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Biomedical subjects

M E Hahn

Publications and source records attributed to M E Hahn.

At least 19 recordsLinked to original sources
PubMed2003

[Radioulnar impingement after distal ulnar resection and ulnar head hemiresection interposition arthroplasty (Bowers procedure). An experimental biomechanical study].

Resection of the distal ulna (Darrach operation) and the ulnar head hemiresection interposition arthroplasty (Bowers procedure) are common methods of treating the arthrotic distal radioulnar joint (DRUJ). The purpose of this study was to evaluate the dynamic effects of both the Bowers operation and the resection of the distal ulna on radioulnar impingement and dorsal-palmar displacement and to compare their biomechanical performances. With a dynamic forearm simulator forearm rotation was performed with simultaneous loading of relevant muscles. Torque along the forearm axis was generated by simulated muscle action with loading of the relevant tendons while the wrist was constrained to prescribed ranges of motion. The instability of the radius relative to the ulna was evaluated using displacement data of digitized landmarks in an ulnar coordinate system. Seven fresh-frozen cadaver upper extremities were used. Resection of the distal ulna created a significant instability of the forearm with movement of the radius ulnarly. Anteroposterior translations in each loading condition could be detected as well but the magnitude of displacement was little and less predictable. The results of the Bowers operation demonstrated significantly less instability compared with the Darrach results. Our data demonstrates that the dynamic behavior of the Bowers procedure is superior to the mechanical performance of complete resection of the distal ulna.

Arthroplasty↗
PubMed2002

[Dynamic radioulnar convergence after Darrach operation, soft tissue stabilizing operations of the distal ulna and ulnar head prosthesis implantation--an experimental biomechanical study].

The most common method of treating the arthrotic distal radioulnar joint (DRUJ) is resection of the entire ulnar head (Darrach procedure). Complications related to instability of the distal forearm resulting from loss of the ulnar head are usually manifested by pain and weak grip strength and have remained the drawbacks of this procedure. In an attempt to mechanically stabilize the distal forearm, an endoprosthesis was developed to replace the ulnar head after Darrach resection. The purpose of this study was to: 1) evaluate the dynamic effects of the Darrach procedure on radioulnar convergence; and 2) evaluate the mechanical efficacy of two soft tissue stabilizing techniques (Pronator quadratus advancement flap and ECU/FCU tenodesis) for the unstable distal ulnar stump and 3) the stability after the implantation of an ulnar head endoprosthesis following a Darrach resection on radioulnar convergence. With a dynamic PC-controled forearm simulator the rotation of 7 fresh-frozen cadaver upper extremities was actively and passively performed while loading relevant muscles. Resultant total forearm torque and the 3-dimensional kinematics of the ulna, radius and third metacarpal were recorded simultaneously. The implantation of the ulnar head endoprosthesis effectively restored the stability of the DRUJ. There were significantly better results after the implantation of the prosthesis compared with the Darrach and the soft tissue stabilization procedures. This study provides laboratory validity to the option of implanting an ulnar head endoprosthesis as an attempt to stabilize the distal forearm after Darrach resection in lieu of performing soft tissue stabilization techniques.

Biomechanical Phenomena↗
PubMed2002

The dynamic radioulnar convergence of the Darrach procedure and the ulnar head hemiresection interposition arthroplasty: a biomechanical study.

Resection of the entire ulnar head (Darrach operation) and the hemiresection interposition arthroplasty are common methods of treating the arthritic distal radioulnar joint (DRUJ). Biomechnical investigation about both of these procedures is lacking. The purpose of this study was to evaluate the dynamic effects of both the hemiresection interposition arthroplasty and the Darrach procedure on radioulnar convergence and dorsal-palmar displacement and to compare their biomechanical behaviours. With a dynamic computer-controlled testing device, cadaveric forearm rotation was performed with simultaneous loading of relevant muscles. Torque along the forearm axis was generated by simulated muscle action through pneumoactuators attached to relevant tendons while the wrist was constrained to prescribed ranges of motion. The instability of the radius relative to the ulna was evaluated using displacement data of digitized landmarks in an ulnar coordinate system. Seven fresh-frozen cadaver upper extremities were used. The Darrach resection created an extreme instability of the forearm with movement of the radius ulnarly. Anteroposterior translations in each loading condition could also be detected, but the magnitude of displacement was small and less predictable. The results of the hemiresection interposition arthroplasty demonstrated significantly less instability compared with the Darrach results.

