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Abstracts from the Eleventh International Symposium on Pollutant Responses in Marine Organisms (PRIMO 11)—Proteomics and Genomics
Marine Environmental Research 54 (2002) 405–412 www.elsevier.com/locate/marenvrev
Abstracts from the Eleventh International Symposium on Pollutant Responses in Marine Organisms (PRIMO 11)—Proteomics and Genomics Molecular characterisation and tissue specific expression of CYP3A30 in killifish (Fundulus heteroclitus) Tove Hegelund Myrba¨ck, Malin C. Celander Go¨teborg University, Department of Zoophysiology, Box 463, SE 405 30 Go¨teborg, Sweden
Abstract The complete coding sequence of CYP3A30 from livers from killifish (Fundulus heteroclitus) was previously isolated (presented at PRIMO 10). Extrahepatic CYP3A cDNA, isolated from intestine, kidney and gills, shared 99% identity with hepatic CYP3A30. CYP3A30 mRNA expression in hepatic and extrahepatic tissue, from adult male and female killifish, was estimated using semi-quantitative RT-PCR. CYP3A protein content and cellular localization were determined using polyclonal antibodies raised against trout CYP3A. Two mammalian fluorescent CYP3A substrates were tested to evaluate CYP3A catalytic activities in postmitochondrial supernatant (PMS) of various tissues from killifish, i.e. 7-benzyloxy-4-(triflouromethyl)-coumarin and 7-benzyloxyquinoline. High levels of CYP3A30 mRNA, CYP3A protein and CYP3A-like catalytic activities were observed in liver and intestine. Low to moderate levels of CYP3A30 mRNA and CYP3A protein expression were observed in gills, kidney, spleen, brain and heart. Low CYP3A expression in these organs was confirmed with lack of detectable CYP3A-like catalytic activities in the PMS of these tissues. Furthermore, males showed slightly higher levels of CYP3A30 mRNA expression in gills, kidney, brain and spleen, compared to that in females. Thus, CYP3A30 expression is prominent in the digestive and respiratory tract in killifish. However, there are substantial gender, individual and tissue differences in the degree of CYP3A/CYP3A30 expression. (Financially supported by grants from Helge Ax:son Johnsons stiftelse, Stiftelsen Wilhelm och Martina Lundgrens vetenskapsfond, Kungliga Vetenskapsakademien och Adlerbertska forskningsfonden to Tove Hegelund Myrba¨ck and from NFR and MISTRA to Malin Celander.)
PII: S0141-1136(02)00232-5
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Abstracts / Marine Environmental Research 54 (2002) 405–412
Gene expression regulation of CYP1A in Nile Tilapia (Oreochromis niloticus): cloning, and sequencing of CYP1A O. Zapata-Pe´reza, G. Gold-Bouchota, A. Ruizb, A. Alboresa, A. Ortegaa a
Marine Resources Department, CINVESTAV. Unidad Me´rida. A.P. 73., Me´rida Yucata´n, Mexico b University of Yucatan, Mexico
Abstract The gene expression regulation of cytochrome P450 (CYP1A) was studied in Tilapia (Oreochromis niloticus) treated with a single i.p. injection of pyrene. The effects of pyrene exposure on CYP content, ethoxyresorufin-O-deethylase (EROD) activity, CYP1A mRNA, and CYP1A protein levels were investigated. A 350 bp fragment of the tilapia CYP1A cDNA was cloned, sequenced, and compared with CYP1A reported sequences in the GeneBank DNA database; the top seven matches corresponded to CYP1A from other teleosts. Specific PCR primers were synthesized and used to quantify tilapia CYP1A mRNA levels. Hepatic CYP1A mRNA levels showed a significant increase at day 1 after pyrene injection and this CYP1A mRNA levels did not return to basal levels for up to 5 days. Immunoblotting analysis of the hepatic microsomal preparations was performed using a polyclonal antibody rabbit-anti-trout-CYP1A. Pyrene administration resulted in a 1.9-fold increase of CYP1A protein and 18-fold induction of EROD activity at day 3 after injection. In the same way, the highest transformation of pyrene was observed as 1-hydroxypyrene (1-OH pyrene) in tilapias sacrificed at day 3 after the injection. The transformed metabolite increased 370-fold with respect to control. A Spearman correlation test showed a significant and good correlation (r=0.85) between EROD activity and 1-OH pyrene concentration.
