Cytochrome P450 1A1 and aromatase (CYP19) in cetaceans : enzyme expression and relationship to contaminant exposure

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 2003Contamnant exposure has been associated with toxic effects in marine mammals. Studies on early biochemical and molecular responses prior to, but associated with, contaminant-induced toxicity will aid in understanding the susceptibility of cetaceans to such exposures. Cytochrome P450 1A1 (CYP1A1) induction by planar halogenated aromatic hydrocarbons, including certain polychlorinated biphenyls (PCBs), has been correlated with toxic effects in rodents. CYP1A1 expression was measured in multiple organs from beluga whales and white-sided dolphins and integument biopsies from bottlenose dolphins. Age and sex do not appear to influence CYP1A1 expression in these species. Mono-artha PCB concentrations were correlated with CYP1A1 in bottlenose dolphin integument and white-sided dolphin liver. White-sided dolphins, with PCBs concentrations six times higher than Arctic beluga, have lower hepatic CYP1A1 expression. Beluga whales thus appear very sensitive to the induction of CYP1A1 by PHAHs. These studies demonstrate that comparative studies between cetacean species, with reference to experimental work in model organisms, could determine the relative susceptibility of cetaceans to contaminant exposures. Aromatase is the enzyme responsible for estrogen production and recent studies suggest that PCBs may alter its activity or expression. We determined the sequence and characterized the catalytic properties of a cetacean aromatase, which was similar to other mammalian aromatases. Evolutionary analyses of vertebrate aromatases revealed striking functional conservation. Although much work is required, these data show that similar to CYP1A1, aromatase studies in other mammals are applicable to cetacean species. Experimental work in model organisms, coupled with careful studies with cetacean tissues, could infer the physiological role and transcriptional control of aromatase in cetacean species.Funding for this research was provided by the Natural Sciences and Engineering Research Council of Canada postgraduate scholarship B, the MIIWOI Joint Program, WHOI Sea Grant Project Number R/-162, NI Superfund Basic Research Program Grant P-42-ES07381, the WHOI Biology Department, and the International Whaling Commission's Pollution 2000+ program

Cytochrome P4501A is Induced in Endothelial Cell Lines From the Kidney and Lung of the Bottlenose Dolphin, \u3ci\u3eTursiops truncatus\u3c/i\u3e

Marine mammals respond to the presence of polycyclic and planar halogenated aromatic hydrocarbons (PAH or PHAH) with the induced expression in endothelium of cytochrome P4501A1, regulated through the aryl hydrocarbon receptor (AHR) transcription factor. Physiological responses in other animals, such as edema and inflammation indicate that the endothelium may be compromised by exposure to AHR agonists, which are ubiquitous in the marine environment. In other mammals and fish the cellular and molecular consequences of exposure to AHR agonists have been elucidated in cultured endothelial cells. We have cultured and characterized cetacean endothelial cells (EC) and used them in induction studies. Endothelial cells were cultured from the lung and kidney of the bottlenose dolphin, Tursiops truncates, and exposed to the AHR agonists β-naphthoflavone (βNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). βNF (1–3 μM) induced significant increases in CYP1A1 (O-deethylation of 7-ethoxyresorufin to resorufin; EROD) activity to 3.6 and 0.92 pmol/mg/min in lung and kidney EC, respectively. TCDD was more potent than βNF, and more efficacious, with maximum induction of CYP1A1 activity of 10.1 and 15.2 pmol/mg/min in lung and kidney EC at 3–10 nM TCDD. The differential response indicates that the lung and kidney endothelial cells in culture retain the ability to respond in a selective manner to specific stimuli. Both the molecular mechanisms of induction and the physiological consequences, especially in the vasculature, of toxicant exposure can be studied in this system

Cytochrome P4501A is induced in endothelial cell lines from the kidney and lung of the bottlenose dolphin, Tursiops truncatus

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Aquatic Toxicology 76 (2006): 295-305, doi:10.1016/j.aquatox.2005.10.005.Marine mammals respond to the presence of polycyclic and planar halogenated aromatic hydrocarbons (PAH or PHAH) with the induced expression in endothelium of cytochrome P4501A1, regulated through the aryl hydrocarbon receptor (AHR) transcription factor. Physiological responses in other animals, such as edema and inflammation indicate that the endothelium may be compromised by exposure to AHR agonists, which are ubiquitous in the marine environment. In other mammals and fish the cellular and molecular consequences of exposure to AHR agonists have been elucidated in cultured endothelial cells. We have cultured and characterized cetacean endothelial cells (EC) and used them in induction studies. Endothelial cells were cultured from the lung and kidney of the bottlenose dolphin Tursiops truncatus and exposed to the AHR agonists β-naphthoflavone (βNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). βNF (1-3 μM) induced significant increases in CYP1A1(O-deethylation of 7-ethoxyresorufin to resorufin;EROD) activity to 3.6 and 0.92 pmol/mg/min in lung and kidney EC, respectively. TCDD was more potent than βNF, and more efficacious, with maximum induction of CYP1A1activity of 10.1 and 15.2 pmol/mg/min in lung and kidney EC at 3-10 nM TCDD. The differential response indicates that the lung and kidney endothelial cells in culture retain the ability to respond in a selective manner to specific stimuli. Both the molecular mechanisms of induction and the physiological consequences, especially in the vasculature, of toxicant exposure can be studied in this system.Part of this work was completed during a faculty fellowship from Fordham University for RAG. The Faculty Research Council of Fordham University provided partial support for RAG. This research was supported by NIH grant 5- P42-ES07381 and by U.S.EPA grant R827102-01-0. This research is an outgrowth and continuing impact of Sea Grant Number Grant No. NA90- AA-D-SG480, project NA86RG0075-R/P61

