoupling of CYP1A by TCB in many species, and when compared between species, the rates of CYP1A inactivation correlated inversely with rates of TCB oxidation. Some feature(s) of binding/active site topology may hinder TCB oxidation, enhancing the likelihood for attack of an oxidizing species in the active site.
Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus,... more Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor kB (NF-kB) that rapidly activates genes encoding cytokines, cell surface receptors, cell adhesion molecules and acute phase proteins. Evidence suggests that xenobiotic compounds also may alter the activation of NF-kB. This study had a dual objective of identifying members of the Rel family and examining their activation by xenobiotic compounds in a marine fish model, scup (Stenotomus chrysops). A DNA-protein crosslinking technique demonstrated that liver, kidney and heart each had at least three nuclear proteins that showed specific binding to an NF-kB consensus sequence, with molecular weights suggesting that the proteins potentially corresponded to mouse p50, p65 (RelA) and c-rel. In addition, an :35kD NF-kB binding protein was evident in liver and kidney. The 50 kD protein was immunoprecipitated by mammalian p50-specific antibodies. The presence of Rel members in fish implied by those results was confirmed by RT-PCR cloning of a Rel homology domain
The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) su... more The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3',4,4',5pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6-and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26-and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 µM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 µM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene-and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions.
Abstract Reproductively mature winter flounder (Pseudopleuronectes americanus) were collected fro... more Abstract Reproductively mature winter flounder (Pseudopleuronectes americanus) were collected from three Northeastern US sites with different degrees of polychlorinated biphenyl (PCB) and polynuclear aromatic hydrocarbon (PAH) contamination. Liver PCB concentrations (measured by capillary electron-capture gas chromatography) in fish collected in 1987 and 1988 at New Bedford Harbor (NBH) ranged from 7·4 to 191 μg/g dry wt; at Gaspee Point 3·9–17·7 μg/g; and at Fox Island 1·6–15·1 μg/g. Levels of ethoxyresorufin O-deethylase (EROD) activity were similar in fish of the same reproductive status from the three sites; however, immunoquantitated P-450E homolog (the EROD catalyst) content was significantly higher in NBH fish. This suggests that P-450E catalytic activity is being suppressed in the livers of the NBH animals. Recent studies in our laboratory indicate that competitive inhibition of P-450E catalytic activity by specific PCB congeners is one likely mechanism of this suppression. Hepatic EROD activity and P-450E content were significantly lower in gravid females (EROD, 0·10–0·69 units per nmol P-450; P-450E, 8·4–19% of spectral P-450) than in spent females (EROD, 1·94–3·49; P-450E, 48–109%), and ripe males (EROD, 1·86–3·41; P-450E, 48–84%) at all sites. This is consistent with a hormonal effect on P-450E expression, and thus EROD activity, in gravid females. The data indicate a complex relationship between levels of EROD activity, or P-450E, and tissue PCB concentrations in highly contaminated fish. How these variables are linked to altered endocrine or gonadal function is not yet known.
Antibodies prepared against the major (3-naphthoflavone (BNF)-inducible cytochrome P450 (P450) fo... more Antibodies prepared against the major (3-naphthoflavone (BNF)-inducible cytochrome P450 (P450) forms from three species of fish (rainbow trout, Atlantic cod, and scup) well separated in teleost phylogeny, were used to investigate the immunochemical relatedness of liver microsomal P450 in different species of BNFtreated fish and rat. Rabbit polyclonal IgG against all three P450s and mouse monoclonal antibodies prepared against scup P450E were employed in this study. Liver microsomes were prepared from BNF-treated specimens of hagfish, herring, rainbow trout, cod, scup, perch, plaice and rat. With Western blotting it was shown that the various antibodies cross-reacted with a protein band in liver microsomes in the P450-region of each of the BNF-treated fish species. The apparent molecular weight of the cross-reacting proteins showed differences within the range 54,000-59,000 daltons. The effects of the different antibodies on the microsomal BNF-inducible 7-ethoxyresorufin 0-deethylase (EROD) activity gave inhibition patterns that reflected to a certain extent the phylogenetic relationship of the species investigated. In rat microsomes a protein band of relative molecular mass similar to rat P450c (Mr = 54,000) was recognized by all antibodies. In addition, a second band of lower molecular mass was strongly recognized by anti-cod P450c antibodies, and faintly stained with anti-rainbow trout P450LM4b IgG and anti-scup P450E MAb 1-12-3. This band could correspond to rat P450d, the isosafrole-inducible rat isoenzyme. Considering the long separate evolutionary history of some of these fishes (50-200 million years), the results demonstrate that certain antigenic epitopes in the BNF-inducible P450 isoenzymes have been strongly conserved during the evolution of fish species. These conserved epitopes seem however not to be directly involved in the measured EROD activities. Furthermore, the results suggest that the BNF-inducible P450s in fish contain regions with structural similarity to the homologous counterpart that has evolved through gene duplication into a P450 family in mammals containing at least two gene products (the P4501A gene family) .
