Tributyltin disrupts fin development in Fundulus heteroclitus from both PCB-sensitive and resistant populations: Investigations of potential interactions between AHR and PPARγ.

Tributyltin (TBT) and dioxin-like polychlorinated biphenyls (PCBs) are environmental contaminants that are highly toxic to fish and co-occur in New Bedford Harbor (NBH), an estuarine Superfund site located in Massachusetts, USA. Atlantic killifish (Fundulus heteroclitus) that reside in NBH (and other highly contaminated sites along the east coast of the United States) have developed resistance to activation of the aryl hydrocarbon receptor (AHR) pathway and the toxicity of dioxin-like chemicals, such as 3,3',4,4',5-pentachlorobiphenyl, PCB126. In many biological systems, TBT disregulates adipose and bone development via the PPARγ-RXR pathway; AHR activation also disrupts adipose and bone homeostasis, potentially through molecular crosstalk between AHR and PPARγ. However, little is known about how co-exposure and the interaction of these pathways modulate the toxicological effects of these contaminants. Here, we tested the hypotheses that TBT would induce teratogenesis in killifish via activation of PPARγ and that PCB126 co-exposure would suppress PPARγ pathway activation in PCB-sensitive killifish from a reference site (Scorton Creek, SC, PCB-sensitive) but not in PCB-tolerant NBH killifish. Killifish embryos from both populations exposed to TBT (50 and 100 nM) displayed caudal fin deformities. TBT did not change the expression of pparg or its target genes related to adipogenesis (fabp11a and fabp1b) in either population. However, expression of osx/sp7, an osteoblast marker gene, and col2a1b, a chondroblast marker gene, was significantly suppressed by TBT only in SC killifish. An RXR-specific agonist, but not a PPARγ-specific agonist, induced caudal fin deformities like those observed in TBT-treated embryos. PCB126 did not induce caudal fin deformities and did not exacerbate TBT-induced fin deformities. Further, PCB126 increased expression of pparg in SC embryos and not NBH embryos, but did not change the expression of fabp1b. Taken together, these results suggest that in killifish embryos the PPARγ pathway is regulated in part by AHR, but is minimally active at least in this early life stage. In killifish, RXR activation, rather than PPARγ activation, appears to be the mechanism by which TBT induces caudal fin teratogenicity, which is not modulated by AHR responsiveness.

The chemical defensome: environmental sensing and response genes in the Strongylocentrotus purpuratus genome.

Metazoan genomes contain large numbers of genes that participate in responses to environmental stressors. We surveyed the sea urchin Strongylocentrotus purpuratus genome for homologs of gene families thought to protect against chemical stressors; these genes collectively comprise the 'chemical defensome.' Chemical defense genes include cytochromes P450 and other oxidases, various conjugating enzymes, ATP-dependent efflux transporters, oxidative detoxification proteins, and transcription factors that regulate these genes. Together such genes account for more than 400 genes in the sea urchin genome. The transcription factors include homologs of the aryl hydrocarbon receptor, hypoxia-inducible factor, nuclear factor erythroid-derived 2, heat shock factor, and nuclear hormone receptors, which regulate stress-response genes in vertebrates. Some defense gene families, including the ABCC, the UGT, and the CYP families, have undergone expansion in the urchin relative to other deuterostome genomes, whereas the stress sensor gene families do not show such expansion. More than half of the defense genes are expressed during embryonic or larval life stages, indicating their importance during development. This genome-wide survey of chemical defense genes in the sea urchin reveals evolutionary conservation of this network combined with lineage-specific diversification that together suggest the importance of these chemical stress sensing and response mechanisms in early deuterostomes. These results should facilitate future studies on the evolution of chemical defense gene networks and the role of these networks in protecting embryos from chemical stress during development.

Estrogen receptor-related receptors in the killifish Fundulus heteroclitus: diversity, expression, and estrogen responsiveness.

The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to the estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the diversity, function, and regulation of ERRs in non-mammalian species are not well understood. In this study, we report the cloning of four ERR cDNAs from the Atlantic killifish, Fundulus heteroclitus, along with adult tissue expression and estrogen responsiveness. Phylogenetic analysis indicates that F. heteroclitus (Fh)ERRalpha is an ortholog of the single ERRalpha identified in mammals, pufferfish, and zebrafish. FhERRbetaa and FhERRbetab are co-orthologs of the mammalian ERRbeta. Phylogenetic placement of the fourth killifish ERR gene, tentatively identified as FhERRgammab, is less clear. The four ERRs showed distinct, partially overlapping mRNA expression patterns in adult tissues. FhERRalpha was broadly expressed. FhERRbetaa was expressed at apparently low levels in eye, brain, and ovary. FhERRbetab was expressed more broadly in liver, gonad, eye, brain, and kidney. FhERRgammab was expressed in multiple tissues including gill, heart, kidney, and eye. Distinct expression patterns of FhERRbetaa and FhERRbetab are consistent with subfunctionalization of the ERRbeta paralogs. Induction of ERRalpha mRNA by exogenous estrogen exposure has been reported in some mammalian tissues. In adult male killifish, ERR expression did not significantly change following estradiol injection, but showed a trend toward a slight induction (three- to five-fold) of ERRalpha expression in heart. In a second, more targeted experiment, expression of ERRalpha in adult female killifish was downregulated 2.5-fold in the heart following estradiol injection. In summary, our results indicate that killifish contain additional ERR genes relative to mammals, including ERRbeta paralogs. In addition, regulation of ERRalpha expression in killifish apparently differs from regulation in mammals. Together, these features may facilitate determination of both conserved and specialized ERR gene functions.