Systemic Effects of Arctic Pollutants in Beluga Whales Indicated by CYP1A1 Expression

Cytochrome P450 1A1 (CYP1A1) is induced by exposure to polycyclic aromatic hydrocarbons (PAHs) and planar halogenated aromatic hydrocarbons (PHAHs) such as non-ortho polychlorinated biphenyls (PCBs). In this study, we examined CYP1A1 protein expression immunohistochemically in multiple organs of beluga whales from two locations in the Arctic and from the St. Lawrence estuary. These beluga populations have some of the lowest (Arctic sites) and highest (St. Lawrence estuary) concentrations of PCBs in blubber of all cetaceans. Samples from these populations might be expected to have different contaminant-induced responses, reflecting their different exposure histories. The pattern and extent of CYP1A1 staining in whales from all three locations were similar to those seen in animal models in which CYP1A has been highly induced, indicating a high-level expression in these whales. CYP1A1 induction has been related to toxic effects of PHAHs or PAHs in some species. In St. Lawrence beluga, the high level of CYP1A1 expression coupled with high levels of contaminants (including CYP1A1 substrates, e.g., PAH procarcinogens potentially activated by CYP1A1) indicates that CYP1A1 could be involved in the development of neoplastic lesions seen in the St. Lawrence beluga population. The systemic high-level expression of CYP1A1 in Arctic beluga suggests that effects of PAHs or PHAHs may be expected in Arctic populations, as well. The high-level expression of CYP1A1 in the Arctic beluga suggests that this species is highly sensitive to CYP1A1 induction by aryl hydrocarbon receptor agonists

Epigenetic Regulation of Histone H3 Serine 10 Phosphorylation Status by HCF-1 Proteins in C. elegans and Mammalian Cells

BACKGROUND: The human herpes simplex virus (HSV) host cell factor HCF-1 is a transcriptional coregulator that associates with both histone methyl- and acetyltransferases, and a histone deacetylase and regulates cell proliferation and division. In HSV-infected cells, HCF-1 associates with the viral protein VP16 to promote formation of a multiprotein-DNA transcriptional activator complex. The ability of HCF proteins to stabilize this VP16-induced complex has been conserved in diverse animal species including Drosophila melanogaster and Caenorhabditis elegans suggesting that VP16 targets a conserved cellular function of HCF-1. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of HCF proteins in animal development, we have characterized the effects of loss of the HCF-1 homolog in C. elegans, called Ce HCF-1. Two large hcf-1 deletion mutants (pk924 and ok559) are viable but display reduced fertility. Loss of Ce HCF-1 protein at reduced temperatures (e.g., 12 degrees C), however, leads to a high incidence of embryonic lethality and early embryonic mitotic and cytokinetic defects reminiscent of mammalian cell-division defects upon loss of HCF-1 function. Even when viable, however, at normal temperature, mutant embryos display reduced levels of phospho-histone H3 serine 10 (H3S10P), a modification implicated in both transcriptional and mitotic regulation. Mammalian cells with defective HCF-1 also display defects in mitotic H3S10P status. CONCLUSIONS/SIGNIFICANCE: These results suggest that HCF-1 proteins possess conserved roles in the regulation of cell division and mitotic histone phosphorylation

Regulation of healthcare ethics committees in Europe

In this article, the question is discussed if and how Healthcare Ethics Committees (HECs) should be regulated. The paper consists of two parts. First, authors from eight EC member countries describe the status quo in their respective countries, and give reasons as to the form of regulation they consider most adequate. In the second part, the country reports are analysed. It is suggested that regulation of HECs should be central and weak. Central regulation is argued to be apt to improve HECs’ accountability, relevance and comparability. To facilitate biomedical citizenship and ethical reflection, regulation should at the same time be weak rather than strict. Independence of HECs to deliberate about ethical questions, and to give solicited and unsolicited advice, should be supported and only interfered with by way of exception. One exception is when circumstances become temporary adversarial to ethical deliberation in healthcare institutions. In view of European unification, steps should be taken to develop consistent policies for both Eastern and Western European countries