Marine mammals may be important indicators of effects of contaminants that are globally distribut... more Marine mammals may be important indicators of effects of contaminants that are globally distributed. Recently the authors described apparent environmental induction of hepatic CYPLA in beluga whales. Here they describe the localization and extent of CYPLA expression in organs of the pilot whale. Tissues from 18 pilot whales stranded on Cape Cod in 1990/91 were frozen in liquid N2 or fixed in formalin and embedded. Liver microsomal EROD activity were comparable to results with other cetaceans. Immunohistochemical analysis showed a periportal localization of CYPLA in liver parenchyma, and staining in the endothelium. Renal staining was strong in brush border and endothelium. Testis, ovary, and spleen showed CYPLA staining only in endothelium. Adrenal zona fasciculata and zona reticularis stained more weakly than did endothelium. In lung there was mild staining of bronchiolar epithelium and strong staining of endothelium. The results indicate that active concentrations of inducer have penetrated throughout the body. CYPLA stained in dermal endothelium, indicating that analysis of skin biopsies could allow nondestructive analysis of CYPLA induction in marine mammals. CYPLA expression in these whales was surprisingly strong, suggesting the possibility of chemical effects related to CYPLA induction.
Environmental Toxicology and Chemistry, May 1, 1997
The induction of CYP1A by the polycyclic aromatic hydrocarbon (PAH)‐type inducer β‐naphthoflavone... more The induction of CYP1A by the polycyclic aromatic hydrocarbon (PAH)‐type inducer β‐naphthoflavone (BNF) in the Poeciliopsis‐lucida hepatocellular carcinoma cell line (PLHC‐1), and the effects of the glucocorticoid receptor (GR) agonist dexamethasone (DEX) on this response were examined. Dose‐response studies revealed that BNF is three orders of magnitude less potent than the planar halogenated aromatic hydrocarbon 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) as an inducer of the CYP1A activity ethoxyresorufin‐O‐deethylase (EROD), and that the apparent efficacy for the induction by BNF is 50% of that obtained with TCDD. Addition of 10 μM DEX resulted in potentiation of CYP1A induction at all doses of BNF tested. The degree of that potentiation of induction of CYP1A protein levels and EROD activity differed substantially between doses of BNF and at different times of exposure. For example, the maximal degree of potentiation of EROD induction by DEX was 12‐fold in PLHC‐1 cells treated with 0.1 μM BNF, 19‐fold in cells treated with 1 μM BNF, and 8‐fold in cells treated with 10 μM BNF. These maximal degrees of potentiation of EROD induction were obtained after 30 h with 0.1 μM BNF, 48 h with 1 μM BNF, and 72 h with 10 μM BNF. These results demonstrate interactions between GR and aryl hydrocarbon receptor pathways that could influence the response of fish to xenobiotic exposure.