A DNA array to monitor gene expression in european flounder (Platichthys flesus) T.D. Williams, S. Minchin, J.K. Chipman School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Abstract We designed degenerate primer pairs for 70 genes of toxicological interest. Gene fragments were amplified from flounder genomic DNA, ovary cDNA and liver cDNA by PCR. As anticipated, many non-targeted sequences were also amplified. DNA fragments were cloned into plasmid vectors and sequenced automatically. Seventy-four fragments of different genes were identified by homology, whereas 34 remained unidentified. All sequences were
Abstracts / Marine Environmental Research 54 (2002) 405–412
407
submitted to EMBL. PCR products generated from these clones were arrayed onto glass slides using a Biorobotics arrayer. mRNA was prepared using livers of flounder from the polluted UK estuaries of the Tyne and Clyde and the relatively unpolluted Alde estuary. RT-PCR was used to label cDNA with Cy5-dCTP for polluted samples, Cy3-dCTP for Alde controls. cDNAs were hybridized to the array, the ratio of Cy5 to Cy3 was quantified for each spot using an Axon scanner. Many of the gene fragments gave reproducible results. Those transcripts more abundant (1.5- to 4-fold) in polluted samples were metallothionein, transferrin, glucose-6-phosphatase, 60S ribosomal protein S7 and six unknown transcripts. Those transcripts less abundant (< 0.66-fold) in polluted samples were CYP3A, complement component C3, a ZPC-like transcript, peroxin, chromobox protein, gamma-fibrinogen, alpha2HS-glycoprotein, UDPGT, 40S ribosomal protein S8, inter-alpha trypsin inhibitor and one unknown transcript.
Identification of a putative metallothionien promoter from the Mediterrenean mussel Mytilus galloprovincialis Francesco Dondero, Aldo Viarengo Deptartment of Science and Advanced Technology, University of Piemonte Orientale ‘‘A. Avogadro’’, Corso Borsalino 54, I-15100 Alessandria, Italy
Abstract A putative metallothionein promoter has been identified by cloning a sequence upstream the first functional exon of a mussel (Mytilus galloprovincialis Lam.) metallothionein gene. Amplification of mussel genomic DNA was achieved by a Metal Responsive Element (MRE)mediated anchored polymerase chain reaction (PCR) strategy. We have used a reverse primer that anneals within the coding sequence of a mussel metallothionein gene that we have recently cloned (MT20III) and a forward primer that was deduced from the MRE consensus sequence. A PCR fragment of about 1.5 Kb has been obtained. DNA sequencing has revealed a high A/T ratio. The sequence was then been subjected to functional analysis for the identification of the putative trans-factor binding sites, using specific algorithms. In analogy with other metallothionien promoters some typical elements have been identified. At least five MRE are distributed along the proximal, middle and distal part of the promoter. Six AP-1 binding sites, having different orientations have been found in the middle part of the sequence. Other putative elements are enhancer binding sites, transcription starting sites (TSS) and a heat shock factor (HSF) binding site that has been found in the distal part of the promoter. For the assessment of the putative elements found along the mussel promoter we are actually generating reporter-constructs having various deletions to be transfected in mussel hepatocyte primary culture for measuring their transcriptional activities.
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Abstracts / Marine Environmental Research 54 (2002) 405–412
The cloning and characterization of metallothionein isoforms from oyster embryos, Crassostrea virginica Jenny J. Matthewa, Amy H. Ringwoodb, Greg Warrc, Robert Chapmanb, Alan Lewitusb, Jason Kemptonb a Marine Biomedical and Environmental Studies, Medical University of SC, Charleston, SC, USA Marine Resources Research Institute, SC Department of Natural Resources, Charleston, SC, USA c Department of Biochemistry, MUSC, Charleston, SC, USA
b
Abstract Metallothioneins (MT) are low-molecular-weight proteins with characteristic repeating cysteine motifs. They function in metal metabolism, homeostasis and detoxification. To date only one MT protein isoform has been identified in Crassostrea virginica. To further understand the role of metallothioneins in oysters, cDNA libraries were constructed from control, Cd-, and Cu-treated C virginica embryos. Random probes were generated from a vector containing the Cd-MT of C. virginica (provided by G. Roesijadi) and used to probe the respective libraries. Preliminary sequencing has identified multiple MT isoforms from all three libraries. In addition to the expected 7 kDa protein, we have identified nucleotide sequences coding for 4–5 kDa as well as 15 kDa proteins. Unique to the Cd-treated library are cDNAs representing splice variants of the original oyster MT which code primarily for the b-domain. This suggests the formation of one-domain MT proteins that may be energetically favorable detoxification pathways. Using random EST screening techniques, a novel MT whose primary structure is 40% homologous to the original oyster MT. These results suggest the presence of at least two distinct MT gene families in oysters.