Changes in persistent contaminant concentration and CYP1A1 protein expression in biopsy samples from northern bottlenose whales, Hyperoodon ampullatus, following the onset of nearby oil and gas development

Author Posting. © Elsevier B.V., 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Environmental Pollution 152 (2008): 205-216, doi:10.1016/j.envpol.2007.05.027.A small population of endangered northern bottlenose whales (Hyperoodon ampullatus) inhabits “The Gully” Marine Protected Area on the Scotian Shelf, eastern Canada. Amid concerns regarding nearby oil and gas development, we took 36 skin and blubber biopsy samples in 1996-97 (prior to major development) and 2002-03 (five years after development began), and 3 samples from a population in the Davis Strait, Labrador in 2003. These were analysed for cytochrome P4501A1 (CYP1A1) protein expression (n=36), and for persistent contaminants (n=23). CYP1A1 showed generally low expression in whales from The Gully, but higher levels during 2003, potentially co-incident with recorded oil spills, and higher levels in Davis Strait whales. A range of PCB congeners and organochlorine compounds were detected, with concentrations similar to other North Atlantic odontocetes. Concentrations were higher in whales from The Gully than from the Davis Strait, with significant increases in 4,4’-DDE and trans-nonachlor in 2002-03 relative to 1996-97.Research was funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada, World Wildlife Fund Canada Endangered Species Recovery Fund, Fisheries and Oceans Canada, the National Geographic Society, the Canadian Federation of Humane Societies and two U.K. Royal Society International Collaborative Awards. S.K.H. was supported by a Canadian Commonwealth Scholarship and Royal Society Dorothy Hodgkin Research Fellowship. C.D.M. was awarded a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada. J.Y.W was supported by an NSERC PGS B fellowship and the Woods Hole Oceanographic Institution

Fatal Cases of Influenza A(H3N2) in Children: Insights from Whole Genome Sequence Analysis

During the Northern Hemisphere winter of 2003–2004 the emergence of a novel influenza antigenic variant, A/Fujian/411/2002-like(H3N2), was associated with an unusually high number of fatalities in children. Seventeen fatal cases in the UK were laboratory confirmed for Fujian/411-like viruses. To look for phylogenetic patterns and genetic markers that might be associated with increased virulence, sequencing and phylogenetic analysis of the whole genomes of 63 viruses isolated from fatal cases and non fatal “control” cases was undertaken. The analysis revealed the circulation of two main genetic groups, I and II, both of which contained viruses from fatal cases. No associated amino acid substitutions could be linked with an exclusive or higher occurrence in fatal cases. The Fujian/411-like viruses in genetic groups I and II completely displaced other A(H3N2) viruses, but they disappeared after 2004. This study shows that two A(H3N2) virus genotypes circulated exclusively during the winter of 2003–2004 in the UK and caused an unusually high number of deaths in children. Host factors related to immune state and differences in genetic background between patients may also play important roles in determining the outcome of an influenza infection

Complex Adaptations Can Drive the Evolution of the Capacitor [PSI+], Even with Realistic Rates of Yeast Sex

The [PSI+] prion may enhance evolvability by revealing previously cryptic genetic variation, but it is unclear whether such evolvability properties could be favored by natural selection. Sex inhibits the evolution of other putative evolvability mechanisms, such as mutator alleles. This paper explores whether sex also prevents natural selection from favoring modifier alleles that facilitate [PSI+] formation. Sex may permit the spread of “cheater” alleles that acquire the benefits of [PSI+] through mating without incurring the cost of producing [PSI+] at times when it is not adaptive. Using recent quantitative estimates of the frequency of sex in Saccharomyces paradoxus, we calculate that natural selection for evolvability can drive the evolution of the [PSI+] system, so long as yeast populations occasionally require complex adaptations involving synergistic epistasis between two loci. If adaptations are always simple and require substitution at only a single locus, then the [PSI+] system is not favored by natural selection. Obligate sex might inhibit the evolution of [PSI+]-like systems in other species

Neogenin May Functionally Substitute for Dcc in Chicken

Dcc is the key receptor that mediates attractive responses of axonal growth cones to netrins, a family of axon guidance cues used throughout evolution. However, a Dcc homolog has not yet been identified in the chicken genome, raising the possibility that Dcc is not present in avians. Here we show that the closely related family member neogenin may functionally substitute for Dcc in the developing chicken spinal cord. The expression pattern of chicken neogenin in the developing spinal cord is a composite of the distribution patterns of both rodent Dcc and neogenin. Moreover, whereas the loss of mouse neogenin has no effect on the trajectory of commissural axons, removing chicken neogenin by RNA interference results in a phenotype similar to the functional inactivation of Dcc in mouse. Taken together, these data suggest that the chick neogenin is functionally equivalent to rodent Dcc