Biological activity and physicochemical parameters of marine halogenated natural products 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole and 2,4,6-tribromoanisole.

Physicochemical parameters (vapor pressure, water solubility, Henry's law constant) and biological activities of two halogenated natural products frequently detected in marine samples and food were determined. Synthetic 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1) and 2,4,6-tribromoanisole (TBA) were available in pure form. The physicochemical parameters were in the range of anthropogenic chlorinated compounds of concern. The aqueous solubilities at 25 degrees C (S(w,25)) of Q1 and TBA were 4.6 microg/L and 12,200 microg/L, respectively, whereas subcooled liquid vapor pressures were 0.00168 Pa (Q1) and 0.06562 Pa (TBA) as measured by the gas chromatographic-retention time technique. Q1 was negative by established test systems for the determination of ethoxyresorufin-O-deethylase (EROD) induction and by sulforhodamine B assay. EROD induction potency was at least 10(-7) times lower than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). At a relatively high concentration (20 microM), Q1 inhibited specific binding of 2 nM [(3)H]TCDD to the in vitro-expressed human aryl hydrocarbon receptor (AHR) by 18%; lower concentrations showed no effect. Molecular modeling showed that Q1 is nonplanar, consistent with its relatively modest affinity as an AHR ligand. When tested for cell-growth inhibitory/cytocidal activity in human tumor cells, Q1 was only marginally, if at all, active with an IC(50) value >50 microM compared with five to ten times lower IC(50) values for potent cytotoxins tested in the test system used. Furthermore, standard pesticide tests on insecticidal, herbicidal, and fungicidal activity did not provide any significant activity at highest concentrations. For TBA, the results in all tests were comparable with Q1. The SRB assay was also applied to the halogenated natural product 4,6-dibromo-2-(2',4'-dibromo)phenoxyanisole, but no toxic response was found. Although it was apparent that Q1 and TBA had been proven to have relatively low biological activity in all tests performed, further research is necessary to clarify whether metabolites of the compounds eventually may possess a risk to humans or other living organisms. Nevertheless, the role of Q1 in nature remains uncertain.

[Radioulnar impingement after distal ulnar resection and ulnar head hemiresection interposition arthroplasty (Bowers procedure). An experimental biomechanical study]. / Radioulnares Impingement nach Resektion der distalen Ulna und Ulnakopfhemiresektions-Interpositionsarthroplastik nach Bowers - Eine experimentelle biomechanische Studie.

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.

[Dynamic radioulnar convergence after Darrach operation, soft tissue stabilizing operations of the distal ulna and ulnar head prosthesis implantation--an experimental biomechanical study]. / Dynamische radioulnare Konvergenz nach Darrach-Operation, Weichteilstabilisierungsoperationen der distalen Ulna und Ulnakopfprothesenimplantation--Eine experimentelle biomechanische Studie.

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.

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.

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.

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.

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.

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.

Dioxin toxicology and the aryl hydrocarbon receptor: insights from fish and other non-traditional models.

Fish are established models in biology. Recent findings suggest that fish and other nontraditional species also may serve as valuable model systems for understanding receptor-dependent signaling pathways and their interactions with environmental chemicals. Because they are highly sensitive to chlorinated dioxins and related halogenated aromatic hydrocarbons, fish are being used to elucidate the role of chemical signaling pathways in the developmental and molecular toxicology of dioxin-like compounds. Much of this work is focused on the aryl hydrocarbon receptor (AHR), a ligand-activated, bHLH-PAS transcription factor through which dioxins cause altered gene expression and toxicity. In contrast to mammals, which appear to express a single dioxin-binding AHR, many fish species possess at least two AHR genes. Studies of these two fish AHRs may help to reveal the multiple functions of the single mammalian "AHR," its physiological ligand, and the molecular mechanisms involved in differential sensitivity to dioxin-like compounds. In addition, fish have great potential as models for understanding the in vivo functions and interactions of bHLH-PAS proteins, the evolutionary history of their diversification in vertebrates, and their role in human physiology and disease.

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.

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.

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.

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.

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.

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.

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.