Endothelium is a common site of cytochrome P450 1A (CYP1A) induction in vertebrates, and endothel... more Endothelium is a common site of cytochrome P450 1A (CYP1A) induction in vertebrates, and endothelial CYP1A could affect the distribution and toxicity of CYP1A substrates. We investigated CYP1A induction in organs rich in endothelium, gill, heart, and a microvascular model, the swimbladder rete mirabile, in the eel. Benzo[a]pyrene (BP) and 3, 3',4,4'-tetrachlorobiphenyl (TCB), radiolabeled and injected intraperitoneally, showed similar distribution in eels, with dose-dependent increases in concentration in heart and rete mirabile. BP [given at 0.1, 1, and 10 mg/kg (0.4, 4, and 40 micromol/kg)], TCB [given at 0.1, 1, and 10 mg/kg (0.3, 3, 30, and 60 micromol/kg)], and beta-naphthoflavone (BNF) [given at 0.1, 1, 5, 10, and 100 mg/kg (0.4, 4, 20, 40, and 400 micromol/kg)] induced microsomal CYP1A and ethoxyresorufin O-deethylase in heart and rete mirabile. Immunohistochemical analysis confirmed that induction of CYP1A in heart and rete mirabile occurs in the endothelium. Increasing doses of each compound caused increasing penetration of induction into the vascular bed of the rete, but with BNF and BP induction penetrated further than with TCB. At high doses of BNF there also was induction in epithelial cells adjacent to endothelium in gill and kidney. CYP1A also was induced in heart and rete mirabile of eels from sites heavily contaminated by aryl hydrocarbon receptor (AHR) agonists. The penetration of CYP1A induction into capillaries of the rete mirabile reflects the penetration of the inducer itself, consistent with the idea that endothelial CYP1A can indicate the local distribution of AHR agonists. The microvascular rete mirabile in the eel provides a model system to explore further a hypothesis that endothelial CYP1A participates in removal of some AHR agonists from the circulation and to examine the consequences of CYP1A induction to the vascular system.
Biosynthesis of steroid hormones in vertebrates involves three cytochrome P450 hydroxylases, CYP1... more Biosynthesis of steroid hormones in vertebrates involves three cytochrome P450 hydroxylases, CYP11A1, CYP17A1 and CYP19A1, which catalyze sequential steps in steroidogenesis. These enzymes are conserved in the vertebrates, but their origin and existence in other chordate subphyla (Tunicata and Cephalochordata) have not been clearly established. In this study, selected protein sequences of CYP11A1, CYP17A1 and CYP19A1 were compiled and analyzed using multiple sequence alignment and phylogenetic analysis. Our analyses show that cephalochordates have sequences orthologous to vertebrate CYP11A1, CYP17A1 or CYP19A1, and that echinoderms and hemichordates possess CYP11-like but not CYP19 genes. While the cephalochordate sequences have low identity with the vertebrate sequences, reflecting evolutionary distance, the data show apparent origin of CYP11 prior to the evolution of CYP19 and possibly CYP17, thus indicating a sequential origin of these functionally related steroidogenic CYPs. Co-occurrence of the three CYPs in early chordates suggests that the three genes may have coevolved thereafter, and that functional conservation should be reflected in functionally important residues in the proteins. CYP19A1 has the largest number of conserved residues while CYP11A1 sequences are less conserved. Structural analyses of human CYP11A1, CYP17A1 and CYP19A1 show that critical substrate binding site residues are highly conserved in each enzyme family. The results emphasize that the steroidogenic pathways producing glucocorticoids and reproductive steroids are several hundred million years old and that the catalytic structural elements of the enzymes have been #
Environmental Toxicology and Chemistry, Apr 1, 1996
Page 1. 582 Environmental Toxicology and Chemistry, Vol. 15, No. 4, pp. 582591, 1996 1996 SETAC ... more Page 1. 582 Environmental Toxicology and Chemistry, Vol. 15, No. 4, pp. 582591, 1996 1996 SETAC Printed in the USA 0730-7268/96 $6.00 .00 RAPID ASSESSMENT OF INDUCED CYTOCHROME P4501A PROTEIN AND ...
Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochro... more Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochrome P450 (CYP) genes, which thus far are poorly known in bivalve mollusks. We searched for CYP sequences in EST databases for Mytilus and Crassostrea species, lophotrochozoan representatives of the protostomes. From ESTs averaging ca. 924 bp, we identified 58 CYP genes in Mytilus californianus and 39 CYP genes in Crassostrea gigas. The sequences fell in all known animal CYP clans, and collectively they clustered in phylogenetic analysis with vertebrate CYP families 1, 2, 3, 4, 17, 20, 26 and 27. As in deuterostomes, a majority of the sequences fell in Clan 2. The CYP sequences found thus far in bivalves suggest a diversity consistent with that found in many other animal species. The present description of mollusk genes provides the overall framework for classification of any additional bivalve sequences. The sequences identified also will be useful in obtaining full-length sequences and in designing primers for analysis of expression of mussel and oyster CYP genes, or for recombinant protein expression to identify potential substrates for the bivalve CYP proteins, and understand their roles in xenobiotic detoxification and physiology of bivalves.
The regulation of different cytochrome P-450 forms and their functions in different organs and ce... more The regulation of different cytochrome P-450 forms and their functions in different organs and cell types could determine the susceptibility of those cells and organs to toxic effects of xenobiotics, including chemical carcinogenesis. Here we describe the cellular localization of cytochrome P-450E (P-450IA1) induced in 10 major organs or organ systems of a marine vertebrate species, the fish, Stenotomus chrysops (scup). Scup were injected ip with 3,3',4,4'-tetrachlorobiphenyl (TCB) at 1 mg/kg, or with 2,3,7,8-tetrachlorodibenzofuran (TCDF) at 3 micrograms/kg. Induction was verified by Western blot analysis of microsomes from selected organs (liver, kidney, and gill) using monoclonal antibody (MAb) 1-12-3 to scup P-450IA1. The localization of P-450IA1 was subsequently determined in sections prepared by standard histological methods (10% buffered formalin fixation, paraffin embedding), and stained with MAb 1-12-3 and peroxidase-labeled second antibody. P-450IA1 was induced in epithelial and endothelial cells in liver (including pancreatic tissue), kidney, gill, gut, spleen, testis, and ovary. Induction also was detected in endothelial cells, but not other types, in heart, brain, and red muscle. In heart, the staining was present in the endocardium as well as in the endothelium of the coronary vasculature and great vessels. Although TCDF and TCB both induced P-450IA1 in various cells of all organs examined, the effect of TCB was in most cases greater than that of TCDF. This may be due to a relatively higher TCB dosage. A wider staining distribution was seen in gut, gill, kidney, and gonad of TCB-treated fish, which might be explained by a greater penetration, or by excretion of parent TCB, as opposed to TCDF. In any case, the results show that these important environmental agents induce P-450IA1 in generally similar patterns in all organs examined. The common finding of a strong induction of P-450IA1 in endothelial cells in all organs examined supports the suggestion that the endothelium may be a primary site of P-450IA1 induction.
Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental to... more Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap'n'collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects.
Viruses generally are defined as lacking the fundamental properties of living organisms in that t... more Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and β-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.
Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbrevia... more Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14a-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an "orphan" P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.
Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several ... more Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.
Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxici... more Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between β-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the β-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). This led us to investigate AHR interaction with β-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of β-catenin signaling. We examined phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a β-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6formylindolo[3,2-b]carbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryotoxic, and we found that in presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholine-oligonucleotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of β-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of β-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating β-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes.