Aged↗
PubMed2001

An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding.

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.

Amino Acid Sequence↗
PubMed2001

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos.

Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC(50) and LD(50) of TCDD for Fundulus embryos were approximately 19.7+/-9.5 pg TCDD/microl (water bath) and 0.25+/-0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction.

Animals↗
PubMed2001

Acquired resistance to Ah receptor agonists in a population of Atlantic killifish (Fundulus heteroclitus) inhabiting a marine superfund site: in vivo and in vitro studies on the inducibility of xenobiotic metabolizing enzymes.

New Bedford Harbor (NBH), MA, is a federal Superfund site that is heavily contaminated with polychlorinated biphenyls (PCBs) and other halogenated aromatic hydrocarbons (HAHs), including some potent aryl hydrocarbon receptor (AhR) agonists. A population of Atlantic killifish (Fundulus heteroclitus) continues to inhabit this site, despite accumulating extraordinarily high concentrations of PCBs (272 microg/g dry weight). To determine if NBH killifish have developed resistance to HAHs that act through the AhR, we examined the inducibility of cytochrome P4501A1 (CYP1A1), UDP glucuronosyl transferase (UGT), and glutathione S-transferase (GST) in fish from NBH and a reference site, Scorton Creek (SC, Cape Cod, MA; PCB concentrations 0.177 microg/g dry weight). 2,3,7,8-Tetrachlorodibenzofuran (TCDF) induced CYP1A1 mRNA, protein, and activity in SC fish in all tissues examined (liver, heart, gut, gill, kidney, spleen, and gonad). In contrast, NBH fish expressed low levels of CYP1A1 and showed no induction of CYP1A1 mRNA, protein, or activity by TCDF, or induction that was lower in magnitude or required higher doses of inducer. p-Nitrophenol UGT activity was not induced by TCDF in either population, while GST activity with 1-chloro-2,4-dinitrobenzene as substrate was induced only in NBH fish in one experiment. Inducibility of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or beta-naphthoflavone (BNF) was measured in primary hepatocyte cultures prepared from SC and NBH fish. TCDD induced CYP1A1 activity (ethoxyresorufin O-deethylase) to the same degree in hepatocytes from both populations, demonstrating the functionality of the AhR signaling pathway in NBH fish. However, hepatocytes from NBH fish were 14-fold less sensitive to TCDD than were those from SC fish. The nonhalogenated AhR agonist BNF also induced CYP1A1 in cells from both populations, although with only a 3-fold difference in sensitivity (NBH < SC). These results indicate that chronic exposure to high levels of HAHs has led to a reduction in the sensitivity of NBH killifish to AhR agonists. The resistance is systemic and pretranslational, and exhibits compound-specific differences in magnitude. These findings suggest an alteration in the AhR signal transduction pathway in NBH fish.

Animals↗
PubMed2001

cDNA cloning and characterization of a high affinity aryl hydrocarbon receptor in a cetacean, the beluga, Delphinapterus leucas.

Some cetaceans bioaccumulate substantial concentrations of planar halogenated aromatic hydrocarbons (PHAHs) in their tissues, but little is known about the effects of such burdens on cetacean health. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related PHAHs cause toxicity via activation of the aryl hydrocarbon receptor (AHR), a member of the bHLH-PAS family of transcription factors. Differences in AHR structure and function are known to contribute to species-specific differences in susceptibility to PHAH toxicity. To ascertain the potential for PHAH effects in a cetacean, we characterized an AHR from the beluga whale, Delphinapterus leucas. The 3.2 kb cDNA encodes an 845-amino acid protein with a predicted size of 95.5 kDa. Overall, the beluga AHR shares 85% amino acid sequence identity with the human AHR and 75% identity with the mouse AHR Ah(b-1) allele. Beluga AHR protein synthesized in a rabbit reticulocyte lysate system demonstrated specific, high-affinity [(3)H]TCDD binding. Saturation binding analysis was used to compare the [(3)H]TCDD binding affinity of the in vitro-expressed beluga AHR with affinities of in vitro-expressed AHRs from a dioxin-sensitive mouse strain (Ah(b-1) allele) and humans. The beluga AHR bound [(3)H]TCDD with an affinity (K(d)= 0.43 +/- 0.16 nM) that was at least as high as that of the mouse AHR (K(d)= 0.68 +/- 0.23 nM), and significantly greater than that of the human AHR (K(d)= 1.63 +/- 0.64 nM). In electrophoretic mobility shift assays, the beluga AHR exhibited sequence-specific, Arnt-dependent binding to a dioxin responsive enhancer (DRE). Upon transient transfection into mammalian cells, the beluga AHR activated transcription of a luciferase reporter under control of a DRE-containing fragment of the mouse Cyp1a1 promoter. These results show that in an in vitro system, the beluga AHR possesses characteristics similar to those of AHRs from other mammals that are considered sensitive to toxic effects of PHAHs. Together, these results demonstrate that the use of in vitro-expressed proteins is a promising approach for addressing molecular and biochemical questions concerning PHAH toxicity in endangered or protected species.