A quantitative competitive RT-PCR assay to measure CYP1A and metallothionein gene expression in liver of the European flounder (Platichthys flesus) D.L. Sheadera, K. Gensberga, B.P. Lyonsb, J.K. Chipmana a
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Centre for Environment, Fisheries and Aquaculture Science, Pakefield Road, Lowestoft, Suffolk NR33 0HT, UK
Abstract Quantitative competitive (qc) RT-PCR is a sensitive method for detecting changes in the levels of specific mRNA transcripts. It is a powerful tool for measuring the induction of metallothionein (MTT) and CYP1A, biomarkers of exposure to heavy metal and polycyclic aromatic hydrocarbons (PAHs) respectively. RNA competitors for qcRT-PCR were generated from previously cloned fragments of flounder MTT and CYP1A genes using the extended primer method. The cDNA competitors were ligated into suitable vectors and transcribed in vitro to yield the RNA competitors. A dilution series of wt RNA, transcribed in vitro from
Abstracts / Marine Environmental Research 54 (2002) 405–412
409
the gene fragments, was used to produce a standard curve by co-reverse transcription and coamplification with a constant amount of competitor RNA. Liver samples were obtained from flounder exposed to PAH in controlled tank studies. Total RNA was extracted and levels of MTT and CYP1A transcripts quantified by co-reverse transcription and co-amplification with competitor RNA using the standard curve. A clear dose response relationship between PAH exposure and both DNA adduct and micronuclei levels has previously been demonstrated for the tank-exposed samples. However, ethoxyresorufin O-deethylase (EROD) activity showed no such relationship. The qcRT-PCR analysis of CYP1A has been compared with EROD to determine the balance between induction and enzyme inhibition by PAH.
Aryl hydrocarbon receptor polymorphisms and dioxin resistance in Atlantic killifish (Fundulus heteroclitus) Mark E. Hahn, Sibel I. Karchner, Diana G. Franks Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to fish. However, some fish populations subjected to multi-generational exposure to high levels of PHAHs have developed resistance to these compounds through physiological or genetic adaptation. Atlantic killifish (Fundulus heteroclitus) inhabiting New Bedford Harbor (MA, USA) have developed heritable resistance to PHAHs [Mar. Biol. 134 (1999) 9; Toxicol. Sci. 60 (2001) 77]. We are examining the mechanism of resistance, focusing on the aryl hydrocarbon receptor (AHR)-dependent signaling pathway. We have cloned multiple components of the Fundulus AHR pathway, including the bHLH-PAS proteins AHR1, AHR2, ARNT2, and AHR repressor. AHR2 is a novel AHR gene [Proc. Nat. Acad. Sci. 94 (1997) 13743] that is now known to occur widely in fish [J. Biol. Chem. 274 (1999) 33814]. AHR1, but not AHR2 or ARNT, is differentially expressed between dioxin-sensitive and -resistant fish [Toxicol. Sci. 57 (2000) 229]. Recently, we identified two AHR1 alleles and observed differences in their frequency between the dioxin-sensitive andresistant populations. When expressed by in vitro transcription and translation, these alleles do not differ in binding affinity for [3H]TCDD. Other functional and regulatory properties of these two alleles are being assessed. [Superfund ES07381.]
Interaction of the aryl hydrocarbon receptor and the retinoblastoma tumor suppressor gene product in the Atlantic killifish (Fundulus heteroclitus) Rebeka R. Merson, Sibel I. Karchner, Mark E. Hahn Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
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Abstracts / Marine Environmental Research 54 (2002) 405–412
Abstract Many of the toxic effects of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and related compounds are mediated by activation of the aryl hydrocarbon receptor (AHR). The physiological role of the AHR is unknown, but evidence suggests the AHR is involved in the cell cycle and this may be related to its ability to complex with the retinoblastoma tumor suppressor gene product (pRB). Activated pRB downregulates genes necessary for cell cycle progression through G1 phase, thus preventing uncontrolled cell growth. Investigating the functional significance of the AHR-pRB interaction may provide insight to the physiological role of the AHR. Fundulus heteroclitus expresses two paralogs of the AHR, whereas mammals express only one AHR. Fundulus AHR1 contains the LXCXE-motif and the glutamine-rich region that interact with pRB in mammals, whereas Fundulus AHR2 has neither pRB-binding site. To test the hypothesis that pRB interacts with AHR1 but not AHR2, we are cloning Fundulus pRB and evaluating its interaction with the two AHRs. We have obtained a nearly full-length sequence of Fundulus pRB, including the A/B-pocket (important for pRB function), by RTPCR and RACE. Fundulus A/B-pocket shares 60 and 78% amino acid identity with human and medaka, respectively. AHR-pRB interaction studies soon will be initiated. [NIH Grants 1F32ES05935, ES07381 (Superfund).]