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, Mar 1, 2000
Previously we showed that the polychlorinated biphenyl 3,3%,4,4%-tetrachlorobiphenyl (TCB) caused... more Previously we showed that the polychlorinated biphenyl 3,3%,4,4%-tetrachlorobiphenyl (TCB) caused a release of reactive oxygen species (ROS) from cytochrome P450 1A (CYP1A) of the fish scup (Stenotomus chrysops), and from rat and human CYP1A1. This was linked to a TCB-and NADPH-dependent oxidative inactivation of the enzyme, which in scup and rat was inversely related to the rates of TCB oxidation. We examined the relationship between rates of TCB oxidation, CYP1A inactivation and ROS production in liver microsomes from additional vertebrate species, including skate (Raja erinacea), eel (Anguilla rostrata), killifish (Fundulus heteroclitus), winter flounder (Pleuronectes americanus), chicken (Gallus domesticus), cormorant (Phalacrocorax auritus), gull (Larus argentatus), and turtle (Chrysemys picta picta). TCB oxidation rates were induced in all fish and birds treated with aryl hydrocarbon receptor agonists. Induced rates of TCB oxidation were B1 pmol/min/mg microsomal protein in all fish, and 6-14 pmol/min/mg in the birds. In all species but one, TCB oxidation rates correlated positively with EROD rates, indicating likely involvement of CYP1A in TCB oxidation. Incubation of liver microsomes of most species with TCB + NADPH resulted in an immediate (TCB-dependent) inhibition of EROD, and a progressive loss of EROD capacity, indicating an oxidative inactivation of CYP1A like that in scup. NADPH stimulated production of ROS (H 2 O 2 and/or O 2 −) by liver microsomes, slightly in some species (eel) and greatly in others (chicken, turtle). Among the birds and the fish, NADPH-stimulated ROS production correlated positively with EROD activity. TCB caused a significant stimulation of ROS production by liver microsomes of flounder, killifish, cormorant and gull, as well as scup. The stimulation of CYP1A inactivation and ROS generation indicates an uncoupling of CYP1A by TCB in many species, and when compared between species, the rates of CYP1A inactivation correlated inversely with rates of TCB oxidation. Some feature(s) of binding/active site topology may hinder TCB oxidation, enhancing the likelihood for attack of an oxidizing species in the active site.
Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus,... more Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor kB (NF-kB) that rapidly activates genes encoding cytokines, cell surface receptors, cell adhesion molecules and acute phase proteins. Evidence suggests that xenobiotic compounds also may alter the activation of NF-kB. This study had a dual objective of identifying members of the Rel family and examining their activation by xenobiotic compounds in a marine fish model, scup (Stenotomus chrysops). A DNA-protein crosslinking technique demonstrated that liver, kidney and heart each had at least three nuclear proteins that showed specific binding to an NF-kB consensus sequence, with molecular weights suggesting that the proteins potentially corresponded to mouse p50, p65 (RelA) and c-rel. In addition, an :35kD NF-kB binding protein was evident in liver and kidney. The 50 kD protein was immunoprecipitated by mammalian p50-specific antibodies. The presence of Rel members in fish implied by those results was confirmed by RT-PCR cloning of a Rel homology domain
The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) su... more The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3',4,4',5pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6-and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26-and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 µM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 µM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene-and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions.
Abstract Reproductively mature winter flounder (Pseudopleuronectes americanus) were collected fro... more Abstract Reproductively mature winter flounder (Pseudopleuronectes americanus) were collected from three Northeastern US sites with different degrees of polychlorinated biphenyl (PCB) and polynuclear aromatic hydrocarbon (PAH) contamination. Liver PCB concentrations (measured by capillary electron-capture gas chromatography) in fish collected in 1987 and 1988 at New Bedford Harbor (NBH) ranged from 7·4 to 191 μg/g dry wt; at Gaspee Point 3·9–17·7 μg/g; and at Fox Island 1·6–15·1 μg/g. Levels of ethoxyresorufin O-deethylase (EROD) activity were similar in fish of the same reproductive status from the three sites; however, immunoquantitated P-450E homolog (the EROD catalyst) content was significantly higher in NBH fish. This suggests that P-450E catalytic activity is being suppressed in the livers of the NBH animals. Recent studies in our laboratory indicate that competitive inhibition of P-450E catalytic activity by specific PCB congeners is one likely mechanism of this suppression. Hepatic EROD activity and P-450E content were significantly lower in gravid females (EROD, 0·10–0·69 units per nmol P-450; P-450E, 8·4–19% of spectral P-450) than in spent females (EROD, 1·94–3·49; P-450E, 48–109%), and ripe males (EROD, 1·86–3·41; P-450E, 48–84%) at all sites. This is consistent with a hormonal effect on P-450E expression, and thus EROD activity, in gravid females. The data indicate a complex relationship between levels of EROD activity, or P-450E, and tissue PCB concentrations in highly contaminated fish. How these variables are linked to altered endocrine or gonadal function is not yet known.
