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Characterization of the aryl hydrocarbon receptor repressor and comparison of its expression in Atlantic tomcod from resistant and sensitive populations
Abstract
Atlantic tomcod from the Hudson River, USA, are resistant to cytochrome P4501A1 (CYP1A1) mRNA induction and early life stage toxicities induced by coplanar polychlorinated biphenyls (PCBs) or tetrachlorodibenzo-p-dioxins but not polycyclic aromatic hydrocarbons. We sought to determine if basal expression or inducibility of aryl hydrocarbon receptor repressor (AHRR) mRNA is higher in tomcod from the resistant Hudson River population than in those from sensitive populations. Tomcod AHRR cDNA was characterized and its expression quantified in different tissues and life stages of tomcod from the Hudson River, Miramichi River, Canada (sensitive), and among environmentally exposed tomcod from these two sources and the St. Lawrence River, Canada. Phylogenetic analysis revealed that tomcod AHRR falls within the clade of other vertebrate aryl hydrocarbon receptors (AHRs) but is most closely related to the four previously identified AHRR genes. Induction of AHRR mRNA was observed in all tissues of PCB77-treated juvenile tomcod of Miramichi River descent, and expression differed among tissues and was significantly related to levels of CYPIAI mRNA expression. Aryl hydrocarbon receptor repressor mRNA was similarly inducible in F2 embryos of Miramichi and Hudson River descent by benzo[a]pyrene but less by PCB77 in Hudson River offspring. A significant, positive correlation was observed between CYP1A1 mRNA and AHRR mRNA concentrations in environmentally exposed tomcod from the three rivers. We conclude that differences in basal expression or inducibility of AHRR mRNA are not the mechanistic basis of resistance but that levels of AHRR often mirror those of CYP1A1, suggesting that a common AHR pathway-related mechanism may modulate expression of both genes.
... AHRR orthologs have been identified and characterized in several mammalian species [68,71,73,74], in an amphibian [75], and in bony fishes [69,70,76]. Zebrafish is notable for possessing two AHRR paralogs, which, based on phylogenetic analysis and conserved synteny, appear to be co-orthologs of the mammalian AHRR [76]. ...
... AHRR orthologs have been identified and characterized in several mammalian species [68,71,73,74], in an amphibian [75], and in bony fishes [69,70,76]. Zebrafish is notable for possessing two AHRR paralogs, which, based on phylogenetic analysis and conserved synteny, appear to be co-orthologs of the mammalian AHRR [76]. An AHRR gene has not been identified in any earlier diverging vertebrates or invertebrates, including an invertebrate chordate (Ciona intestinalis [77]) or a deuterostome (sea urchin Strongylocentrotus purpuratus [78]. ...
... AHRR is expressed in fish and amphibian embryos [70,75,76], and in mammalian fetuses, where expression appears to be low relative to that in adult tissues [83,90,91]. The embryo-fetal expression suggests a possible developmental role for the AHRR. ...
The AHR is well known for regulating responses to an array of environmental chemicals. A growing body of evidence supports the hypothesis that the AHR also plays perhaps an even more important role in modulating critical aspects of cell function including cell growth, death, and migration. As these and other important AHR activities continue to be elucidated, it becomes apparent that attention now must be directed towards the mechanisms through which the AHR itself is regulated. Here, we review what is known of and what biological outcomes have been attributed to the AHR repressor (AHRR), an evolutionarily conserved bHLH-PAS protein that inhibits both xenobiotic-induced and constitutively active AHR transcriptional activity in multiple species. We discuss the structure and evolution of the AHRR and the dominant paradigm of a xenobiotic-inducible negative feedback loop comprised of AHR-mediated transcriptional up-regulation of AHRR and the subsequent AHRR-mediated suppression of AHR activity. We highlight the role of the AHRR in limiting AHR activity in the absence of xenobiotic AHR ligands and the important contribution of constitutively repressive AHRR to cancer biology. In this context, we also suggest a new hypothesis proposing that, under some circumstances, constitutively active AHR may repress AHRR transcription, resulting in unbridled AHR activity. We also review the predominant hypotheses on the molecular mechanisms through which AHRR inhibits AHR as well as novel mechanisms through which the AHRR may exert AHR-independent effects. Collectively, this discussion emphasizes the importance of this understudied bHLH-PAS protein in tissue development, normal cell biology, xenobiotic responsiveness, and AHR-regulated malignancy.
... Evidence from other fishes, suggested that AHR2 was a more functional form of AHR in mediating developmental toxicities (Prasch et al. 2003), including cardiac teratogenesis (Clark et al. 2010), because it was more widely expressed across tissues (Karchner et al. 1999) and bound AHR ligands more efficiently than AHR1 (Andreasen et al. 2002). Roy and colleagues cloned AHR2 (Roy and Wirgin 1997), ARNT (Roy and Wirgin, unpublished), and AHRR (Roy et al. 2006) from tomcod and compared their expression in tomcod treated with PCB77, B[a]P, or vehicle solvent, and in environmentally exposed tomcod from the Hudson River and other cleaner rivers. They hypothesized that AHR2 or ARNT expression would be downregulated and AHRR expression upregulated in Hudson River compared to tomcod from cleaner locales. ...
... They hypothesized that AHR2 or ARNT expression would be downregulated and AHRR expression upregulated in Hudson River compared to tomcod from cleaner locales. These hypotheses were not supported as neither AHR2 nor ARNT expression were downregulated in Hudson River tomcod and AHRR expression was lower, rather than higher in Hudson River tomcod compared to tomcod from elsewhere (Roy and Wirgin 1997;Roy et al. 2006). ...
The Hudson River (HR) Estuary has a long history of pollution with a variety of contaminants including PCBs and dioxins. In fact, 200 miles of the mainstem HR is designated a U.S. federal Superfund site, the largest in the nation, because of PCB contamination. The tidal HR hosts the southernmost spawning population of Atlantic tomcod, Microgadus tomcod, and studies revealed a correlation between exposure of juveniles to warm water temperature during summer to abundance of spawning adults of the same cohort in the following winter. Further, a battery of mechanistically linked biomarkers, ranging from the molecular to the population levels, were significantly impacted from contaminant exposures of the HR tomcod population. In response to xenobiotic insult, the HR tomcod population developed resistance to PCB and TCDD toxicity resulting from a deletion in the aryl hydrocarbon receptor2 (AHR2) gene. Furthermore, RNA-Seq analysis of global gene expression demonstrated that effects of the AHR2 polymorphism were far more pervasive than anticipated. The most highly PCB-contaminated sediments in the upper HR were dredged between 2009 and 2015 with the objective of lowering PCB concentrations in fishes in the lower HR. Success of the remediation project has been controversial. These observations suggest that tomcod provides an informative model to evaluate the efficacy of HR PCB remediation efforts on downriver fish populations and possible interactive effects between contaminant exposure and a warming environment.
... In this case, lack of CYP1A inducibility may not be a toxicologically protective adaptation but rather a marker of AHR pathway inhibition. While regulation of other AHR pathway genes has thus far received less attention in resistant fish populations, mRNA inducibility of at least one additional AHR-regulated gene, AHRR, is diminished in at least two resistant populations (Meyer et al., 2003b;Roy et al., 2006). mRNA expression levels in the absence of a laboratory exposure were not significantly different from those observed in a reference population in any of three tested resistant populations (Karchner et al., 2002;Meyer et al., 2003b;Roy et al., 2006). ...
... While regulation of other AHR pathway genes has thus far received less attention in resistant fish populations, mRNA inducibility of at least one additional AHR-regulated gene, AHRR, is diminished in at least two resistant populations (Meyer et al., 2003b;Roy et al., 2006). mRNA expression levels in the absence of a laboratory exposure were not significantly different from those observed in a reference population in any of three tested resistant populations (Karchner et al., 2002;Meyer et al., 2003b;Roy et al., 2006). ...
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. Traditionally, much of the research has
focused on the carcinogenic potential of specific PAHs, such as benzo(a)pyrene, but recent studies using sensitive fish models have shown that exposure to PAHs alters normal fish development. Some
PAHs can induce a teratogenic phenotype similar to that caused by planar halogenated aromatic hydrocarbons, such as dioxin.
Consequently, mechanism of action is often equated between the two classes of compounds. Unlike dioxins, however, the developmental
toxicity of PAH mixtures is not necessarily additive. This is likely related to their multiple mechanisms of toxicity and
their rapid biotransformation by CYP1 enzymes to metabolites with a wide array of structures and potential toxicities. This
has important implications for risk assessment and management as the current approach for complex mixtures of PAHs usually
assumes concentration addition. In this review we discuss our current knowledge of teratogenicity caused by single PAH compounds
and by mixtures and the importance of these latest findings for adequately assessing risk of PAHs to humans and wildlife.
Throughout, we place particular emphasis on research on the early life stages of fish, which has proven to be a sensitive
and rapid developmental model to elucidate effects of hydrocarbon mixtures.
... Karchner et al. (2002) determined that the expression and tissue distribution of AHRR mRNA from TCDD-and PCB-resistant killfish (Fundulus heteroclitus) from New Bedford Harbor, MA resembled fish from a reference site, and Meyer et al. (2003) made similar observations of PAH-resistant killifish from Virginia's Elizabeth River. Basal AHRR expression in HAH-resistant Atlantic tomcod (Microgadus tomcod) from the highly polluted Hudson River (New York) was not altered and could not explain the CYP1A-refractory phenotype in this population (Roy et al. 2006). Rather, there was a positive correlation between CYP1A and AHRR expression in both sensitive and resistant populations (Roy et al. 2006). ...
... Basal AHRR expression in HAH-resistant Atlantic tomcod (Microgadus tomcod) from the highly polluted Hudson River (New York) was not altered and could not explain the CYP1A-refractory phenotype in this population (Roy et al. 2006). Rather, there was a positive correlation between CYP1A and AHRR expression in both sensitive and resistant populations (Roy et al. 2006). Our observations of AHRR expression in frogs reinforce the notion that AHRR is regulated in coordinate fashion with CYP1A. ...