Are sharks susceptible to dioxins? cDNA cloning and characterization of elasmobranch aryl hydrocarbon receptors Rebeka R. Merson, Mark E. Hahn Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
Abstract Anthropogenic pollutants in marine environments include many planar halogenated aromatic hydrocarbons (PHAHs). These compounds are toxic and carcinogenic, and bioaccumulate in sharks. The toxic effects of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and related PHAHs (e.g. non-ortho-substituted PCBs) are mediated by activation of the aryl hydrocarbon receptor (AHR). Differences in ligand binding affinity of AHRs explain some species- and strain-specific differences in TCDD toxicity. Our objectives are to clone and characterize AHRs in elasmobranchs (sharks, skates and rays) and to assess their TCDD-binding affinities. We used reverse transcription polymerase chain reaction (RT-PCR) with degenerate primers to isolate fragments of the AHRs. Previous work in this lab identified partial sequences of two AHR forms from spiny dogfish Squalus acanthias and smooth dogfish Mustelus canis, and one AHR from little skate Raja erinacea. Recently we cloned fragments of two AHR forms each from Greenland shark Somniosus microcephalus and sandbar shark Carcharhinus plumbeus. Rapid amplification of cDNA ends (RACE) is being used to obtain the 50 - and 30 -ends of AHRs from all five species. The divergence and conservation of AHR genes in elasmobranchs will be presented in the context of functional domains of the receptor. [Supported in part by NIH Grants ES06272, ES05935.]
Abstracts / Marine Environmental Research 54 (2002) 405–412
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Proteomics and gene expression in zebrafish exposed to endocrine disrupting chemicals E.A. Shradera, P.R. Hoytb, M.J. Doktycz2, B.P. Bradleya, M.S. Greeley Jr.b a
University of Maryland (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250 USA Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 6038 37831, USA
b
Abstract In experiments designed to understand complex response pathways, RNA and proteins were isolated at intervals during embryonic development from zebrafish embryos exposed to non-lethal concentrations of the estrogen mimic nonylphenol and estradiol. Protein expression signatures (PES) of embryo homogenates were derived by proteome analysis using 2D gel electrophoresis and digital imaging. RNA expression was analyzed by a zebrafish DNA microarray constructed from zebrafish expressed sequence tags (ESTs) corresponding to known estrogen-responsive genes, among others. The 55 and 76 proteins, respectively found in the PES for nonylphenol and estradiol exceeded the estimated number of changes in gene expression found with the initial microarrays. Identification of the unique and excluded proteins in the PES of exposed zebrafish embryos will allow us to better direct further development of the DNA microarray with the addition of genes for the proteins demonstrating toxicant- or hormone-responsiveness in this specific model system. Thus proteome analysis can complement DNA microarray analysis both by adding an additional level of expression data, including post-translationally modified proteins, and by focusing gene selection during array development on those genes actually expressing as proteins. (Research sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory. managed by UT-Battelle, LLC, for the US Department of Energy under Contract No. DE-AC05-00OR22725.)
Generation of zebrafish cDNA microarrays for investigation of cardiovascular embryo toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin Heather Handleya, Matthew Growb, Mark Fishmanb, John Stegemana a Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA
b
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a powerful teratogen that targets the developing cardiovascular system of fish. Effects of early TCDD exposure typically include pericardial and yolk sac edema, localized hemorrhaging, altered heart morphology, weak heart beat, and reduced regional blood flow. It is widely held that these symptoms result from TCDD-induced changes in gene expression. However, the identities of impacted genes and
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their potential roles in cardiovascular embryo toxicity are largely unknown. We have begun generating zebrafish cDNA microarrays that could be used to address this information deficit. Arrays consisting of 1200 embryonic (72 hpf) heart clones were used to develop protocols and identify novel cardiac-specific markers. Based on our protocol testing, we have assembled a thorough quality control scheme for the generation and use of cDNA microarrays. Finally, we are arraying cDNA libraries from additional zebrafish tissues, including adult heart. These arrays will be used to identify TCDD-responsive cardiovascular and developmental genes that may be involved in cardiovascular embryo toxicity.
