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.

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.

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.

Halogenated aromatic hydrocarbon-mediated porphyrin accumulation and induction of cytochrome P4501A in chicken embryo hepatocytes.

Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), and a commercial mixture of PCBs (Aroclor 1254). For these halogenated aromatic hydrocarbons (HAHs), or mixture, maximal CYP1A activity [measured as ethoxyresorufin-O-deethylase (EROD) activity] and immunodetectable protein were observed at concentrations just prior to, or coincident with, the concentrations at which porphyrin accumulation became evident. Both immunodetectable CYP1A protein and catalytic activity decreased at high concentrations of these compounds, but the rate and extent of decrease of immunodetectable CYP1A protein varied. Time-course studies with PCB 77 indicated a decrease in potency and an increase in maximal CYP1A induction between 24 and 48 hr of exposure which may indicate in vitro metabolism of this HAH. Intracellular accumulation of total porphyrins without CYP1A induction, was observed for 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54), 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',4,5,5'-pentachlorobiphenyl (PCB 101), 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136), and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Overall, these results are consistent with a role for CYP1A induction and/or Ah receptor activation in porphyrin accumulation mediated by HAHs with a planar configuration, whereas those that are not planar may mediate porphyrin accumulation by a mechanism not involving induction of CYP1A.

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.

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.

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.

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.

Low inducibility of CYP1A activity by polychlorinated biphenyls (PCBs) in flounder (Platichthys flesus): characterization of the Ah receptor and the role of CYP1A inhibition.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.

Genetic selection disrupts stability of mouse brain weight development.

Fuller Brain Weight Selection lines are well differentiated for 42-day brain weight and a high degree of genetic homogeneity for trait-relevant genes is indicated. Using these lines, random bred descendants of the foundation population and an inbred line, biometrical analysis of brain growth from birth to 23 days was attempted. While strain means differ as expected, within and between litter variances for genetically heterogeneous mice were typically no greater than for more genetically homogeneous selected lines. Possible explanations involving gestational age, intra-uterine and postnatal competition and ontogenetic buffering are discussed.

Lead effects on food competition and predatory aggression in Binghamton HET mice.

The ubiquity of lead in our environment, its toxic nature and its potential to alter behavior of humans and animals has stimulated much research. In addition to the well known, but complex, changes in activity and performance of learned tasks following exposure to lead, an increasing body of literature suggests that changes in social behavior also occur. This study examined the impact of ingesting a 0.5% lead acetate solution (as the only available fluid)--a protocol that results in ca. 100 micrograms/dl blood-levels in our Binghamton Heterogenous (HET) mice--on food competition in both male and female HET mice. Effects of this lead exposure on cricket predation by the same HET mice also were observed. Our results from the food competition study (Experiment I) show, compared with water controls, that such exposure to lead increases both food possession time and amount of social contact after food consumption; but very little agonistic behavior took place in either the water control or lead-exposed competition testing sessions. In the cricket predation observations (Experiment II) there was a tendency for lead exposure to reduce the latency of males to attack, and the indication that lead-exposed females initially attacked a cricket's legs more often than any of the other groups. In fact, suggestions of such gender x treatment interactions occurred in both the food competition and the predation work. Overall, we believe our results, in conjunction with other relevant literature, suggest that exposure to lead reduced the individual's general fitness, even when levels are relatively low.

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.

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.

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.

The aryl hydrocarbon receptor: a comparative perspective.

The aryl hydrocarbon receptor (Ah receptor or AHR) is a ligand-activated transcription factor involved in the regulation of several genes, including those for xenobiotic-metabolizing enzymes such as cytochrome P450 1A and 1B forms. Ligands for the AHR include a variety of aromatic hydrocarbons, including the chlorinated dioxins and related halogenated aromatic hydrocarbons whose toxicity occurs through activation of the AHR. The AHR and its dimerization partner ARNT are members of the emerging bHLH-PAS family of transcriptional regulatory proteins. In this review, our current understanding of the AHR signal transduction pathway in non-mammalian and other non-traditional species is summarized, with an emphasis on similarities and differences in comparison to the AHR pathway in rodents and humans. Evidence and prospects for the presence of a functional AHR in early vertebrates and invertebrates are also examined. An overview of the bHLH-PAS family is presented in relation to the diversity of bHLH-PAS proteins and the functional and evolutionary relationships of the AHR and ARNT to the other members of this family. Finally, some of the most promising directions for future research on the comparative biochemistry and molecular biology of the AHR and ARNT are discussed.

Aryl hydrocarbon receptor function in early vertebrates: inducibility of cytochrome P450 1A in agnathan and elasmobranch fish.

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that controls the expression of cytochrome P450 1A (CYP1A) genes in response to halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand and normal physiologic function of this protein are as yet unknown. One approach to understanding AHR function and significance is to determine the evolutionary history of this receptor and of processes such as CYP1A induction that are controlled by the AHR in mammals. In these studies, AHR function was evaluated in representative cartilaginous fish (little skate, Raja erinacea) and jawless fish (sea lamprey, Petromyzon marinus and Atlantic hagfish, Myxine glutinosa), using CYP1A induction as a model AHR-dependent response. Treatment of skate with beta-naphthoflavone (BNF) caused an 8-fold increase in hepatic ethoxyresorufin O-deethylase (EROD) activity as well as a 37-fold increase in the content of immunodetectable CYP1A protein. Evidence of CYP1A inducibility was also obtained for another cartilaginous fish, the smooth dogfish Mustelus canis. In contrast, hepatic EROD activity was not detected in untreated lamprey nor in lamprey treated with 3,3'4,4'-tetrachlorobiphenyl (TCB), a potent AHR agonist in teleosts. A possible CYP1A homolog was detected in lamprey hepatic microsomes by one of three antibodies to teleost CYP1A, but expression of this protein was not altered by TCB treatment. CYP1A protein and catalytic activity were measurable in hagfish, but neither was induced after treatment with TCB. These results suggest that the AHR-CYP1A signal transduction pathway is highly conserved in gnathostomes, but that there may be fundamental differences in AHR signaling or AHR-CYP1A coupling in agnathan fish. Agnathan fish such as hagfish and lamprey may be interesting model species for examining possible ancestral AHR functions not related to CYP1A regulation.