Amino Acid Sequence↗
PubMed2000

Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency.

Models of receptor action are valuable for describing properties of ligand-receptor interactions and thereby contribute to mechanism-based risk assessment of receptor-mediated toxic effects. In order to build such a model for the aryl hydrocarbon receptor (AHR), binding affinities and CYP1A induction potencies were measured in PLHC-1 cells and were used to determine intrinsic efficacies for 10 halogenated aromatic hydrocarbons (HAH): 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7, 8-tetrachlorodibenzofuran (TCDF), and eight polychlorinated biphenyls (PCB). TCDD, TCDF, and non-ortho-substituted PCBs 77, 81, 126, and 169 behaved as full agonists and displayed high-intrinsic efficacy. In contrast, the mono- and di-ortho-substituted PCBs bound to the AHR but displayed lower or no intrinsic efficacy. PCB 156 was a full agonist, but with an intrinsic efficacy 10- to 50-fold lower than non-ortho-substituted PCBs. PCB 118 was a very weak partial agonist. PCBs 105 and 128 were shown to be competitive antagonists in this system. The model was then used to predict CYP1A induction by binary mixtures. These predictions were tested with binary mixtures of PCB 126, 128, or 156 with TCDD. Both PCB 156 (a low-intrinsic efficacy agonist) and PCB 128 (a competitive antagonist) inhibited the response to TCDD, while the response to TCDD and PCB126 was additive. These data support the following conclusions: 1) only 1-2% of the receptors in the cell need be occupied to achieve 50% of maximal CYP1A induction by one of the high-intrinsic efficacy agonists, demonstrating the existence of "spare" receptors in this system; 2) the insensitivity of fish to ortho-substituted PCBs is due to both reduced affinity and reduced intrinsic efficacy compared to non-ortho-substituted PCBs; 3) PCB congeners exhibit distinct structure-affinity and structure-efficacy relationships. Separation of AHR ligand action into the properties of affinity and intrinsic efficacy allows for improved prediction of the behavior of complex mixtures of ligands, as well as mechanistic comparisons across species and toxic endpoints.

Animals↗
PubMed2000

Cytochrome P4501A induction and porphyrin accumulation in PLHC-1 fish cells exposed to sediment and oil shale extracts.

The present study describes the use of a fish hepatoma cell line (PLHC-1) in monitoring the biological effects of sediments collected from recipient waters of the oil shale industry. Sampling sites were located in River Purtse and River Kohtla in northeast Estonia. The effects of pure oil shale on the PLHC-1 cells were also studied. The cells were exposed to n-hexane-extracted samples in 48-well plates for 24 h, and 7-ethoxyresorufin O-deethylase (EROD) activity, total protein, and porphyrin content were measured in the exposed cells. Polycyclic aromatic hydrocarbon (PAH) contents in the samples were measured by high-performance liquid chromatography (HPLC). All the sediment and oil shale samples induced CYP1A activity and led to porphyrin accumulation in the cells. The most potent inducers were the sediments collected near the oil shale processing plants (site Lüganuse in River Purtse and Kohtla in River Kohtla), as well as those at the most downstream site in River Purtse (Purtse). These samples possessed high total PAH contents, ranging from 4,270 to nearly 150,000 microg/kg dry sediment. The presence of other lipophilic organic contaminants in the samples was not determined in this study. Both EROD activity and porphyrin content exhibited biphasic induction curves, and the ED(50)(1) values for EROD activity were lower than the ED(50)s for porphyrin content. 2,3,7, 8-Tetrachlorodibenzo-p-dioxin induction equivalents (TCDD-EQs) calculated from EROD induction potencies correlated well with total PAHs (r(2) = 0.827 and p = 0.003 for log-transformed data) and also with individual PAHs. TCDD-EQs for porphyrin content did not correlate significantly with total PAHs (log-log r(2) = 0.785, p = 0. 116). The biological potency and PAH contamination of the samples showed the same rank order, except at Lüganuse, where sediment extracts induced CYP1A and porphyrins more than could have been expected based on PAH contents. Bioassay-derived induction EQs (normalized to dibenz(a,h)anthracene) were 20- to 3,200-fold greater than EQs calculated from the concentrations of five PAHs, suggesting important contributions from other compounds or nonadditive effects. The PLHC-1 cells proved to be a sensitive bioanalytical tool for sediments contaminated with PAH-type pollutants in the oil shale processing area. We suggest further use of this bioassay in screening and monitoring waters with similar background of pollution as in northeast Estonia.