Abstracts from the Eleventh International Symposium on Pollutant Responses in Marine Organisms (PRIMO 11)—Proteomics and Genomics Molecular characterisation and tissue specific expression of CYP3A30 in killifish (Fundulus heteroclitus) Tove Hegelund Myrba¨ck, Malin C. Celander Go¨teborg University, Department of Zoophysiology, Box 463, SE 405 30 Go¨teborg, Sweden
Abstract The complete coding sequence of CYP3A30 from livers from killifish (Fundulus heteroclitus) was previously isolated (presented at PRIMO 10). Extrahepatic CYP3A cDNA, isolated from intestine, kidney and gills, shared 99% identity with hepatic CYP3A30. CYP3A30 mRNA expression in hepatic and extrahepatic tissue, from adult male and female killifish, was estimated using semi-quantitative RT-PCR. CYP3A protein content and cellular localization were determined using polyclonal antibodies raised against trout CYP3A. Two mammalian fluorescent CYP3A substrates were tested to evaluate CYP3A catalytic activities in postmitochondrial supernatant (PMS) of various tissues from killifish, i.e. 7-benzyloxy-4-(triflouromethyl)-coumarin and 7-benzyloxyquinoline. High levels of CYP3A30 mRNA, CYP3A protein and CYP3A-like catalytic activities were observed in liver and intestine. Low to moderate levels of CYP3A30 mRNA and CYP3A protein expression were observed in gills, kidney, spleen, brain and heart. Low CYP3A expression in these organs was confirmed with lack of detectable CYP3A-like catalytic activities in the PMS of these tissues. Furthermore, males showed slightly higher levels of CYP3A30 mRNA expression in gills, kidney, brain and spleen, compared to that in females. Thus, CYP3A30 expression is prominent in the digestive and respiratory tract in killifish. However, there are substantial gender, individual and tissue differences in the degree of CYP3A/CYP3A30 expression. (Financially supported by grants from Helge Ax:son Johnsons stiftelse, Stiftelsen Wilhelm och Martina Lundgrens vetenskapsfond, Kungliga Vetenskapsakademien och Adlerbertska forskningsfonden to Tove Hegelund Myrba¨ck and from NFR and MISTRA to Malin Celander.)
PII: S0141-1136(02)00232-5
406
Abstracts / Marine Environmental Research 54 (2002) 405–412
Gene expression regulation of CYP1A in Nile Tilapia (Oreochromis niloticus): cloning, and sequencing of CYP1A O. Zapata-Pe´reza, G. Gold-Bouchota, A. Ruizb, A. Alboresa, A. Ortegaa a
Marine Resources Department, CINVESTAV. Unidad Me´rida. A.P. 73., Me´rida Yucata´n, Mexico b University of Yucatan, Mexico
Abstract The gene expression regulation of cytochrome P450 (CYP1A) was studied in Tilapia (Oreochromis niloticus) treated with a single i.p. injection of pyrene. The effects of pyrene exposure on CYP content, ethoxyresorufin-O-deethylase (EROD) activity, CYP1A mRNA, and CYP1A protein levels were investigated. A 350 bp fragment of the tilapia CYP1A cDNA was cloned, sequenced, and compared with CYP1A reported sequences in the GeneBank DNA database; the top seven matches corresponded to CYP1A from other teleosts. Specific PCR primers were synthesized and used to quantify tilapia CYP1A mRNA levels. Hepatic CYP1A mRNA levels showed a significant increase at day 1 after pyrene injection and this CYP1A mRNA levels did not return to basal levels for up to 5 days. Immunoblotting analysis of the hepatic microsomal preparations was performed using a polyclonal antibody rabbit-anti-trout-CYP1A. Pyrene administration resulted in a 1.9-fold increase of CYP1A protein and 18-fold induction of EROD activity at day 3 after injection. In the same way, the highest transformation of pyrene was observed as 1-hydroxypyrene (1-OH pyrene) in tilapias sacrificed at day 3 after the injection. The transformed metabolite increased 370-fold with respect to control. A Spearman correlation test showed a significant and good correlation (r=0.85) between EROD activity and 1-OH pyrene concentration.