Antibodies prepared against the major (3-naphthoflavone (BNF)-inducible cytochrome P450 (P450) fo... more Antibodies prepared against the major (3-naphthoflavone (BNF)-inducible cytochrome P450 (P450) forms from three species of fish (rainbow trout, Atlantic cod, and scup) well separated in teleost phylogeny, were used to investigate the immunochemical relatedness of liver microsomal P450 in different species of BNFtreated fish and rat. Rabbit polyclonal IgG against all three P450s and mouse monoclonal antibodies prepared against scup P450E were employed in this study. Liver microsomes were prepared from BNF-treated specimens of hagfish, herring, rainbow trout, cod, scup, perch, plaice and rat. With Western blotting it was shown that the various antibodies cross-reacted with a protein band in liver microsomes in the P450-region of each of the BNF-treated fish species. The apparent molecular weight of the cross-reacting proteins showed differences within the range 54,000-59,000 daltons. The effects of the different antibodies on the microsomal BNF-inducible 7-ethoxyresorufin 0-deethylase (EROD) activity gave inhibition patterns that reflected to a certain extent the phylogenetic relationship of the species investigated. In rat microsomes a protein band of relative molecular mass similar to rat P450c (Mr = 54,000) was recognized by all antibodies. In addition, a second band of lower molecular mass was strongly recognized by anti-cod P450c antibodies, and faintly stained with anti-rainbow trout P450LM4b IgG and anti-scup P450E MAb 1-12-3. This band could correspond to rat P450d, the isosafrole-inducible rat isoenzyme. Considering the long separate evolutionary history of some of these fishes (50-200 million years), the results demonstrate that certain antigenic epitopes in the BNF-inducible P450 isoenzymes have been strongly conserved during the evolution of fish species. These conserved epitopes seem however not to be directly involved in the measured EROD activities. Furthermore, the results suggest that the BNF-inducible P450s in fish contain regions with structural similarity to the homologous counterpart that has evolved through gene duplication into a P450 family in mammals containing at least two gene products (the P4501A gene family) .
Marine mammals may be important indicators of effects of contaminants that are globally distribut... more Marine mammals may be important indicators of effects of contaminants that are globally distributed. Recently the authors described apparent environmental induction of hepatic CYPLA in beluga whales. Here they describe the localization and extent of CYPLA expression in organs of the pilot whale. Tissues from 18 pilot whales stranded on Cape Cod in 1990/91 were frozen in liquid N2 or fixed in formalin and embedded. Liver microsomal EROD activity were comparable to results with other cetaceans. Immunohistochemical analysis showed a periportal localization of CYPLA in liver parenchyma, and staining in the endothelium. Renal staining was strong in brush border and endothelium. Testis, ovary, and spleen showed CYPLA staining only in endothelium. Adrenal zona fasciculata and zona reticularis stained more weakly than did endothelium. In lung there was mild staining of bronchiolar epithelium and strong staining of endothelium. The results indicate that active concentrations of inducer have penetrated throughout the body. CYPLA stained in dermal endothelium, indicating that analysis of skin biopsies could allow nondestructive analysis of CYPLA induction in marine mammals. CYPLA expression in these whales was surprisingly strong, suggesting the possibility of chemical effects related to CYPLA induction.
Environmental Toxicology and Chemistry, May 1, 1997
The induction of CYP1A by the polycyclic aromatic hydrocarbon (PAH)‐type inducer β‐naphthoflavone... more The induction of CYP1A by the polycyclic aromatic hydrocarbon (PAH)‐type inducer β‐naphthoflavone (BNF) in the Poeciliopsis‐lucida hepatocellular carcinoma cell line (PLHC‐1), and the effects of the glucocorticoid receptor (GR) agonist dexamethasone (DEX) on this response were examined. Dose‐response studies revealed that BNF is three orders of magnitude less potent than the planar halogenated aromatic hydrocarbon 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) as an inducer of the CYP1A activity ethoxyresorufin‐O‐deethylase (EROD), and that the apparent efficacy for the induction by BNF is 50% of that obtained with TCDD. Addition of 10 μM DEX resulted in potentiation of CYP1A induction at all doses of BNF tested. The degree of that potentiation of induction of CYP1A protein levels and EROD activity differed substantially between doses of BNF and at different times of exposure. For example, the maximal degree of potentiation of EROD induction by DEX was 12‐fold in PLHC‐1 cells treated with 0.1 μM BNF, 19‐fold in cells treated with 1 μM BNF, and 8‐fold in cells treated with 10 μM BNF. These maximal degrees of potentiation of EROD induction were obtained after 30 h with 0.1 μM BNF, 48 h with 1 μM BNF, and 72 h with 10 μM BNF. These results demonstrate interactions between GR and aryl hydrocarbon receptor pathways that could influence the response of fish to xenobiotic exposure.