Xenopus laevis and other frogs are extremely insensitive to the toxicity of xenobiotic ligands of the aryl hydrocarbon receptor (AHR), including
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Premetamorphic life stages are especially insensitive, and they are reported to be refractory to induction
of Cytochrome P4501As, which are readily induced in older animals. The AHR repressor (AHRR) is a member of the AHR gene family.
AHRR expression is induced by TCDD; it then represses AHR in an apparent negative feedback loop. In this study, we sought
to test the hypothesis that constitutive AHRR expression underlies the lack of TCDD responsiveness in frog early life stages.
We determined the sequence of an AHRR complimentary DNA encoding an 85.3-kDa protein sharing 52–55% identity with the bHLH/PAS
domains of other AHRRs. In transient transfection assays, X. laevis AHRR inhibited TCDD-induced reporter gene expression mediated by either X. laevis AHR paralog, AHR1α or AHR1β. AHRR messenger RNA was expressed at low levels in embryos (Nieuwkoop-Faber stage 33–38; approximately
52 h.p.f.) and was induced approximately twofold following TCDD exposure (42 ng/g wet weight). In contrast, AHRR exhibited
higher constitutive expression and was induced more than threefold in tadpoles at stage 52–55 (prometamorphic; ∼4 weeks postfertilization)
and in isolated viscera of stage 62 tadpoles (in the metamorphic climax; ∼7 weeks postfertilization). Although the magnitude
of induction was smaller, the temporal pattern of AHRR expression and inducibility resembled that of CYP1A6. Thus, attenuated transcriptional activation of AHR target genes and low TCDD toxicity in X. laevis embryos cannot be explained by constitutive, high-level expression of AHRR.
... After being exported from the nucleus, mammalian AhR is degraded in the cytoplasm (Pollenz, 2002). In contrast to mammals, fish have at least two AhR genes, AhR1 and AhR2 (Hahn et al., 2006;Hansson et al., 2003;Roy et al., 2006). Two forms of the AhR2 gene have been identified in rainbow trout (␣ and ) (Abnet et al., 1999) and in Atlantic salmon (␥ and ␦) (Hansson et al., 2003). ...
The fate of pharmaceuticals in aquatic environments is an issue of concern. Current evidence indicates that the risks to fish greatly depend on the nature and concentrations of the pharmaceuticals and might be species-specific. Assessment of risks associated with the presence of pharmaceuticals in water is hindered by an incomplete understanding of the metabolism of these pharmaceuticals in aquatic species. In mammals and fish, pharmaceuticals are primarily metabolized by cytochrome P450 enzymes (CYP450). Thus, CYP450 activity is a crucial factor determining the detoxification abilities of organisms. Massive numbers of toxicological studies have investigated the interactions of human pharmaceuticals with detoxification systems in various fish species. In this paper, we review the effects of pharmaceuticals found in aquatic environments on fish hepatic CYP450. Moreover, we discuss the roles of nuclear receptors in cellular regulation and the effects of various groups of chemicals on fish, presented in the recent literature.
Copyright © 2015 Elsevier B.V. All rights reserved.
... Both populations had similarly increased levels of AHR expression, a normal response to PAHs, but their CYP1A expression was recalcitrant (Roy and Wirgin 1997). The downregulation of the pathway could not be attributed to the AHR repressor (AHRR), which was also found to be downregulated in all tissues, similar to CYP1A (Roy et al. 2006). Following these results, researchers sequenced AHR2, the more active form in fish, for tomcod from four reference populations and two populations in the polluted Hudson River. ...
Anthropogenic activity has affected nearly every environment on the planet. The changes that have occurred as a consequence of human activities have altered aquatic habitats by exacerbating already existing extreme environments and by introducing novel stressors. In some cases, particularly adjacent to heavily industrialized areas, these changes have introduced sufficient novel selective pressures to drive resident populations to genetically adapt in order to survive in the altered habitats, while species that were unable to adapt have been extirpated from these extreme environments. In this chapter, we aim to explore the effects of natural and novel stressors, resulting from anthropogenic activity, on fish populations. We will provide an overview of the possible multi-generational outcomes of anthropogenic contamination, as well as explore documented examples of population-wide changes that have occurred. We present case studies, including population responses to UV light, radionuclides, and metals contamination, as well as adaptational responses to persistent organic pollutants. Through this examination, we aim to not only give an overview of the existing evolutionary changes in fish populations in response to anthropogenic contamination, but also identify future areas of research on the impacts, long-term persistence, and ecological significance of these effects.
... Investigations into the possible molecular mechanisms of resistance in HR tomcod led to the characterization of several key molecules in the aryl hydrocarbon receptor (AhR) pathway including tomcod AhR2 [9], AhR nuclear translocator1 (ARNT1; N.K. Roy and I.I. Wirgin, New York University, Tuxedo, NY, USA, unpublished observations), and AhR repressor (AhRR) [10]. In vertebrates, AhR is a cytosolic transcription factor that exhibits a high binding affinity for various planar AHs including PCBs [11]. ...
Several populations of fishes inhabiting contaminated Atlantic Coast estuaries exhibit resistance to early life-stage (ELS) toxicities induced by halogenated aromatic hydrocarbons such as coplanar polychlorinated biphenyls (PCBs). These toxicities include mortality, circulatory failure, edema, and craniofacial malformations. The mechanisms behind resistance to HAH toxicity in these populations are unknown. First and second generation Atlantic tomcod Microgadus tomcod embryos derived from the Hudson River (HR; NY, USA) population are highly resistant to PCB -induced cytochrome P450 1A (CYP1A) expression and ELS toxicity when compared to embryos of Miramichi River (MR; NB, Canada) and Shinnecock Bay (SB; NY, USA) origin. The present study sought to identify novel genes involved in population differences in response to PCB exposure using custom microarrays. Microarray probes consisted of un-sequenced inserts of randomly-picked clones from a tomcod cardiac cDNA library. Tomcod embryos from three populations (HR, MR, and SB) were exposed to two doses of an environmentally-relevant mixture of coplanar PCBs and screened for dose- and population-specific patterns of gene expression. Clones displaying significant differences between populations exposed to the high dose of PCBs were identified by DNA sequencing. Of the 28 identified non-ribosomal protein clones, none displayed expression patterns highly similar to CYP1A (altered in MR and SB, but not in HR). However, several transcripts representing biomarkers of cardiomyopathy in mammals (cardiac troponin T2, cathepsin L, and atrial natriuretic peptide) were differentially altered among the 3 tomcod populations by PCBs. Although the present study did not identify any novel genes associated with PCB resistance in tomcod, several potential molecular biomarkers of PCB exposure were revealed.
... These results suggest that both cyp1a1 and ahrr mRNA inducibility is part of a mechanistic basis for resistance of fish larvae against compounds in dispersed oil, explaining the simultaneous induction of cyp1a1 and ahrr mRNA. A similar finding has been reported for Atlantic tomcod (Microgadus tomcod), with a positive correlation between ahrr and cyp1a1 mRNA levels in fish exposed to AH-responsive compounds [30]. Another explanation for this finding could also be that the dispersed oil mediated different effects in different organs, e.g. ...
Background
The use of dispersants can be an effective way to deal with acute oil spills to limit environmental damage, however very little is known about whether chemically dispersed oil have the same toxic effect on marine organisms as mechanically dispersed oil. We exposed Atlantic cod larvae to chemically and mechanically dispersed oil for four days during the first-feeding stage of development, and collected larvae at 14 days post hatch for transcriptional analysis. A genome-wide microarray was used to screen for effects and to assess whether molecular responses to chemically and mechanically dispersed oil were similar, given the same exposure to oil (droplet distribution and concentration) with and without the addition of a chemical dispersant (Dasic NS).
Results
Mechanically dispersed oil induced expression changes in almost three times as many transcripts compared to chemically dispersed oil (fold change >+/−1.5). Functional analyses suggest that chemically dispersed oil affects partly different pathways than mechanically dispersed oil. By comparing the alteration in gene transcription in cod larvae exposed to the highest concentrations of either chemically or mechanically dispersed oil directly, the chemically dispersed oil affected transcription of genes involved nucleosome regulation, i.e. genes encoding proteins participating in DNA replication and chromatin formation and regulation of cell proliferation, whereas the mechanically dispersed oil most strongly affected genes encoding proteins involved in proteasome-mediated protein degradation. Cyp1a was the transcript that was most strongly affected in both exposure groups, with a 60-fold induction in the two high-exposure groups according to the RT-qPCR data, but no significant difference in transcriptional levels was observed between the two treatments.
Conclusions
In summary, dispersants do not appear to add to the magnitude of transcriptional responses of oil compounds but rather appear to lower or modify the transcriptional effect on cod larvae.
... In transient transfection assays, AHRR exhibited repression activity in AHR-dependent transactivation of CYP1A that was induced by TCDD (Evans et al., 2005;Karchner et al., 2002;Mimura et al., 1999;Zimmermann et al., 2008). In addition, AHRR was transcriptionally induced by ligand-activated AHR in vivo and in vitro (Evans et al., 2005;Hanno et al., 2010;Roy et al., 2006) via XREs located in the promoter region of AHRR (Karchner et al., 2002). Hence, TCDD-induced expression of AHRR and CYP1A and the following repression of induced CYP1A transactivation by AHRR imply the existence of a negative feedback loop of AHR signaling pathway by AHRR. ...