A DNA array to monitor gene expression in european flounder (Platichthys flesus) T.D. Williams, S. Minchin, J.K. Chipman School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Abstract We designed degenerate primer pairs for 70 genes of toxicological interest. Gene fragments were amplified from flounder genomic DNA, ovary cDNA and liver cDNA by PCR. As anticipated, many non-targeted sequences were also amplified. DNA fragments were cloned into plasmid vectors and sequenced automatically. Seventy-four fragments of different genes were identified by homology, whereas 34 remained unidentified. All sequences were
Abstracts / Marine Environmental Research 54 (2002) 405–412
407
submitted to EMBL. PCR products generated from these clones were arrayed onto glass slides using a Biorobotics arrayer. mRNA was prepared using livers of flounder from the polluted UK estuaries of the Tyne and Clyde and the relatively unpolluted Alde estuary. RT-PCR was used to label cDNA with Cy5-dCTP for polluted samples, Cy3-dCTP for Alde controls. cDNAs were hybridized to the array, the ratio of Cy5 to Cy3 was quantified for each spot using an Axon scanner. Many of the gene fragments gave reproducible results. Those transcripts more abundant (1.5- to 4-fold) in polluted samples were metallothionein, transferrin, glucose-6-phosphatase, 60S ribosomal protein S7 and six unknown transcripts. Those transcripts less abundant (< 0.66-fold) in polluted samples were CYP3A, complement component C3, a ZPC-like transcript, peroxin, chromobox protein, gamma-fibrinogen, alpha2HS-glycoprotein, UDPGT, 40S ribosomal protein S8, inter-alpha trypsin inhibitor and one unknown transcript.
Identification of a putative metallothionien promoter from the Mediterrenean mussel Mytilus galloprovincialis Francesco Dondero, Aldo Viarengo Deptartment of Science and Advanced Technology, University of Piemonte Orientale ‘‘A. Avogadro’’, Corso Borsalino 54, I-15100 Alessandria, Italy
Abstract A putative metallothionein promoter has been identified by cloning a sequence upstream the first functional exon of a mussel (Mytilus galloprovincialis Lam.) metallothionein gene. Amplification of mussel genomic DNA was achieved by a Metal Responsive Element (MRE)mediated anchored polymerase chain reaction (PCR) strategy. We have used a reverse primer that anneals within the coding sequence of a mussel metallothionein gene that we have recently cloned (MT20III) and a forward primer that was deduced from the MRE consensus sequence. A PCR fragment of about 1.5 Kb has been obtained. DNA sequencing has revealed a high A/T ratio. The sequence was then been subjected to functional analysis for the identification of the putative trans-factor binding sites, using specific algorithms. In analogy with other metallothionien promoters some typical elements have been identified. At least five MRE are distributed along the proximal, middle and distal part of the promoter. Six AP-1 binding sites, having different orientations have been found in the middle part of the sequence. Other putative elements are enhancer binding sites, transcription starting sites (TSS) and a heat shock factor (HSF) binding site that has been found in the distal part of the promoter. For the assessment of the putative elements found along the mussel promoter we are actually generating reporter-constructs having various deletions to be transfected in mussel hepatocyte primary culture for measuring their transcriptional activities.
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Abstracts / Marine Environmental Research 54 (2002) 405–412
The cloning and characterization of metallothionein isoforms from oyster embryos, Crassostrea virginica Jenny J. Matthewa, Amy H. Ringwoodb, Greg Warrc, Robert Chapmanb, Alan Lewitusb, Jason Kemptonb a Marine Biomedical and Environmental Studies, Medical University of SC, Charleston, SC, USA Marine Resources Research Institute, SC Department of Natural Resources, Charleston, SC, USA c Department of Biochemistry, MUSC, Charleston, SC, USA
b
Abstract Metallothioneins (MT) are low-molecular-weight proteins with characteristic repeating cysteine motifs. They function in metal metabolism, homeostasis and detoxification. To date only one MT protein isoform has been identified in Crassostrea virginica. To further understand the role of metallothioneins in oysters, cDNA libraries were constructed from control, Cd-, and Cu-treated C virginica embryos. Random probes were generated from a vector containing the Cd-MT of C. virginica (provided by G. Roesijadi) and used to probe the respective libraries. Preliminary sequencing has identified multiple MT isoforms from all three libraries. In addition to the expected 7 kDa protein, we have identified nucleotide sequences coding for 4–5 kDa as well as 15 kDa proteins. Unique to the Cd-treated library are cDNAs representing splice variants of the original oyster MT which code primarily for the b-domain. This suggests the formation of one-domain MT proteins that may be energetically favorable detoxification pathways. Using random EST screening techniques, a novel MT whose primary structure is 40% homologous to the original oyster MT. These results suggest the presence of at least two distinct MT gene families in oysters.