Animals↗
PubMed2000

The effects of prenatal cocaine exposure and genotype on the ultrasonic calls of infant mice.

Estimates are that as many as 44,000 humans are exposed to cocaine in utero per year. In this study we examined the effects of prenatal cocaine exposure on one aspect of the mother-infant relationship in mice, infant ultrasonic calls. We mated C57BL/10J female mice with males of three different inbred strains (producing pups of three different F(1) genotypes). We injected those females, subcutaneously, with saline or 20 mg/kg of cocaine hydrochloride on days 7-17 of gestation. That dosage did not compromise mother or pup viability, weight, or gestation length. On postnatal days 2-4, we recorded and measured the calls of pups while they were separated from their nest and slightly chilled. The results indicate changes in the ultrasonic calls as a function of cocaine and genotype. Overall, cocaine reduced the number of calls and increased the beginning pitch of calls. Pups of one genotype, a C57BL/10JxSJL/J hybrid were unaffected by cocaine exposure. The effects of cocaine, though reliable, were small, explaining only 1-2% of the total sum of squares. The size of the effect is in part due to the differential effect of cocaine on different genotypes.

Animals↗
PubMed2000

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression.

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.

Animals↗
PubMed2000

Serum alters the uptake and relative potencies of halogenated aromatic hydrocarbons in cell culture bioassays.

The effects of many chemicals on cellular processes are governed by their ability to enter the cell, which is in turn a function of the composition of the cell's external environment. To examine this relationship, the effect of serum in cell culture medium on the bioavailability of cytochrome P450 1A (CYP1A)-inducing compounds was determined in PLHC-1 (Poeciliopsis lucida hepatocellular carcinoma) cells. The presence of 10% calf serum in the medium increased the EC50 (effective concentration to achieve 50% maximal response) for induction of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 20-fold as compared to treatment in serum-free medium. Measurement of [3H]TCDD uptake and Ah receptor binding indicated that the apparent difference in potencies was a result of decreased bioavailability in the presence of serum, effectively reducing the concentration of TCDD within the cells. Induction of EROD and CYP1A protein in response to treatment with each of three coplanar polychlorinated biphenyls (PCB congeners 77, 126, and 169) was similarly affected by serum, although the magnitude varied among inducers and assays. Relative potencies (calculated as EC50TCDD / EC50PCB) for EROD induction by the three PCBs were significantly higher in the absence of serum. However, serum showed no significant effect on the relative potencies for CYP1A protein induction. These results demonstrate that measured inducing potencies, and relative potencies for EROD induction, by halogenated aromatic hydrocarbons are strongly dependent on the composition of culture medium, which can lead to artificial differences in comparisons among cell types.

Animals↗
PubMed2000

Developmental and tissue-specific expression of AHR1, AHR2, and ARNT2 in dioxin-sensitive and -resistant populations of the marine fish Fundulus heteroclitus.