The aryl hydrocarbon receptor (AHR) repressor (AHRR) has been recognized as a negative feedback modulator of AHR-mediated
responses in fish and mammals. However, the repressive mechanism by the AHRR has not been investigated in other animals. To
understand the molecular mechanism of dioxin toxicity and the evolutionary history of the AHR signaling pathway in avian species,
the present study addresses chicken AHRR (ckAHRR). The complementary DNA sequence of ckAHRR encodes an 84-kDa protein sharing 29–52% identities with other AHRRs. High levels of ckAHRR messenger RNA were recorded in the kidney and intestine of nontreated chicks. In hepatoma LMH cells, the 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) 50% effective concentration value for ckAHRR induction (0.0016nM) was the same as that for chicken cytochrome P450 1A5 (ckCYP1A5), implying a shared transcriptional regulation of ckAHRR and ckCYP1A5 by chicken AHR (ckAHR). In ckAHRR transient transfection assays, ckAHRR repressed both ckAHR1- and ckAHR2-mediated transcriptional activities. Deletion and mutation assays revealed that basic helix-loop-helix/Per-ARNT-Sim A
domains of ckAHRR, particularly 217–402 amino acid residues, are indispensable for the repression, but the AHR nuclear translocator sequestration
by ckAHRR and SUMOylation of ckAHRR are not involved in its repressive mechanism. Additionally, subcellular localization assay of ckAHR1-enhanced green fluorescent protein fusion protein showed that ckAHRR did not affect nuclear translocation of the ckAHR1. Furthermore, ckAHRR inhibited the TCDD- and 17β estradiol-enhanced ckCYP1A5 transcription through AHR-estrogen receptor α (ERα) cross talk. Taken together, the function of AHRR is conserved in
chicken in terms of the negative regulation of AHR and ERα activities, but its functional mechanism is likely distinct from
those of the mammalian and fish homologues.
... Moreover, appear to be co-orthologs of the mammalian AHRR; it is maybe the result of the fish-specific whole genome duplication (Evans et al., 2005). Not unexpectedly, the zebrafish AHRR (zfAHRR) genes clustered most closely with other fishes AHRR compared to amphibian and mammalian AHRRs, which received the support from Roy et al. (2006). Furthermore, zfAHRR1 more tightly clustered with the other fish AHRR genes than zfAHRR2 gene, with 97% bootstrap support using our methods. ...
Phylogenetic analysis of AhR pathway genes and their evolutionary rate variations were studied on aquatic animals. The gene sequences for the proteins involved in this pathway were obtained from four major phylogenetic groups, including bivalvia, amphibian, teleostei and mammalia. These genes were distributed under four major steps of toxicology regulation: formation of cytosolic complex, translocation of AhR, heterodimerization of AhR and induction of CYP1A. The NJ, MP, and ML algorithm were used on protein coding DNA sequences to deduce the evolutionary relationship for the respective AhR pathway gene among different aquatic animals. The rate of non-synonymous nucleotide substitutions per non-synonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S)) were calculated for different clade of the respective phylogenetic tree for each AhR pathway gene. The phylogenetic analysis suggests that evolutionary pattern of AhR pathway genes in aquatic animals is characterized mainly through gene duplication events or alterative splicing. The d(N) values indicate that all AhR pathway genes are well conserved in aquatic animals, except for CYP1A gene. Furthermore, compare with other aquatic animals, the d(N) value indicates that AhR pathway genes of fish are less conserved, and these genes likely go through an adaptive evolution within aquatic animals.
... Es konnte gezeigt werden, dass der AhRR in die Regulation von Wachstum und Differenzierung dieser Zellen involviert ist. Das AhRR Gen wurde bisher im Erbgut von Mensch (Watanabe et al., 2001), Maus (Mimura et al., 1999), Ratte (Korkalainen et al., 2004) und diversen Fischarten nachgewiesen (Karchner et al., 2002; Evans et al., 2005; Roy et al., 2006). Durch die voranschreitende ...
Mensch und Tier sind täglich einer Vielzahl von chemischen Substanzen ausgesetzt, mit denen sie über Nahrung, Wasser und Luft in Kontakt geraten. Einige dieser Stoffe wirken relativ unspezifisch auf die exponierten Gewebe bzw. Zellen ein und schädigen biologische Strukturen und Makromoleküle. Andere Substanzen wiederum sind in der Lage, an intrazelluläre Rezeptormoleküle zu binden und dadurch die Signaltransduktion einer Zelle zu beeinflussen. Diese Stoffe lösen somit ein charakteristisches Spektrum von biochemischen und toxischen Wirkungen in einem Organismus aus. Gut untersuchte fremdstoffbindende Rezeptoren sind z.B. der Pregnan X Rezeptor (PXR), der konstitutive Androstan Rezeptor (CAR) oder der in dieser Arbeit im Mittelpunkt stehende Arylhydrocarbon Rezeptor (AhR).
Zu der Gruppe der AhR-bindenden Chemikalien (Liganden) gehören u.a. die in der Umwelt ubiquitär verbreiteten, persistenten halogenierten aromatischen Kohlenwasserstoffe und die polyzyklischen aromatischen Kohlenwasserstoffe. Diese Stoffe entstehen bei Verbrennungsprozessen und gelangen somit permanent in unsere Umwelt. Unter diesen lipophilen Chemikalien finden sich einige Vertreter mit hohem toxischem Potential wieder. Der wohl prominenteste Vertreter dieser Gruppe von Umweltchemikalien ist das 1976 bei einem tragischen Chemieunfall als „Seveso-Gift“ bekannt gewordene 2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD). Das Spektrum der durch diese Substanz hervorgerufenen toxischen Effekte ist breit gefächert und umfasst beispielsweise Prozesse wie Immunsuppression, Teratogenese und Kanzerogenese. Des Weiteren kommt es durch die Exposition mit TCDD und verwandten Chemikalien zu einer adaptiven Induktion des fremdstoffmetabolisierenden Enzymsystems. Sowohl Toxizität als auch Enzyminduktion werden von dem zytoplasmatisch lokalisierten Transkriptionsfaktor AhR vermittelt. Durch Bindung eines Liganden wird das AhR-Molekül aktiviert und dringt in den Zellkern ein, wo es ein Heterodimer mit seinem Partnermolekül ARNT (AhR Nuklear Translokator) bildet. Der AhR/ARNT-Komplex ist in der Lage durch Bindung an spezifische DNA-Motive die Expression von Genen zu modulieren. Die auf diese Weise regulierten Gene bzw. deren Genprodukte üben verschiedene Funktionen innerhalb des Fremdstoffwechsels aus, können aber auch Prozesse des Zellwachstums und der Zelldifferenzierung beeinflussen.
Vor einigen Jahren konnte ein weiteres Mitglied der AhR-Signalkaskade identifiziert werden, der AhR Repressor (AhRR). Die Expression des AhRR wird durch den ligandenaktivierten AhR kontrolliert. Mit Hilfe von Überexpressionsstudien konnte gezeigt werden, dass der AhRR in der Lage ist den Ablauf AhR-abhängiger Signalwege zu supprimieren. Über die molekulare Funktionsweise und die Bedeutung des AhRR für die AhR-vermittelten biologischen und toxischen Prozesse ist bis heute wenig bekannt.
Ziel dieser Arbeit war es, die Mechanismen der transkriptionellen Regulation sowie die intrazellulären Funktionen des humanen AhRR zu erforschen. Für die Untersuchungen standen vier humane Zelllinien zur Verfügung. Neben den drei transformierten Zelllinien A549, HepG2 und HeLa, wurde eine primäre Fibroblasten Kultur in die Studien einbezogen.
In der vorliegenden Arbeit wurden folgende wesentliche Ergebnisse erzielt:
1. Die untersuchten Zelllinien zeigen eine zellspezifische AhRR Genexpression auf. Die Höhe der basalen AhRR Transkription korreliert mit der Responsivität des CYP1A1 Gens gegenüber AhR-Agonisten.
2. Das AhRR Gen wird durch einen regulatorisch aktiven Bereich in der Intron 1 Region moduliert. In diesem Bereich ist ein responsives XRE-Motiv lokalisiert, welches durch AhR und ARNT angesteuert wird. Das AhRR Protein bindet ebenfalls an dieses XRE-Motiv und moduliert somit seine eigene Expression autoregulativ.
3. In direkter Nachbarschaft des XREs ist eine funktionelle GC Box lokalisiert. Dieses DNA-Bindemotiv für Sp1-verwandte Transkriptionsfaktoren beeinflusst sowohl die basale als auch die 3-MC-induzierbare AhRR Genexpression.
4. Die Aktivierung der Hypoxie-sensitiven Signalkaskade führt zu einer NF-κB-vermittelten Induktion der AhRR Transkription. Ein im Intron 1 lokalisiertes NF-κB-Bindemotiv zeigt DFX- bzw. Hypoxie-responsive Aktivitäten auf.
5. Ein Ausschalten der AhRR Expression in den nicht-responsiven HeLa Zellen geht mit einem Anstieg der basalen CYP1A1 mRNA Expression einher. Der AhRR spielt somit in der konstitutiven Regulation der CYP1A1 Transkription eine funktionelle Rolle.
6. Die fehlende CYP1A1 Responsivität in Fibroblasten und HeLa Zellen wird durch den konstitutiv XRE-gebundenen AhRR verursacht. Dieser rekrutiert HDAC-Moleküle wodurch es zu einer nachgeschalteten Hemmung der CYP1A1 Genexpression kommt.
Die gewonnen Befunde lassen auf eine komplexe Steuerung der AhRR Transkription schließen und deuten auf mögliche AhR-unabhängige Wege der AhRR-Aktivierung hin. Der AhRR spielt eine Rolle in der konstitutiven Regulation von AhR-Zielgenen und stellt somit vermutlich Teil eines zellulären Schutzmechanismus dar. Die postulierte Funktion des AhRR als negativer Regulator der AhR-Signalkaskade konnte nicht belegt werden.
... Investigations into the possible molecular mechanisms of resistance in HR tomcod led to the characterization of several key molecules in the aryl hydrocarbon receptor (AhR) pathway including tomcod AhR2 [9], AhR nuclear translocator1 (ARNT1; N.K. Roy and I.I. Wirgin, New York University, Tuxedo, NY, USA, unpublished observations), and AhR repressor (AhRR) [10]. In vertebrates, AhR is a cytosolic transcription factor that exhibits a high binding affinity for various planar AHs including PCBs [11]. ...