A fish hepatoma cell line (PLHC-1) as a tool to study cytotoxicity and CYP1A induction properties of cellulose and wood chip extracts.

Cytotoxicity and CYP1A induction properties of celluloses and wood chips were studied with a teleost liver cell line, PLHC-1. Cells were exposed to acetone extracts of celluloses produced using new bleaching techniques (elemental chlorine free, ECF; totally chlorine free, TCF) in two sulphate mills or without any bleaching (unbleached, UB) in a sulphite mill. In another set of exposures, celluloses (ECF and TCF bleached) and wood chips (from pine and birch) were collected from a sulphate mill, extracted with acetone, and the extracts used to treat the cells. After exposure, O-deethylation of 7-ethoxyresorufin (EROD, a measure of cytochrome P4501A (CYP1A) catalytic activity), and total protein content, a measure of cytotoxicity, were assayed. The presence of the CYP1A protein in the exposed cells was assessed by immunoblotting. The cellulose and wood chip extracts were able to cause both cytotoxicity and EROD induction in the PLHC-1 cells. In the exposures conducted with the material from three different mills, the celluloses made of birch were more cytotoxic and more potent inducers of EROD activity than were the celluloses of pine. Further, UB celluloses increased EROD activity and caused cytotoxicity at lower doses than material bleached with modern bleaching techniques. In the exposures made with material from one single mill, there were no clear trends between the celluloses made of pine or birch. Wood chips of pine, however, were more cytotoxic than wood chips of birch. Especially with pine wood chips, cytotoxicity interfered with the induction of EROD activity, thus complicating the evaluation of CYP1A induction. CYP1A protein content was not detected in cells exposed to extracts of celluloses or wood chips, possibly due to low amounts of protein available for the assay. Wood and pulp processing, like bleaching, may change the chemical composition of the raw material in a way that reduces the potency for biological effects of the final product, cellulose. This could explain why both UB celluloses and wood chips were more potent in the cells than ECF or TCF bleached celluloses. In this study the PLHC-1 cell line showed its potential for use in evaluating the biological activity existing in pulp and paper mill products and raw materials. The identity and source of the compounds that were able to affect the PLHC-1 cell line remain to be determined.

The evolution of aryl hydrocarbon signaling proteins: diversity of ARNT isoforms among fish species.

The aryl hydrocarbon receptor nuclear translocator (ARNT) mediates aryl hydrocarbon signaling and toxicity by dimerizing with the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that binds specific DNA elements and alters transcription of target genes. Two genes encode different forms of ARNT in rodents: ARNT1, which is widely expressed, and ARNT2, which exhibits a very restricted expression pattern. In an effort to characterize aryl hydrocarbon signaling mechanisms in fishes, we previously isolated an ARNT cDNA from Fundulus heteroclitus and discovered that this species expresses ARNT2 ubiquitously. This situation differs not only from mammals, but also from rainbow trout, which expresses a divergent ARNT gene that we hypothesized was peculiar to salmonids (rtARNTa/b). In this communication, we examine the ARNT sequences of multiple fish species, including a newly isolated cDNA from scup (Stenotomus chrysops). Our phylogenetic analysis demonstrates that zebrafish ARNT, like the Fundulus protein, is an ARNT2. Contrary to expectations, the scup ARNT is closely related to the rainbow trout protein, demonstrating that the existence of this ARNT isoform predates the divergence of salmonids from the other teleosts. Thus, different species of fish express distinct and highly conserved isoforms of ARNT. The number, type, and expression pattern of ARNT proteins may contribute to interspecies differences in aryl hydrocarbon toxicity, possibly through distinct interactions with additional PAS-family proteins.

Towards molecular understanding of species differences in dioxin sensitivity: initial characterization of Ah receptor cDNAs in birds and an amphibian.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to most vertebrate animals, but there are dramatic species differences in sensitivity, both within and among vertebrate classes. For example, studies in cultured avian hepatocytes have revealed differential sensitivity of birds to PHAHs [Kennedy et al. (1996). Toxicol. Appl. Pharmacol., 141, 214-230]. Differences in the characteristics or expression of the aryl hydrocarbon receptor (AHR) could contribute to these species differences in PHAH responsiveness. To investigate the molecular mechanism of differential PHAH sensitivity, we have begun to characterize the AHR in white leghorn chicken (Gallus gallus), Pekin duck (Anas platyrhynchos), and common tern (Sterna hirundo), as well as an amphibian, mudpuppy (Necturus maculosus). Partial AHR cDNAs encompassing the helix-loop-helix and PAS domains were cloned and sequenced. Comparison of amino acid sequences in this region indicated a high degree of sequence conservation among the bird species (97% amino acid identity). The percent identity between bird sequences and either mouse or mudpuppy was lower (79%); the mudpuppy AHR was 74% identical to the mouse AHR. Phylogenetic analysis of these and other AHR amino acid sequences showed that the bird and mudpuppy AHRs were more closely related to mammalian and fish AHR1 forms than to fish AHR2. Future studies include the in vitro expression and functional characterization of AHRs from these and other non-mammalian vertebrates.

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.