Endothelium is a common site of cytochrome P450 1A (CYP1A) induction in vertebrates, and endothel... more Endothelium is a common site of cytochrome P450 1A (CYP1A) induction in vertebrates, and endothelial CYP1A could affect the distribution and toxicity of CYP1A substrates. We investigated CYP1A induction in organs rich in endothelium, gill, heart, and a microvascular model, the swimbladder rete mirabile, in the eel. Benzo[a]pyrene (BP) and 3, 3',4,4'-tetrachlorobiphenyl (TCB), radiolabeled and injected intraperitoneally, showed similar distribution in eels, with dose-dependent increases in concentration in heart and rete mirabile. BP [given at 0.1, 1, and 10 mg/kg (0.4, 4, and 40 micromol/kg)], TCB [given at 0.1, 1, and 10 mg/kg (0.3, 3, 30, and 60 micromol/kg)], and beta-naphthoflavone (BNF) [given at 0.1, 1, 5, 10, and 100 mg/kg (0.4, 4, 20, 40, and 400 micromol/kg)] induced microsomal CYP1A and ethoxyresorufin O-deethylase in heart and rete mirabile. Immunohistochemical analysis confirmed that induction of CYP1A in heart and rete mirabile occurs in the endothelium. Increasing doses of each compound caused increasing penetration of induction into the vascular bed of the rete, but with BNF and BP induction penetrated further than with TCB. At high doses of BNF there also was induction in epithelial cells adjacent to endothelium in gill and kidney. CYP1A also was induced in heart and rete mirabile of eels from sites heavily contaminated by aryl hydrocarbon receptor (AHR) agonists. The penetration of CYP1A induction into capillaries of the rete mirabile reflects the penetration of the inducer itself, consistent with the idea that endothelial CYP1A can indicate the local distribution of AHR agonists. The microvascular rete mirabile in the eel provides a model system to explore further a hypothesis that endothelial CYP1A participates in removal of some AHR agonists from the circulation and to examine the consequences of CYP1A induction to the vascular system.
Biosynthesis of steroid hormones in vertebrates involves three cytochrome P450 hydroxylases, CYP1... more Biosynthesis of steroid hormones in vertebrates involves three cytochrome P450 hydroxylases, CYP11A1, CYP17A1 and CYP19A1, which catalyze sequential steps in steroidogenesis. These enzymes are conserved in the vertebrates, but their origin and existence in other chordate subphyla (Tunicata and Cephalochordata) have not been clearly established. In this study, selected protein sequences of CYP11A1, CYP17A1 and CYP19A1 were compiled and analyzed using multiple sequence alignment and phylogenetic analysis. Our analyses show that cephalochordates have sequences orthologous to vertebrate CYP11A1, CYP17A1 or CYP19A1, and that echinoderms and hemichordates possess CYP11-like but not CYP19 genes. While the cephalochordate sequences have low identity with the vertebrate sequences, reflecting evolutionary distance, the data show apparent origin of CYP11 prior to the evolution of CYP19 and possibly CYP17, thus indicating a sequential origin of these functionally related steroidogenic CYPs. Co-occurrence of the three CYPs in early chordates suggests that the three genes may have coevolved thereafter, and that functional conservation should be reflected in functionally important residues in the proteins. CYP19A1 has the largest number of conserved residues while CYP11A1 sequences are less conserved. Structural analyses of human CYP11A1, CYP17A1 and CYP19A1 show that critical substrate binding site residues are highly conserved in each enzyme family. The results emphasize that the steroidogenic pathways producing glucocorticoids and reproductive steroids are several hundred million years old and that the catalytic structural elements of the enzymes have been #
Environmental Toxicology and Chemistry, Apr 1, 1996
Page 1. 582 Environmental Toxicology and Chemistry, Vol. 15, No. 4, pp. 582591, 1996 1996 SETAC ... more Page 1. 582 Environmental Toxicology and Chemistry, Vol. 15, No. 4, pp. 582591, 1996 1996 SETAC Printed in the USA 0730-7268/96 $6.00 .00 RAPID ASSESSMENT OF INDUCED CYTOCHROME P4501A PROTEIN AND ...
Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochro... more Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochrome P450 (CYP) genes, which thus far are poorly known in bivalve mollusks. We searched for CYP sequences in EST databases for Mytilus and Crassostrea species, lophotrochozoan representatives of the protostomes. From ESTs averaging ca. 924 bp, we identified 58 CYP genes in Mytilus californianus and 39 CYP genes in Crassostrea gigas. The sequences fell in all known animal CYP clans, and collectively they clustered in phylogenetic analysis with vertebrate CYP families 1, 2, 3, 4, 17, 20, 26 and 27. As in deuterostomes, a majority of the sequences fell in Clan 2. The CYP sequences found thus far in bivalves suggest a diversity consistent with that found in many other animal species. The present description of mollusk genes provides the overall framework for classification of any additional bivalve sequences. The sequences identified also will be useful in obtaining full-length sequences and in designing primers for analysis of expression of mussel and oyster CYP genes, or for recombinant protein expression to identify potential substrates for the bivalve CYP proteins, and understand their roles in xenobiotic detoxification and physiology of bivalves.
The regulation of different cytochrome P-450 forms and their functions in different organs and ce... more The regulation of different cytochrome P-450 forms and their functions in different organs and cell types could determine the susceptibility of those cells and organs to toxic effects of xenobiotics, including chemical carcinogenesis. Here we describe the cellular localization of cytochrome P-450E (P-450IA1) induced in 10 major organs or organ systems of a marine vertebrate species, the fish, Stenotomus chrysops (scup). Scup were injected ip with 3,3',4,4'-tetrachlorobiphenyl (TCB) at 1 mg/kg, or with 2,3,7,8-tetrachlorodibenzofuran (TCDF) at 3 micrograms/kg. Induction was verified by Western blot analysis of microsomes from selected organs (liver, kidney, and gill) using monoclonal antibody (MAb) 1-12-3 to scup P-450IA1. The localization of P-450IA1 was subsequently determined in sections prepared by standard histological methods (10% buffered formalin fixation, paraffin embedding), and stained with MAb 1-12-3 and peroxidase-labeled second antibody. P-450IA1 was induced in epithelial and endothelial cells in liver (including pancreatic tissue), kidney, gill, gut, spleen, testis, and ovary. Induction also was detected in endothelial cells, but not other types, in heart, brain, and red muscle. In heart, the staining was present in the endocardium as well as in the endothelium of the coronary vasculature and great vessels. Although TCDF and TCB both induced P-450IA1 in various cells of all organs examined, the effect of TCB was in most cases greater than that of TCDF. This may be due to a relatively higher TCB dosage. A wider staining distribution was seen in gut, gill, kidney, and gonad of TCB-treated fish, which might be explained by a greater penetration, or by excretion of parent TCB, as opposed to TCDF. In any case, the results show that these important environmental agents induce P-450IA1 in generally similar patterns in all organs examined. The common finding of a strong induction of P-450IA1 in endothelial cells in all organs examined supports the suggestion that the endothelium may be a primary site of P-450IA1 induction.
Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental to... more Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap'n'collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects.
Viruses generally are defined as lacking the fundamental properties of living organisms in that t... more Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and β-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.
Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbrevia... more Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14a-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an "orphan" P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.
Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several ... more Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.
Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxici... more Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between β-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the β-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). This led us to investigate AHR interaction with β-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of β-catenin signaling. We examined phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a β-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6formylindolo[3,2-b]carbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryotoxic, and we found that in presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholine-oligonucleotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of β-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of β-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating β-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes.
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Papers by John Stegeman