Several populations of fishes inhabiting contaminated Atlantic Coast estuaries exhibit resistance to early life-stage (ELS) toxicities induced by halogenated aromatic hydrocarbons such as coplanar polychlorinated biphenyls (PCBs). These toxicities include mortality, circulatory failure, edema, and craniofacial malformations. The mechanisms behind resistance to halogenated aromatic hydrocarbon toxicity in these populations are unknown. First and second generation Atlantic tomcod Microgadus tomcod embryos derived from the Hudson River ([HR]; New York, USA) population are highly resistant to PCB-induced cytochrome P4501A (CYP1A) expression and ELS toxicity when compared to embryos of Miramichi River ([MR]; New Brunswick, Canada) and Shinnecock Bay ([SB]; New York, USA) origin. The present study sought to identify novel genes involved in population differences in response to PCB exposure using custom microarrays. Microarray probes consisted of unsequenced inserts of randomly picked clones from a tomcod cardiac cDNA library. Tomcod embryos from three populations (HR, MR, and SB) were exposed to two doses of an environmentally relevant mixture of coplanar PCBs and screened for dose- and population-specific patterns of gene expression. Clones displaying significant differences between populations exposed to the high dose of PCBs were identified by DNA sequencing. Of the 28 identified nonribosomal protein clones, none displayed expression patterns highly similar to CYP1A (altered in MR and SB, but not in HR). However, several transcripts representing biomarkers of cardiomyopathy in mammals (cardiac troponin T2, cathepsin L, and atrial natriuretic peptide) were differentially altered among the three tomcod populations by PCBs. Although the present study did not identify any novel genes associated with PCB resistance in tomcod, several potential molecular biomarkers of PCB exposure were revealed.
... The function of the AhRR was described as a negative feedback modulator of the AhR pathway, doing this by competing with the AhR for ARNT-and XRE-binding, thereby blocking AhR-dependent gene expression (Mimura et al., 1999). The AhRR gene was detected in several species, including fish, rat, mice and humans (Tsuchiya et al., 2003a;Korkalainen et al., 2004;Yamamoto et al., 2004;Evans et al., 2005;Bernshausen et al., 2006;Nishihashi et al., 2006;Roy et al., 2006). Expression analysis of AhRR mRNA in different organs of mice, rats, and humans revealed a tissue-specific expression of the AhRR (Tsuchiya et al., 2003a;Korkalainen et al., 2004;Yamamoto et al., 2004;Bernshausen et al., 2006;Nishihashi et al., 2006). ...
The aryl hydrocarbon receptor repressor (AhRR) is a member of the aryl hydrocarbon receptor (AhR) signaling cascade, which mediates dioxin toxicity and is involved in regulation of cell growth and differentiation. The AhRR was described as a feedback modulator, which counteracts AhR-dependent gene expression. We investigated the molecular mechanisms of transcriptional regulation of the human AhRR by cloning its regulatory DNA region located in intron I of the AhRR. By means of reporter gene analyses and generation of deletion variants, we identified a functional, 3-methylcholanthrene-sensitive xenobiotic response element (XRE) site. Chromatin immunoprecipitation analyses revealed that the AhRR binds to this XRE, displaying an autoregulatory loop of AhRR expression. In addition we show that an adjacent GC-box is of functional relevance for AhRR transcription, since blocking of this GC-box resulted in a decrease of constitutive and inducible AhRR gene activity. The differences in constitutive AhRR mRNA level observed in HepG2, primary fibroblast, and HeLa cells are directly correlated with CYP1A1 inducibility. We show that the nonresponsiveness of high AhRR-expressing cells toward AhR-agonists is associated with a constitutive binding of the AhRR to XRE sites of CYP1A1. Treatment with the histone deacetylase inhibitor sodium butyrate restored the responsiveness of CYP1A1 in these cell lines, due to the dissociation of AhRR from the XREs. Furthermore, transient AhRR mRNA silencing in untreated HeLa cells was accompanied by an increase of basal CYP1A1 expression, pointing to a constitutive role of the AhRR in regulation of CYP1A1. The functional relevance of the AhRR in high AhRR-expressing primary fibroblasts is discussed.
... The AHR2 isoform-and tissue-specific differences in gene expression support this scenario. The AHRs' functions are also likely to be affected by their partners in toxicity mediation and regulation, especially ARNT but also the aryl hydrocarbon receptor repressor (AHRR) genes, which have been demonstrated to effectively repress activation of AHR target genes in fish (Evans et al. 2005;Karchner et al. 2002;Roy et al. 2006). How many ARNT and AHRR genes exist in Atlantic salmon is currently unknown. ...
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which organochlorine contaminants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and some polycyclic aromatic hydrocarbons induce toxicity and altered gene expression. Atlantic salmon has multiple AHR genes, of which two belong to the AHR1 clade and four belong to the AHR2 clade. The four AHR2 forms (alpha, beta, gamma, delta) are more highly expressed than the AHR1 (alpha, beta,) forms and all six AHRs are highly similar in pairs, likely originating from a whole-genome duplication in the salmonid ancestor. It has been speculated that having multiple AHRs contributes to the very high sensitivity of salmonid species to TCDD and related chemicals. To test the hypothesis that all four salmon AHR2 proteins are expressed and functional, we measured mRNA transcription for each AHR2 in several tissues, cloned the cDNAs and evaluated the functional properties of the expressed proteins. Analysis by real-time PCR revealed that the receptors showed differences in transcript levels among salmon tissues and that in general AHR2alpha was transcribed at higher levels than the other three AHR2s. Velocity sedimentation analysis showed that all four in vitro-expressed AHR2 proteins exhibit specific, high-affinity binding of [(3)H]TCDD. When expressed in COS-7 cells, all four AHR2 proteins were able to drive the expression of a reporter gene under control of murine CYP1A1 enhancer elements. From EC(50) values determined in TCDD concentration-response experiments, all four salmon AHR2s show similar sensitivity to TCDD. In summary, all four Atlantic salmon AHR2 appear to function in AHR-mediated signaling, suggesting that all four proteins are involved in TCDD-mediated toxicity.
The aryl hydrocarbon receptor repressor (AhRR) was first described as a specific competitive repressor of aryl hydrocarbon receptor (AhR) activity based on its ability to dimerize with the AhR nuclear translocator (ARNT) and through direct competition of AhR/ARNT and AhRR/ARNT complexes for binding to dioxin-responsive elements (DREs). Like AhR, AhRR belongs to the basic Helix-Loop-Helix/Per-ARNT-Sim (bHLH/PAS) protein family but lacks functional ligand-binding and transactivation domains. Transient transfection experiments with ARNT and AhRR mutants examining the inhibitory mechanism of AhRR suggested a more complex mechanism than the simple mechanism of negative feedback through sequestration of ARNT to regulate AhR signaling. Recently, AhRR has been shown to act as a tumor suppressor gene in several types of cancer cells. Furthermore, epidemiological studies have found epigenetic changes and silencing of AhRR associated with exposure to cigarette smoke and cancer development. Additional studies from our laboratories have demonstrated that AhRR represses other signaling pathways including NF-κB and is capable of regulating inflammatory responses. A better understanding of the regulatory mechanisms of AhRR in AhR signaling and adverse outcome pathways leading to deregulated inflammatory responses contributing to tumor promotion and other adverse health effects is expected from future studies. This review article summarizes the characteristics of AhRR as an inhibitor of AhR activity and highlights more recent findings pointing out the role of AhRR in inflammation and tumorigenesis.
European eel (Anguilla anguilla) elvers were intraperitoneally injected with different doses of 3,3',4,4'-tetrachlorobiyphenyl (PCB77) to examine and characterize the inductive effect of coplanar PCBs on CYP1A1 gene expression in liver and gills by using a semi-quantitative RT-PCR analysis. The influence of PCB77 injection on transcription activity of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was tested to determine its suitability as a reference gene for further quantitative gene expression analyses. Our results clearly indicate a significant dose-dependent increase in CYP1A1 gene expression in the gills of European eel, while in liver tissues a significant elevation in CYP1A1 gene expression was only detectable at highest contamination rates, indicating the potential of CYP1A1 differential gene expression analysis in gills as a biomarker for PCB contamination in eels. PCB77 contamination did not affect GAPDH transcription in gills but, at highest doses, resulted in a significant elevation in liver, speaking against GAPDH as a reference housekeeping gene after PCB exposure.
The arylhydrocarbon receptor (AhR) pathway is known to be critical for cellular events, especially for those evoked by several environmental chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Whereas the function of the AhR in TCDD toxicity is well analyzed, the importance of the recently cloned AhRR in the TCDD-stimulated AhR signaling cascade is still unclear. In mammalian tissues, the AhRR gene seems to be ubiquitously expressed and its expression is altered by various AhR ligands. Basal and induced AhRR mRNA levels were found to be highly cell-, tissue- and species-specific. An inhibitory activity of the AhRR on AhR signaling was proposed from overexpression studies. However, there are not sufficient data showing such functional activity of the AhRR in vivo. This short overview summarizes the present knowledge about the AhRR and should stimulate research in the AhRR field to elucidate its physiological function and its toxicological importance in dioxin toxicity.
An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in viva evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties. VA:IBN
The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library.
The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at
human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative
regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-κB-binding sites have been identified
in the 5′-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain
deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR
gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible
expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt
heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence.
We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility
shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-κB site when the cells
has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.
Unlabelled:
We have developed a new software package, Molecular Evolutionary Genetics Analysis version 2 (MEGA2), for exploring and analyzing aligned DNA or protein sequences from an evolutionary perspective. MEGA2 vastly extends the capabilities of MEGA version 1 by: (1) facilitating analyses of large datasets; (2) enabling creation and analyses of groups of sequences; (3) enabling specification of domains and genes; (4) expanding the repertoire of statistical methods for molecular evolutionary studies; and (5) adding new modules for visual representation of input data and output results on the Microsoft Windows platform.
Availability:
http://www.megasoftware.net.