Fundulus heteroclitus is a well-characterized marine fish model for studying aryl hydrocarbon toxicity. The F. heteroclitus population in New Bedford Harbor (NBH), a Superfund site in southeastern Massachusetts, exhibits heritable resistance to the toxic effects of planar halogenated aromatic hydrocarbons (PHAHs), including 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs). To investigate the role of the aryl hydrocarbon receptor (AHR) signal transduction pathway in PHAH resistance, we measured the relative levels of AHR1, AHR2, and ARNT2 mRNA in whole embryos at different developmental stages and in dissected tissues of adults, comparing expression of these genes in NBH fish with fish from a reference site (Scorton Creek, MA [SC]). Expression of both AHR1 and AHR2 mRNA increased during development, achieving maximum levels prior to hatching. Maximal embryonic expression of AHR1 was delayed relative to AHR2. Whole NBH and SC embryos exhibited no discernable differences in expression of these genes. As we have previously observed, adult SC fish expressed AHR2 and ARNT2 mRNA in all tissues examined, while AHR1 was expressed predominantly in brain, heart, and gonads. In contrast, AHR1 mRNA was widely expressed in NBH fish, appearing with unusual abundance in gill, gut, kidney, liver, and spleen. This AHR1 expression pattern was not observed in the lab-reared progeny of NBH fish, demonstrating that constitutive AHR1 expression in gill, gut, kidney, liver, and spleen is not a heritable phenotype. Furthermore, widespread AHR1 expression was not induced in reference-site fish by TCDD or PCB mixtures, suggesting that aberrant AHR1 expression is not simply a normal physiological response of contaminant exposure. These results identify ubiquitous AHR1 expression as an attribute unique to feral NBH F. heteroclitus, and they represent a first step in determining the regulatory mechanisms underlying this expression pattern and its possible role in TCDD resistance.

Actins↗
PubMed2000

The bioflavonoid galangin blocks aryl hydrocarbon receptor activation and polycyclic aromatic hydrocarbon-induced pre-B cell apoptosis.

Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancers induced by common environmental chemicals. Much of the biologic activity of one such class of pollutants, polycyclic aromatic hydrocarbons (PAH), is mediated by the aryl hydrocarbon receptor/transcription factor (AhR). For example, the AhR regulates PAH immunotoxicity that manifests as pre-B cell apoptosis in models of B cell development. Because bioflavonoids block PAH-induced cell transformation and are structurally similar to AhR ligands, it was postulated that some of them would suppress PAH-induced, AhR-dependent immunotoxicity, possibly through a direct AhR blockade. This hypothesis was tested using a model of B cell development in which pre-B cells are cultured with and are dependent on bone marrow stromal or hepatic parenchymal cell monolayers. Of seven bioflavonoids screened, galangin (3,5,7-trihydroxyflavone) blocked PAH-induced but not C(2)-ceramide- or H(2)O(2)-induced pre-B cell apoptosis. Because galangin blocked AhR-dependent reporter gene expression, AhR complex-DNA binding, and AhR nuclear translocation, inhibition of a relatively early step in AhR signaling was implicated. This hypothesis was supported by the ability of galangin to bind the AhR and stabilize AhR-90-kDa heat shock protein complexes in the presence of AhR agonists. These studies demonstrate the utility of pre-B cell culture systems in identifying compounds capable of blocking PAH immunotoxicity, define at least one mechanism of galangin activity (i.e., repression of AhR activation), and motivate the use of this and similar dietary bioflavonoids as relatively nontoxic inhibitors of AhR agonist activity and as pharmacologic agents with which to dissect AhR signaling pathways.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the teleost Fundulus heteroclitus. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds cause altered gene expression and toxicity. The AHR belongs to an emerging multigene family of transcription factors possessing basic helix loop helix (bHLH) and Per-ARNT-Sim (PAS) domains. Most bHLH-PAS proteins occur as duplicates or "paralog groups" in mammals, but only a single mammalian AHR has been identified. Here we report the cDNA cloning of two distinct AHRs, designated FhAHR1 and FhAHR2, from a single vertebrate species, the teleost Fundulus heteroclitus (Atlantic killifish). Both Fundulus AHR proteins possess bHLH and PAS domains that are closely related to those of the mammalian AHR. FhAHR1 and FhAHR2 are highly divergent (40% overall amino acid identity; 61% identity in the N-terminal half), suggesting that they arose from a gene duplication predating the divergence of mammals and fish. Photoaffinity labeling with 2-azido-3-[(125)I]iodo-7, 8-dibromodibenzo-p-dioxin and velocity sedimentation analysis using 2,3,7,8-[1,6-(3)H]TCDD showed that both FhAHR1 and FhAHR2 exhibit specific, high-affinity binding of dioxins. Both AHRs also showed specific, TCDD- and ARNT-dependent interactions with a mammalian xenobiotic response element. The two Fundulus AHR genes displayed different tissue-specific patterns of expression; FhAHR1 transcripts were primarily expressed in brain, heart, ovary, and testis, while FhAHR2 transcripts were equally abundant in many tissues. Phylogenetic analysis demonstrated that Fundulus AHR1 is an ortholog of mammalian AHRs, while AHR2 forms in Fundulus and other fish are paralogous to Fundulus AHR1 and the mammalian AHRs and thus represent a novel vertebrate subfamily of ligand-binding AHRs.