A quantitative competitive RT-PCR assay to measure CYP1A and metallothionein gene expression in liver of the European flounder (Platichthys flesus) D.L. Sheadera, K. Gensberga, B.P. Lyonsb, J.K. Chipmana a
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Centre for Environment, Fisheries and Aquaculture Science, Pakefield Road, Lowestoft, Suffolk NR33 0HT, UK
Abstract Quantitative competitive (qc) RT-PCR is a sensitive method for detecting changes in the levels of specific mRNA transcripts. It is a powerful tool for measuring the induction of metallothionein (MTT) and CYP1A, biomarkers of exposure to heavy metal and polycyclic aromatic hydrocarbons (PAHs) respectively. RNA competitors for qcRT-PCR were generated from previously cloned fragments of flounder MTT and CYP1A genes using the extended primer method. The cDNA competitors were ligated into suitable vectors and transcribed in vitro to yield the RNA competitors. A dilution series of wt RNA, transcribed in vitro from
Abstracts / Marine Environmental Research 54 (2002) 405–412
409
the gene fragments, was used to produce a standard curve by co-reverse transcription and coamplification with a constant amount of competitor RNA. Liver samples were obtained from flounder exposed to PAH in controlled tank studies. Total RNA was extracted and levels of MTT and CYP1A transcripts quantified by co-reverse transcription and co-amplification with competitor RNA using the standard curve. A clear dose response relationship between PAH exposure and both DNA adduct and micronuclei levels has previously been demonstrated for the tank-exposed samples. However, ethoxyresorufin O-deethylase (EROD) activity showed no such relationship. The qcRT-PCR analysis of CYP1A has been compared with EROD to determine the balance between induction and enzyme inhibition by PAH.
Aryl hydrocarbon receptor polymorphisms and dioxin resistance in Atlantic killifish (Fundulus heteroclitus) Mark E. Hahn, Sibel I. Karchner, Diana G. Franks Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to fish. However, some fish populations subjected to multi-generational exposure to high levels of PHAHs have developed resistance to these compounds through physiological or genetic adaptation. Atlantic killifish (Fundulus heteroclitus) inhabiting New Bedford Harbor (MA, USA) have developed heritable resistance to PHAHs [Mar. Biol. 134 (1999) 9; Toxicol. Sci. 60 (2001) 77]. We are examining the mechanism of resistance, focusing on the aryl hydrocarbon receptor (AHR)-dependent signaling pathway. We have cloned multiple components of the Fundulus AHR pathway, including the bHLH-PAS proteins AHR1, AHR2, ARNT2, and AHR repressor. AHR2 is a novel AHR gene [Proc. Nat. Acad. Sci. 94 (1997) 13743] that is now known to occur widely in fish [J. Biol. Chem. 274 (1999) 33814]. AHR1, but not AHR2 or ARNT, is differentially expressed between dioxin-sensitive and -resistant fish [Toxicol. Sci. 57 (2000) 229]. Recently, we identified two AHR1 alleles and observed differences in their frequency between the dioxin-sensitive andresistant populations. When expressed by in vitro transcription and translation, these alleles do not differ in binding affinity for [3H]TCDD. Other functional and regulatory properties of these two alleles are being assessed. [Superfund ES07381.]
Interaction of the aryl hydrocarbon receptor and the retinoblastoma tumor suppressor gene product in the Atlantic killifish (Fundulus heteroclitus) Rebeka R. Merson, Sibel I. Karchner, Mark E. Hahn Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
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Abstracts / Marine Environmental Research 54 (2002) 405–412
Abstract Many of the toxic effects of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and related compounds are mediated by activation of the aryl hydrocarbon receptor (AHR). The physiological role of the AHR is unknown, but evidence suggests the AHR is involved in the cell cycle and this may be related to its ability to complex with the retinoblastoma tumor suppressor gene product (pRB). Activated pRB downregulates genes necessary for cell cycle progression through G1 phase, thus preventing uncontrolled cell growth. Investigating the functional significance of the AHR-pRB interaction may provide insight to the physiological role of the AHR. Fundulus heteroclitus expresses two paralogs of the AHR, whereas mammals express only one AHR. Fundulus AHR1 contains the LXCXE-motif and the glutamine-rich region that interact with pRB in mammals, whereas Fundulus AHR2 has neither pRB-binding site. To test the hypothesis that pRB interacts with AHR1 but not AHR2, we are cloning Fundulus pRB and evaluating its interaction with the two AHRs. We have obtained a nearly full-length sequence of Fundulus pRB, including the A/B-pocket (important for pRB function), by RTPCR and RACE. Fundulus A/B-pocket shares 60 and 78% amino acid identity with human and medaka, respectively. AHR-pRB interaction studies soon will be initiated. [NIH Grants 1F32ES05935, ES07381 (Superfund).]