Contact:
s.kumar@asu.edu
The nucleotide sequence of a rat 18 S rRNA gene was determined. The 18 S rRNA encoded in the gene contains 1874 nucleotides, and the molecular weight estimated from the sequence is 6.09 X 10(5). The sequences of rat and Xenopus laevis 18 S rRNAs are very similar; the only differences of consequence are the insertions between nucleotides 197 and 206 and between 258 and 279 of the rat nucleic acid of sequences rich in guanine and cytosine. A proposal is presented for the secondary structure of rat 18 S rRNA based on a comparison with the sequences of 17 other small ribosomal subunit RNAs. While the primary sequences of rat and eubacterial RNAs are different, the deduced secondary structures are remarkably similar.
Relationships between hepatic lesions and chemical contaminant concentrations in sediments, stomach contents, liver tissue, and bile were statistically evaluated in three species of bottomfish, English sole (Pleuronectes vetulus), starry flounder (Platichthys stellatus), and white croaker (Genyonemus lineatus), captured from 27 urban and nonurban sites on the Pacific Coast from Alaska to southern California. Lesions detected were neoplasms, preneoplastic foci of cellular alteration, nonneoplastic proliferative lesions, unique or specific degenerative/necrotic lesions, nonspecific degenerative/necrotic lesions, and hydropic vacuolation of biliary epithelial cells and hepatocytes. In general, lesion prevalences were significantly higher in all three species captured at chemically contaminated urban sites, and certain lesions had significantly higher relative risks of occurrence at urban sites in Puget Sound, San Francisco Bay, the vicinity of Los Angeles, and San Diego Bay. Concentrations of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, DDT and its derivatives, and chlordanes and dieldrin in sediment, stomach contents, liver, and fluorescent aromatic compounds in bile were significant risk factors for the occurrence of neoplastic, preneoplastic, nonneoplastic proliferative, and specific degenerative/necrotic lesions, as well as hydropic vacuolation. Fish age also had a significant influence on occurrence of several hepatic lesions, but gender was rarely a significant risk factor. These relationships provide strong evidence for the involvement of environmental contaminants in the etiology of hepatic lesions in several marine bottomfish species and clearly indicate the utility of these lesions as biomarkers of contaminant-induced effects in wild fish.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.
We determined levels of hepatic cytochrome P4501A (CYP1A) mRNA, hepatic DNA adducts, and fluorescent aromatic compounds (FACs) in bile, a measure of exposure to polyaromatic hydrocarbons, in Atlantic tomcod from six river systems ranging from highly polluted to relatively pristine on the northeast North American coast (the Hudson River, New York; the St. Lawrence River, Quebec; the Miramichi River, New Brunswick; the Saco and Royal rivers, Maine; and the Margaree River, Nova Scotia). Hudson River tomcod showed the greatest response for all parameters, and tomcod from the Margaree River exhibited the least response. Tomcod from the Miramichi River exhibited marked induction of CYP1A mRNA but low levels of hepatic DNA adducts and biliary FACs, whereas fish from the St. Lawrence River showed no induction of CYP1A mRNA and moderately elevated levels of DNA adducts and biliary FACs. In tomcod from the Hudson and Miramichi rivers, the levels of CYP1A mRNA were 28 times and 14 times, respectively, as great as the levels in fish from the St. Lawrence, Saco/Royal, and Margaree rivers. Mean levels of DNA adducts varied from 120 nmol adducts/mol bases in Hudson River tomcod to < 3 nmol adducts/mol bases in fish from the Miramichi and Margaree rivers. Concentrations of FACs in the bile of tomcod from the Hudson and St. Lawrence rivers were 8 and 1.8 times, respectively, as great as the concentrations in tomcod from the Miramichi River and Margaree River. In tomcod from the Hudson River, all three biomarkers were markedly elevated; in the St. Lawrence River two biomarkers were elevated, in the Miramichi River one was elevated, but no biomarker was substantially elevated in fish from the Saco/Royal and Margaree rivers. Elevated levels of hepatic DNA adducts and biliary FACs in tomcod from the Hudson River suggest increased exposure to PAHs, consistent with previous studies.
An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.
Ah receptor (AhR) is a ligand-activated transcription factor that mediates pleiotropic effects of environmental pollutants such as 2,3, 7,8-tetrachlorodibenzo-p-dioxin on host animals. In addition to induction of drug-metabolizing enzymes, the liganded AhR complex was found to activate gene expression of a factor designated AhR repressor (AhRR), which inhibits AhR function by competing with AhR for dimerizing with Arnt and binding to the XRE sequence. Thus, AhR and AhRR form a regulatory circuit in the xenobiotic signal transduction pathway and provide a novel mechanism of regulation of AhR function that may determine tissue-specific sensitivity to environmental pollutants.
Adult Atlantic tomcod, Microgadus tomcod, from the Hudson River, New York State, USA, exhibit reduced inducibility of hepatic cytochrome P4501A1 (CYP1A1) mRNA compared with adult tomcod from the cleaner Miramichi River, New Brunswick, Canada, when treated with coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, little difference in CYP1A1 inducibility is observed between tomcod from these two rivers when treated with polycyclic aromatic hydrocarbons (PAHs). We sought to determine if impaired hepatic CYP1A1 inducibility in Hudson River tomcod results from a multigenerational, genetic adaptation or a single generational, physiological acclimation. Embryos and larvae from controlled experimental crosses of Hudson River and Miramichi River parents were exposed for 24 h to water-borne PCB congener 77 (10 ppm), benzo[a]pyrene (BaP; 10 ppm), or dimethysulfoxide, and CYP1A1 expression was assessed in individual larva using competitive reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The CYP1A1 mRNA was significantly induced in larvae from both populations by BaP (47- and 52-fold) and PCB 77 (9- and 22-fold), although levels of expression were higher in offspring of Miramichi matings. Most important, CYP1A1 mRNA was significantly induced by PCB 77 in larvae from Hudson River parents. Concentrations of dioxin, furan, and PCB congeners were measured in livers and eggs of female tomcod from these two locales to quantify the extent of maternal transfer of contaminants. For both rivers, wet-weight contaminant concentrations were significantly higher (4-7 times) in livers than in eggs of the same females, suggesting that a threshold level of contaminants may have to be reached before CYP1A1 transcription is impaired. We conclude that reduced inducibility of hepatic CYP1A1 mRNA in adult tomcod from the Hudson River is most consistent with single-generational acclimation.
The diversity of biological effects resulting from exposure to dioxin may reflect the ability of this environmental pollutant to alter gene expression by binding to the arylhydrocarbon receptor (AHR) gene and related genes. AHR function may be regulated by structural variations in AHR itself, in the AHR repressor (AHRR), in the AHR nuclear translocator (ARNT), or in AHR target molecules such as cytochrome P-4501A1 (CYP1A1) and glutathione S-transferase. Analysis of the genomic organization of AHRR revealed an open reading frame consisting of a 2094-bp mRNA encoded by ten exons. We found one novel polymorphism, a substitution of Ala by Pro at codon 185 (GCC to CCC), in exon 5 of the AHRR gene; among 108 healthy unrelated Japanese women, genotypes Ala/Ala, Ala/Pro, and Pro/Pro were represented, respectively, by 20 (18.5%), 49 (45.4%), and 39 (36.1%) individuals. We did not detect previously published polymorphisms of ARNT (D511N) or the CYP1A1 promoter (G-469A and C-459T) in our subjects, suggesting that these polymorphisms are rare in the Japanese population. No association was found between uterine endometriosis and any polymorphisms in the AHRR, AHR, ARNT, or CYP1A1 genes analyzed in the present study.
The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds occur via the aryl hydrocarbon receptor (AHR), a member of the basic helix-loop-helix-Per-ARNT-Sim homology (bHLH-PAS) protein superfamily. A single AHR gene has been identified in mammals, whereas many fish species, including the Atlantic killifish (Fundulus heteroclitus) possess two distinct AHR genes (AHR1 and a novel form, AHR2). A mouse bHLH-PAS protein closely related to AHR and designated AHR repressor (AHRR) is induced by 3-methylcholanthrene and represses the transcriptional activity of the AHR. To determine whether AHRR is the mammalian ortholog of fish AHR2 and to investigate the mechanisms by which AHRR regulates AHR function, we cloned an AHRR ortholog in F. heteroclitus with high sequence identity to the mouse and human AHRRs. Killifish AHRR encodes a 680-residue protein with a predicted molecular mass of 75.2 kDa. We show that in vitro expressed AHRR proteins from human, mouse, and killifish all fail to bind [(3)H]TCDD or [(3)H]beta-naphthoflavone. In transient transfection experiments using a luciferase reporter gene under control of AHR response elements, killifish AHRR inhibited the TCDD-dependent transactivation function of both AHR1 and AHR2. AHRR mRNA is widely expressed in killifish tissues and is inducible by TCDD or polychlorinated biphenyls, but its expression is not altered in a population of fish exhibiting genetic resistance to these compounds. The F. heteroclitus AHRR promoter contains three putative AHR response elements. Both AHR1 and AHR2 activated transcription of luciferase driven by the AHRR promoter, and AHRR could repress its own promoter. Thus, AHRR is an evolutionarily conserved, TCDD-inducible repressor of AHR1 and AHR2 function. Phylogenetic analysis shows that AHRR, AHR1, and AHR2 are distinct genes, members of an AHR gene family; these three vertebrate AHR-like genes descended from a single invertebrate AHR.