Amino Acid Sequence↗
PubMed1999

The role of polycyclic aromatic hydrocarbon metabolism in dimethylbenz[a]anthracene-induced pre-B lymphocyte apoptosis.

Previous studies indicated that two prototypic PAH, benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA), suppress the developing immune system by inducing apoptosis in bone marrow pre-B lymphocytes. In bone marrow cultures consisting of pre-B cells growing on bone marrow stromal cell monolayers, pre-B cell apoptosis was shown to be dependent on the aryl hydrocarbon receptor/transcription factor (AhR) expressed in stromal cells. However, it was not determined if AhR activation alone is sufficient or if DMBA metabolism is required for induction of a stromal cell-derived apoptosis signal. To address these issues we assessed: 1) the ability of poorly metabolized AhR ligands to induce pre-B cell apoptosis and 2) the capacity for and the mechanism through which an early DMBA metabolite induces pre-B cell apoptosis. Three poorly metabolized AhR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3,3',4,4',5-pentachlorobiphenyl, and 3,3',4,4'-tetrachlorobiphenyl failed to induce pre-B cell apoptosis in bone marrow cultures, indicating that AhR activation alone is not sufficient to induce apoptosis and suggesting a role for PAH metabolism in induction of an apoptosis signal. Consistent with this hypothesis, DMBA-3, 4-dihydrodiol, an early DMBA metabolite, induced significant pre-B cell apoptosis. The ability of DMBA-3,4-dihydrodiol to activate the AhR, inhibition of DMBA-3,4-dihydrodiol-induced apoptosis by alpha-naphthoflavone, and the significantly lower levels of DMBA-3, 4-dihydrodiol-induced apoposis in pre-B cell populations maintained on AhR(-) stromal cells strongly support a role for the AhR in DMBA-3,4-dihydrodiol-induced apoptosis. Of two DMBA-metabolizing enzymes evaluated, CYP1A1 and CYP1B1, the latter appeared to be the more likely to play a role in DMBA-induced apoptosis. These data confirm a role for the AhR in PAH-induced pre-B cell apoptosis, indicate a role for DMBA metabolism, and suggest a feedback loop in which at least one product of DMBA metabolism augments AhR signaling, leading to induction of an apoptosis stimulus.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

Two forms of aryl hydrocarbon receptor type 2 in rainbow trout (Oncorhynchus mykiss). Evidence for differential expression and enhancer specificity.

Two aryl hydrocarbon receptors (AhRs), rtAhR2alpha and rtAhR2beta, were cloned from rainbow trout (rt) cDNA libraries. The distribution of sequence differences, genomic Southern blot analysis, and the presence of both transcripts in all individual rainbow trout examined suggest that the two forms of rtAhR2 are derived from separate genes. The two rtAhR2s have significant sequence similarity with AhRs cloned from mammalian species, especially in the basic helix-loop-helix and PAS functional domains located in the amino-terminal 400 amino acids of the protein. In contrast, the Gln-rich transactivation domain found in the carboxyl-terminal half of mammalian AhRs is absent from both rtAhR2s. Both clones were expressed by in vitro transcription/translation and proteins of approximately 125 kDa were produced. These proteins bind 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and are able to bind dioxin response elements in gel shift assays. rtAhR2alpha and rtAhR2beta are expressed in a tissue-specific manner with the highest expression of rtAhR2beta in the heart. Expression of rtAhR2alpha and rtAhR2beta mRNAs is positively regulated by TCDD. Both rtAhR2alpha and rtAhR2beta produced TCDD-dependent activation of a reporter gene driven by dioxin response elements. Surprisingly, the two receptors showed distinct preferences for different enhancer sequences. These results suggest that the two receptor forms may regulate different sets of genes, and may play different roles in the toxic responses produced by AhR agonists such as TCDD.

Amino Acid Sequence↗