Are sharks susceptible to dioxins? cDNA cloning and characterization of elasmobranch aryl hydrocarbon receptors Rebeka R. Merson, Mark E. Hahn Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
Abstract Anthropogenic pollutants in marine environments include many planar halogenated aromatic hydrocarbons (PHAHs). These compounds are toxic and carcinogenic, and bioaccumulate in sharks. The toxic effects of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and related PHAHs (e.g. non-ortho-substituted PCBs) are mediated by activation of the aryl hydrocarbon receptor (AHR). Differences in ligand binding affinity of AHRs explain some species- and strain-specific differences in TCDD toxicity. Our objectives are to clone and characterize AHRs in elasmobranchs (sharks, skates and rays) and to assess their TCDD-binding affinities. We used reverse transcription polymerase chain reaction (RT-PCR) with degenerate primers to isolate fragments of the AHRs. Previous work in this lab identified partial sequences of two AHR forms from spiny dogfish Squalus acanthias and smooth dogfish Mustelus canis, and one AHR from little skate Raja erinacea. Recently we cloned fragments of two AHR forms each from Greenland shark Somniosus microcephalus and sandbar shark Carcharhinus plumbeus. Rapid amplification of cDNA ends (RACE) is being used to obtain the 50 - and 30 -ends of AHRs from all five species. The divergence and conservation of AHR genes in elasmobranchs will be presented in the context of functional domains of the receptor. [Supported in part by NIH Grants ES06272, ES05935.]
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Proteomics and gene expression in zebrafish exposed to endocrine disrupting chemicals E.A. Shradera, P.R. Hoytb, M.J. Doktycz2, B.P. Bradleya, M.S. Greeley Jr.b a
University of Maryland (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250 USA Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 6038 37831, USA
b
Abstract In experiments designed to understand complex response pathways, RNA and proteins were isolated at intervals during embryonic development from zebrafish embryos exposed to non-lethal concentrations of the estrogen mimic nonylphenol and estradiol. Protein expression signatures (PES) of embryo homogenates were derived by proteome analysis using 2D gel electrophoresis and digital imaging. RNA expression was analyzed by a zebrafish DNA microarray constructed from zebrafish expressed sequence tags (ESTs) corresponding to known estrogen-responsive genes, among others. The 55 and 76 proteins, respectively found in the PES for nonylphenol and estradiol exceeded the estimated number of changes in gene expression found with the initial microarrays. Identification of the unique and excluded proteins in the PES of exposed zebrafish embryos will allow us to better direct further development of the DNA microarray with the addition of genes for the proteins demonstrating toxicant- or hormone-responsiveness in this specific model system. Thus proteome analysis can complement DNA microarray analysis both by adding an additional level of expression data, including post-translationally modified proteins, and by focusing gene selection during array development on those genes actually expressing as proteins. (Research sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory. managed by UT-Battelle, LLC, for the US Department of Energy under Contract No. DE-AC05-00OR22725.)
Generation of zebrafish cDNA microarrays for investigation of cardiovascular embryo toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin Heather Handleya, Matthew Growb, Mark Fishmanb, John Stegemana a Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA
b
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a powerful teratogen that targets the developing cardiovascular system of fish. Effects of early TCDD exposure typically include pericardial and yolk sac edema, localized hemorrhaging, altered heart morphology, weak heart beat, and reduced regional blood flow. It is widely held that these symptoms result from TCDD-induced changes in gene expression. However, the identities of impacted genes and
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their potential roles in cardiovascular embryo toxicity are largely unknown. We have begun generating zebrafish cDNA microarrays that could be used to address this information deficit. Arrays consisting of 1200 embryonic (72 hpf) heart clones were used to develop protocols and identify novel cardiac-specific markers. Based on our protocol testing, we have assembled a thorough quality control scheme for the generation and use of cDNA microarrays. Finally, we are arraying cDNA libraries from additional zebrafish tissues, including adult heart. These arrays will be used to identify TCDD-responsive cardiovascular and developmental genes that may be involved in cardiovascular embryo toxicity.