Populations of organisms that are chronically exposed to high levels of chemical contaminants may not suffer the same sublethal or lethal effects as naive populations, a phenomenon called resistance. Atlantic tomcod (Microgadus tomcod) from the Hudson River, New York, are exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs). They have developed resistance to PCBs and PCDDs but not to PAHs. Resistance is largely heritable and manifests at early-life-stage toxic end points and in inducibility of cytochrome P4501A (CYP1A) mRNA expression. Because CYP1A induction is activated by the aryl hydrocarbon receptor (AHR) pathway, as are most toxic responses to these compounds, we sought to determine the geographic extent of resistance to CYP1A mRNA induction by PCBs in the Hudson River tomcod population. Samples of young-of-the-year tomcod were collected from seven locales in the Hudson River, extending from the Battery at river mile 1 (RM 1) to RM 90, and from the Miramichi River, New Brunswick, Canada. Laboratory-reared offspring of tomcod adults from Newark Bay, in the western portion of the Hudson River estuary, were also used in this study. Fish were partially depurated in clean water and intraperitoneally injected with 10 ppm coplanar PCB-77, 10 ppm benzo[a]pyrene (BaP), or corn oil vehicle, and levels of CYP1A mRNA were determined. CYP1A was significantly inducible by treatment with BaP in tomcod from the Miramichi River, from laboratory-spawned offspring of Newark Bay origin, and from all Hudson River sites spanning 90 miles of river. In contrast, only tomcod from the Miramichi River displayed significantly induced CYP1A mRNA expression when treated with PCB-77. Our results suggest that the population of tomcod from throughout the Hudson River estuary has developed resistance to CYP1A inducibility and probably other toxicities mediated by the AHR pathway. Tomcod from the Hudson River may represent the most geographically expansive population of vertebrates with resistance to chemical pollutants that has been characterized.
The Hudson River Estuary is a comprehensive look at the physical, chemical, biological and environmental management issues that are important to our understanding of the Hudson River. Chapters cover the entire range of fields necessary to understanding the workings of the Hudson River estuary; the physics, bedrock geological setting and sedimentological processes of the estuary; ecosystem-level processes and biological interactions; and environmental issues such as fisheries, toxic substances, and the effect of nutrient input from densely populated areas. This 2006 book places special emphasis on important issues specific to the Hudson, such as the effect of power plants and high concentrations of PCBs. The chapters are written by specialists at a level that is accessible to students, teachers and the interested layperson. The Hudson River Estuary is a fascinating scientific biography of a major estuary, with relevance to the study of any similar natural system in the world.
Numerous studies have shown that species, strains, sexes, and developmental stages of animals differ in their sensitivity to effects of toxic chemicals. In addition to this innate differential chemical sensitivity, some natural populations chronically exposed to environmental contaminants can acquire increased resistance or tolerance to those contaminants through genetic adaptation or physiological acclimation. Differential sensitivity of experimental animals to 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAH) is well known, but a clear understanding of the underlying mechanisms is lacking. In fish and wildlife, both innate differential sensitivity to dioxins and acquired dioxin resistance have been observed. Most of the biological activity of PHAH is mediated through the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor and member of the bHLH-PAS gene family. The expression and function of the AHR and other components of the AHR-dependent signal transduction pathway are likely to be key factors determining sensitivity of species, populations, and subpopulations to PHAH effects. Similarly, alterations in AHR signaling might be responsible for acquired dioxin resistance. This review begins with an overview of general mechanisms underlying differential chemical sensitivity and acquired tolerance, followed by a summary of our current understanding of mechanisms underlying dioxin sensitivity and resistance in laboratory mammals and mammalian cell lines, in which alterations in certain 'control points' in the AHR signaling pathway have been shown to be responsible for reduced dioxin sensitivity. Differential dioxin sensitivity in aquatic animals is then reviewed, including case histories of adaptation following chronic exposure to dioxin-like compounds. Knowledge from mammalian cellular and organismal models can help guide the investigation of mechanisms underlying dioxin resistance in aquatic species.
Atlantic tomcod (Microgadus tomcod) collected from the Hudson River estuary during the winter spawning season revealed neoplastic and preneoplastic lesions in livers from 44% of the age 1 individuals and 93% of the age 2 individuals collected. These lesions appear as a continuum ranging from basophilic foci with little cellular alterations to hepatocellular carcinoma frequently involving the entire liver. Lesions were more prevalent in larger individuals within the same age group, suggesting a relationship between growth rate and tumor expression. Atlantic tomcod from a relatively unpolluted estuary in Connecticut did not reveal a comparable prevalence of hepatic lesions. Juvenile tomcod collected from the Hudson during the summer, only 4–5 mo prior to the winter spawning season had no hepatic lesions, suggesting that these lesions form rapidly, coincident with gonadal maturation. Chemical analysis of liver tissue revealed high levels of PCBs (2.5–38.2 mg/kg) and the presence of several pesticides including DDT (and metabolites), chlordane, and dieldrin (<0.1–3.0 mg/kg) and several heavy metals (<0.1–6.5 mg/kg). These results suggest that chemical contamination of the lower estuary nursery areas, coupled with high-temperature stresses of summer, may contribute to the high levels of hepatic lesions observed.
More than 500 tomcod were evaluated by other researchers for gross and histological evidence of hepatocellular carcinoma. Incidences from 24% to 100%, depending on age/size class, were reported for the Hudson River population as compared to 3% to 10% for another less impacted population from the Pawcatuck River, CT. However, a clear link with xenobiotics could not be made. Their study expands this work to include evaluation of cancer incidence in tomcod from rivers in Maine, as well as, more detailed analysis of the Hudson River histological data. However, the data presented here emphasizes observations of tomcod from the Hudson and Pawcatuck Rivers. Collected fish were measured and sexed. The livers were excised and examined for gross pathology. Gross evidence of hepatocellular carcinoma was only detected in tomcod from the Hudson River. No gross tumors or small lesions were seen in any of the other rivers. However, histological examination showed two small carcinomas in one fish from the Saco, a 2% incidence. Atlantic tomcod inhabiting tidal rivers of the northeast US, appear to exhibit differential levels of liver disease that may be associated with xenobiotics. Fish from the Hudson River have a very significant incidence of liver cancer compared to less impacted rivers such as the Pawcatuck and Saco Rivers. This study revealed other differences in livers from the Hudson River tomcod compared to other tomcod populations in addition to the high incidence of hepatocellular carcinoma. Of particular noteworthiness was the absence of darkly staining cells from the Hudson River sample. Although the function of these cells is not known, it seems reasonable to assume that the loss of alteration of these elements is not beneficial.
Incidental observations of Atlantic tomcod during routine laboratory processing revealed that a portion of the adult population collected during the 1977–78 spawning season had enlarged livers containing dark coloured tumours and other abnormalities. Of the total of 264 livers collected between 16 January and 27 February 1978 and grossly examined for prevalence of abnormalities, 25% appeared to contain neoplastic nodules and hepatocellular carcinoma. One liver contained a massive tumour (7 × 12 mm) that involved approximately 60% of the liver. The exact causes of the high prevalence of hepatocellular carcinoma are unknown but poly-chlorinated biphenyls (PCBs) are suspected of having a possible role. The Hudson River is known to contain elevated concentrations of PCBs. Twelve tomcod livers from the 1977–78 spawning population representing both normal and hepatoma conditions contained concentrations of PCBs ranging from 10–9 to 98–2 ppm (mean of 37–5 ppm).
Adult Atlantic tomcod, Microgadus tomcod, from the Hudson River, New York State, USA, exhibit reduced inducibility of hepatic cytochrome P4501A1 (CYP1A1) mRNA compared with adult tomcod from the cleaner Miramichi River, New Brunswick, Canada, when treated with coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, little difference in CYP1A1 inducibility is observed between tomcod from these two rivers when treated with polycyclic aromatic hydrocarbons (PAHs). We sought to determine if impaired hepatic CYP1A1 inducibility in Hudson River tomcod results from a multigenerational, genetic adaptation or a single generational, physiological acclimation. Embryos and larvae from controlled experimental crosses of Hudson River and Miramichi River parents were exposed for 24 h to water-borne PCB congener 77 (10 ppm), benzo[a]pyrene (BaP; 10 ppm), or dimethysulfoxide, and CYP1A1 expression was assessed in individual larva using competitive reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The CYP1A1 mRNA was significantly induced in larvae from both populations by BaP (47- and 52-fold) and PCB 77 (9- and 22-fold), although levels of expression were higher in offspring of Miramichi matings. Most important, CYP1A1 mRNA was significantly induced by PCB 77 in larvae from Hudson River parents. Concentrations of dioxin, furan, and PCB congeners were measured in livers and eggs of female tomcod from these two locales to quantify the extent of maternal transfer of contaminants. For both rivers, wet-weight contaminant concentrations were significantly higher (4–7 times) in livers than in eggs of the same females, suggesting that a threshold level of contaminants may have to be reached before CYP1A1 transcription is impaired. We conclude that reduced inducibility of hepatic CYP1A1 mRNA in adult tomcod from the Hudson River is most consistent with single-generational acclimation.
Quantification of cytochrome P4501A1 (CYP1A1) mRNA levels in environmentally exposed Atlantic tomcod (Microgadus tomcod) has revealed significantly induced gene expression in fish from contaminated locales including the Hudson River, New York, and the Miramichi River, New Brunswick. In order to calibrate this response, determine its sensitivity and dose-responsiveness, levels of hepatic CYP1A1 mRNA were quantified in depurated Atlantic tomcod intraperitoneally (i.p.) injected with various concentrations of: β-naphthoflavone (β-NF), the PAH benzo[a]pyrene (B[a]P), the non-ortho coplanar PCB congener-3,3′,4,4′- tetrachlorobiphenyl (IUPAC: PCB-77), and the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). Additionally, the rates of CYP1A1 mRNA induction and disappearance were quantified in depurated Atlantic tomcod i.p. injected with single doses of these chemicals and sacrificed at times ranging up to 72 days. Levels of CYP1A1 mRNA were dose-responsive for all four chemicals with maximum induction ranging from 50- to 460-fold and first significant induction being observed in the low mg per kg fish (wet weight) range for β-NF and B[a]P, μg/kg range for PCB-77 and ng/kg range for 2,3,7,8-TCDD. However, while tomcod from the Miramichi River responded to both PAHs and halogenated aromatic hydrocarbons (HAHs), Hudson River tomcod responded only to PAHs indicating population level differences in CYP1A1 mRNA inducibility in tomcod. Furthermore, differences in the responsiveness to PAHs and HAHs suggest that more than one molecular mechanism mediates CYP1A1 transcription in Atlantic tomcod. Kinetic profiles of CYP1A1 mRNA induction differed greatly between tomcod treated with HAHs and PAHs. Initial induction occurred within hours of treatment with PAHs and peaked after 1–3 days, compared to initial induction 4–7 days after treatment with HAHs, and maximum induction not occurring for up to 72 days after exposure. Quantification of halogenated aromatic hydrocarbons (HAH) in the livers of tomcod caught in the Hudson and Miramichi Rivers confirmed exposure and accumulation of known CYP1A1 inducing chemicals including 2,3,7,8-TCDD at concentrations as high as 1.5 μg/kg lipid (554 ng/kg w.w.) and PCB-77 at concentrations as high as 108 μg/kg lipid (15 μg/kg w.w.). These results suggest that hepatic CYP1A1 mRNA concentration can be a useful bioindicator of exposure to some aromatic hydrocarbon compounds in the aquatic environment and that profiles of gene induction and disappearance may help identify environmental inducers provided that gene responsiveness is also evaluated under controlled laboratory conditions.
Typescript. Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2003. Includes bibliographical references (leaves 152-167 leaves).
Atlantic tomcod (Microgadus tomcod) from the cancer-prone Hudson River population exhibit a genetic polymorphism in the cytochrome P4501A (CYP1A) gene which is evidenced in Northern blot analyses by a truncated transcript and in Southern blot analyses by a deletion in the variant allele. To initially evaluate the functional significance of this polymorphism, we sought to characterize the molecular basis for this polymorphism and to determine its frequency in tomcod from other populations in which the prevalence of neoplasia is low. The common CYP1A allelic sequence was determined from beta-naphthoflavone-induced tomcod cDNA and from tomcod genomic DNA. A sequence of the variant CYP1A allele was obtained by direct sequence analysis of the amplicons of variant tomcod cDNA and genomic DNA. CYP1A exon and intron structure is highly conserved between tomcod and all other teleost and mammalian species compared. Similarity of the deduced tomcod, rainbow trout, and plaice amino acid sequences was 72%, whereas similarity between tomcod and mammalian sequences was approximately 50%. The variant tomcod CYP1A allele results from a 606-bp deletion in the 7th exon of the 3' untranslated region (UTR) of the cDNA. Polymerase chain reaction and Northern blot analyses revealed an absence of this CYP1A polymorphism in tomcod from other rivers. Studies in humans suggest that variation in CYP1A1 cDNA may impact on genetic susceptibility to environmentally induced neoplasia. Furthermore, studies in in vitro mammalian models indicate the importance of 3' UTRs on gene expression by impacting on the stability of transcript. These results suggest that the 3' UTR CYP1A polymorphism in tomcod may have consequences for the genetic susceptibility of Hudson River fish to hepatic neoplasia.
The recent development of techniques to measure levels of carcinogens covalently bound to DNA provides the opportunity to use DNA adducts as molecular dosimeters of exposure to environmental carcinogens and mutagens. This is especially important because epizootiologic studies have shown a positive association between environmental carcinogens, such as polycyclic aromatic hydrocarbons, and increased prevalence of neoplasms and related lesions, primarily in liver, of benthic fish species from a wide range of urban and industrialized areas. In studies with wild fish and mammalian species the 32P-postlabeling assay, as developed for aromatic compounds, has been used most extensively because of its high sensitivity and ability to detect structurally uncharacterized adducts. The results to date of field and laboratory studies show that hepatic DNA adducts detected in fish are associated with increased exposure to environmental polycyclic aromatic compounds in the preponderance of species examined, whereas in the limited studies with wild mammals, such a relationship is equivocal at present. The findings with fish suggest that DNA adducts, as measured by 32P-postlabeling, have the potential to be effective molecular dosimeters of exposure to environmental carcinogenic aromatic compounds and thereby may lead to an improved understanding of the etiology of neoplasia in wild teleosts.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
Cytochrome P4501A1 (CYP1A1) mRNA is not inducible in Atlantic tomcod from the Hudson River that are treated with halogenated aromatic hydrocarbons (HAHs). In contrast, CYP1A1 mRNA is inducible in Hudson River tomcod that are treated with polycyclic aromatic hydrocarbons (PAHs) and in tomcod that are collected from cleaner rivers and treated with HAHs or PAHs. We hypothesize that CYP1A1 transcription is inhibited in Hudson River tomcod because of down-regulation of the aromatic hydrocarbon receptor (AhR) pathway and that separate molecular pathways modulate CYP1A1 transcription in fish treated with HAHs and PAHs. We initially evaluated levels of hepatic nuclear protein binding at enhancer elements (DREs) in the regulatory region of tomcod CYP1A1. No difference in levels of protein binding was observed between tomcod from the Hudson and Miramichi (cleaner) rivers that were untreated or were treated with benzo[a]pyrene. In contrast, levels of protein binding were lower in tomcod from the Hudson River that were treated with TCB than in similarly treated fish from the Miramichi River, suggesting differences between the populations in the structure or expression of AhR pathway molecules. To address this possibility, AhR DNA sequences were characterized from tomcod cDNA and genomic DNA libraries. In tomcod and mammals, AhR is represented by 11 exons, overall peptide sizes are similar, and amino acid sequences at basic, helix-loop-helix, PAAS A, and PAAS B domains are highly conserved. In contrast, little similarity was observed between tomcod and mammals in the sizes or sequences of AhR exons 10 and 11, including the absence in tomcod of glutamine-rich domains. No differences in levels of hepatic AhR mRNA were observed between the two populations or treatment groups when tomcod were untreated or were treated with aromatic hydrocarbons. In contrast, variation in levels of AhR mRNA expression was observed among tomcod tissues; however, no relationship was observed between levels of AhR mRNAs and CYP1A1 mRNAs in tissues from chemically or vehicle control-treated fish. RFLP analysis revealed extensive variation in exons 10 and 11 of AhR cDNA among tomcod from different rivers. Our results suggest that variation between tomcod populations in CYP1A1 mRNA inducibility is reflected by differences in levels of inducible hepatic protein binding to DREs. However, levels of hepatic AhR mRNA are not down-regulated in the Hudson River population, are not affected by AH treatments, and levels of AhR mRNA expression are not responsible for the differential inducibility of CYP1A1 transcription.
The planar polychlorinated biphenyl (PCB) 3,3',4,4'-tetrachlorobiphenyl (TCB) causes dose-dependent induction and post-transcriptional suppression of hepatic cytochrome P450 1A (CYP1A) in the marine teleost scup (Stenotomus chrysops). That suppression is linked to inhibition and oxidative inactivation of CYP1A by TCB. Other planar PCBs, including 3,3',4,4',5-pentachlorobiphenyl (PeCB), inactivate scup CYP1A in vitro leading us to hypothesize that PeCB also will suppress CYP1A in vivo. We examined induction and suppression of CYP1A by PeCB in scup, as related to oxidative stress. PeCB at a low dose (0.01 mg/kg) induced hepatic microsomal spectral P450 and CYP1A protein and catalytic activities (ethoxyresorufin o-deethylase (EROD) and methoxyresorufin o-demethylase (MROD)) over an 18 day period. A high dose (1 mg PeCB/kg) only minimally induced hepatic spectral P450 and CYP1A content, and EROD and MROD rates remained at control levels at all sampling times, while CYP1A mRNA expression was induced strongly (up to 35-fold) at both doses. High dose PeCB had minimal effects on content of P450A (a CYP3A protein), P450B (a CYP2B-like protein) and cytochrome b5 in scup liver, suggesting that the suppression was specific for CYP1A. High dose PeCB suppressed EROD but not CYP1A protein in the kidney but did not strongly suppress either CYP1A or EROD in the heart or gill. PeCB stimulated ROS production (oxidation of dihydroethidium) by liver microsomes from the low dose but not the high dose fish, and the rate of PeCB-stimulated ROS production was correlated with EROD activity (r(2)=0.641, P<0.0005). Oxidative stress, indicated by increased levels of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase activities, was stimulated in the liver by low dose but not high dose PeCB. The results support a hypothesis that many PHAH can inactivate teleost CYP1A in vivo, and that CYP1A is a source of ROS. However, there appears to be a complex balance between the effects of PeCB on the levels of active CYP1A, ROS release and oxidative stress.
The CYP1A1 gene encodes microsomal cytochrome P4501A1 that catalyzes the metabolism of many xenobiotics, including the oxygenation of polycyclic aromatic hydrocarbons (PAH). Induction of CYP1A1 enhances the metabolism of PAHs, and therefore, represents an adaptive response to chemical exposure in mammalian cells. Mechanistic studies reveal an AhR/DRE paradigm for the induction, which involves activation of the aryl hydrocarbon receptor (AhR) by an agonist, dimerization of AhR with the Ah recceptor nuclear translocator (Arnt), followed by binding of the AhR/Arnt heterodimer to the dioxin-responsive enhancer (DRE) and transcription of the gene. The AhR mediated transcription is tightly regulated through, at least, two mechanisms: (a) the cytoplasmic AhR interacts with hsp90 and an immunophilin chaperone AIP for proper folding and receptivity, and (b) the agonist-activated, nuclear AhR is degraded through the ubiquitin-26S proteasome mediated protein turnover, such that the transcription by AhR is controlled at a physiologically adequate level. In addition to CYP1A1 induction, AhR mediates a broad range of biological responses to CYP1A1 inducers, typified by the environmental contaminant dioxin, via modulating gene expression. Thus, mechanistic studies of CYP1A1 induction have provided insights into P450 induction, PAH carcinogenesis, dioxin action, AhR function, and receptor-mediated mammalian gene expression.
Lake trout embryos and sac fry are very sensitive to toxicity associated with maternal exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related chemicals that act through a common aryl hydrocarbon receptor (AHR)-mediated mechanism of action. The loading of large amounts of these chemicals into Lake Ontario during the middle of the 20th century coincided with a population decline that culminated in extirpation of this species around 1960. Prediction of past TCDD toxicity equivalence concentrations in lake trout eggs (TEC(egg)s) relative to recent conditions required fine resolution of radionuclide-dated contaminant profiles in two sediment cores; reference core specific biota--sediment accumulation factors (BSAFs) for TCDD-like chemicals in lake trout eggs; adjustment of the BSAFs for the effect of temporal changes in the chemical distributions between water and sediments; and toxicity equivalence factors based on trout early life stage mortality. When compared to the dose-response relationship for overt early life stage toxicity of TCDD to lake trout, the resulting TEC(egg)s predict an extended period during which lake trout sac fry survival was negligible. By 1940, following more than a decade of population decline attributable to reduced fry stocking and loss of adult lake trout to commercial fishing, the predicted sac fry mortality due to AHR-mediated toxicity alone explains the subsequent loss of the species. Reduced fry survival, associated with lethal and sublethal adverse effects and possibly complicated by other environmental factors, occurred after 1980 and contributed to a lack of reproductive success of stocked trout despite gradually declining TEC(egg)s. Present exposures are close to the most probable no observable adverse effect level (NOAEL TECegg = 5 pg TCDD toxicity equivalence/g egg). The toxicity predictions are very consistent with the available historical data for lake trout population levels in Lake Ontario, stocking programs, and evidence for recent improvement in natural reproduction concomitant with declining levels of persistent bioaccumulative chemicals in sediments and biota.
Wildcaught killifish from a contaminated site on the Elizabeth River (VA, USA) are refractory to induction of cytochrome P4501A (CYP1A, measured as catalytic activity and immunodetectable CYP1A protein) after exposure to typical aryl hydrocarbon receptor (AHR) agonists, as has been reported for fish from other sites highly contaminated with these compounds. In an attempt to understand the molecular basis for the lack of inducibility of CYP1A protein expression and activity in Elizabeth River killifish, we analyzed the expression of CYP1A and four other members of the AHR signal transduction pathway: AHR1, AHR2, AHR repressor (AHRR), and AHR nuclear translocator (ARNT). Gene expression was measured by cycle-optimized reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of messenger ribonucleic acid (mRNA) extracted from livers of killifish from the Elizabeth River and King's Creek (VA, USA) (reference site), 36 h after injection with beta-naphthoflavone (BNF, an AHR agonist) or corn oil (carrier control). Hepatic CYP1A mRNA was inducible in King's Creek killifish. However, in Elizabeth River killifish, no induction of CYP1A mRNA was observed, confirming and extending previous results showing no induction of CYP1A protein or catalytic activity in this population. Similarly, AHRR and AHR2 mRNA levels were induced by BNF in King's Creek but not Elizabeth River killifish. No population or treatment-related differences were observed in expression of AHR1 or ARNT mRNAs. The results reveal in Elizabeth River killifish a consistent lack of inducibility of genes that are normally inducible by AHR agonists (CYP1A, AHRR, AHR2). However, the expression of AHR1, AHR2, and AHRR in vehicle-treated fish did not differ between Elizabeth River and King's Creek killifish, suggesting that altered constitutive expression of AHRs or AHRR does not underlie the refractory CYP1A phenotype in Elizabeth River killifish.
To investigate the expression of aryl hydrocarbon receptor repressor (AhRR) and related molecules in various tissues and the effects of aromatic hydrocarbons (AHs) on their expression, we developed a reliable technique of quantification of human AhRR as well as aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT) and cytochrome P450 1A1 (CYP1A1) mRNA by real-time TaqMan PCR method. First, we examined the expression of these genes in human adult or fetal tissues. The levels of AhRR expression were extremely high in testis, very high in lung, ovary, spleen and pancreas from adults, whereas those were low in those from fetuses. On the other hand, CYP1A1 expression was extremely high in lung, and AhR and ARNT were ubiquitously expressed in almost all tissues. Second, we compared the expression levels of these genes in mononuclear cells (MNCs) from various sources. Comparison of the basal expression levels of these genes in MNCs demonstrated that MNCs from umbilical cord blood showed higher AhRR or CYP1A1 expression than those from adults. The induction of AhRR or CYP1A1 expression by 3-methylcholanthrene (3-MC) was observed in MNCs from adults but not from umbilical cord blood. Consequently, there existed characteristic differences in the basal levels of AhRR and CYP1A1 expression in MNCs, as well as in their inducibility by 3-MC among MNCs from various types of human bloods. These results will provide basic information for a possible application of AhRR and CYP1A1 measurements to evaluate AH exposure in vivo.
Full congener-specific polychorinated biphenyl (PCB) and partial-congener-specific polychorinated dibenzo-p-dioxin/furan (PCDD/F) analyses were performed on livers from young-of-the-year (YOY) and adult Atlantic tomcod from the Hudson River estuary including multiple sites along the main-stem Hudson River and Newark Bay/Hackensack River, NJ, and from a reference river, the Miramichi River, NB. Highest hepatic burdens of PCBs were found in fish collected in the main-stem Hudson River between river miles (RM) 37 and 50 and in Newark Bay/Hackensack River. By far, the highest concentrations of PCDD/Fs were seen in fish from Newark Bay/Hackensack River. The di- to tetrachlorinated biphenyls dominated the PCB composition in YOY tomcod, whereas the penta- to nonachlorinated biphenyls predominated in adults with particular prevalence of the 2,4,5-substituted diortho congeners. Overall, using a direct mixing model an aroclor composition of approximate 1:1:1, A1242:A1254:A1260, was calculated from the hepatic PCB profiles in YOY tomcod. A linear increase in A1242 characteristics with river mile was seen in YOY collected between RM 0 and RM 80, which was likely due to the well-characterized A1242 source from the former capacitor manufacturing plants located upriver. However, tomcod caught upstream of RM 80 exhibited a PCB pattern with decreasing A1242 characteristics, and it was hypothesized that this was due to the increased depuration or decreased uptake of low chlorinated (log K(OW) < 6) congeners upon entry of the fish into freshwater from brackish water. The most abundant tetra-octa PCDD/F chlorohomologue in tomcod collected from the main stem of the Hudson River was TCDF, whereas 2,3,7,8-TCDD was the major congener detected in tomcod from Newark Bay/Hackensack River, which showed elevated total PCDD/F levels compared to tomcod from the main-stem Hudson River.
Fish from urban and industrialized estuaries are exposed among the highest levels of contaminants of any vertebrate populations. As a result, they serve as especially relevant models for determining the toxic effects and mechanisms through which environmental toxicants work. In controlled laboratory experiments, fish from highly contaminated locales sometimes exhibit resistance to contaminant-induced toxicity. Resistance may be due to genetic adaptation or physiological acclimations. Distinguishing between these possibilities is important in predicting the persistence of resistance and its potential costs to affected populations and communities. Along the Atlantic coast of North America, populations of two estuarine species, Atlantic killifish (mummichog) Fundulus heteroclitus and Atlantic tomcod Microgadus tomcod, exhibit phenotypes that are resistant to aromatic hydrocarbon (AH) contaminants, including polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polycyclic aromatic hydrocarbons (PAHs). Populations of these species exhibit resistance to AH-induced lethality, early life-stage toxicities, and expression of cytochrome P4501A (CYP1A). However, some differences among populations in the occurrence and type (genetic or physiological) of AH-resistant phenotypes have been observed. In some instances, resistance was obviously genetic and resulted in its transmission to at least the F2 generation, in others, resistance had a physiological or yet to be identified epigenetic basis. In some cases, resistance was observed for all AH compounds tested, in others, it was seen only for halogenated AHs. As toxic responses to AHs are believed to be mediated by the aryl hydrocarbon receptor pathway (AHR), several studies compared the structure and expression of AHR pathway molecules between resistant and sensitive fish populations. However, no obvious differences in these molecular parameters were observed between resistant and sensitive populations at the transcriptional level. Further studies at the protein level are recommended to further evaluate the role of the AHR pathway in conferring resistance. Open-ended microarray and proteomic approaches may provide additional resolution in determining the molecular mechanisms of resistance. Also, studies that evaluate the prevalence and ecosystem cost of resistance are needed.
The aryl hydrocarbon receptor repressor (AHRR) is a negative regulator of AH receptor (AHR), which mediates most of the toxic and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has been shown to be the major reason for the exceptionally wide (ca. 1000-fold) sensitivity difference in acute toxicity of TCDD between two rat strains, sensitive Long-Evans (Turku/AB) (L-E) and resistant Han/Wistar (Kuopio) (H/W), but there is another, currently unknown contributing factor involved. In the present study, we examined AHRR structure and expression in these rat strains to find out whether AHRR could be this auxiliary factor. Molecular cloning of AHRR coding region showed that consistent with AHRR proteins in other species, the N-terminal end of rat AHRR is highly conserved, but PAS B and Q-rich domains are severely truncated or lacking. Identical structures were recorded in both strains. Next, the time-, dose-, and tissue-dependent expression of AHRR was determined using quantitative real-time RT-PCR. In liver, AHRR expression was very low in untreated rats, but it increased rapidly after TCDD exposure (100microg/kg). Testis exhibited the highest constitutive expression of AHRR, whereas kidney, spleen, and heart showed the highest induction of AHRR in response to TCDD treatment. Again, no marked differences were found between H/W and L-E rats, implying that AHRR is not the auxiliary contributing factor to the strain difference in TCDD sensitivity. However, simultaneous measurement of CYP1A1 mRNA reinforced the view that AHRR is an important determinant of tissue-specific responsiveness to TCDD.
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Fig. 5. Box plots comparing cytochrome P4501A1 (CYP1A1) mRNA (A) and aryl hydrocarbon receptor repressor (AHRR) mRNA (B) ex-pression in eight tissues of laboratory-reared juvenile F1 tomcod of Miramichi River (Canada) ancestry that were intraperitoneally in-jected with 10 ppm of polychlorinated biphenyl 77 (hatched bars) or 102:200–215.
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