RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone
C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses...
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description | C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the H |
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D ; SCHMID, J. E ; BROWN, J. G ; WOOD, C. R ; WHITE, R. D ; BUCKALEW, A. R ; HELD, G. A</creator><creatorcontrib>ABBOTT, B. D ; SCHMID, J. E ; BROWN, J. G ; WOOD, C. R ; WHITE, R. D ; BUCKALEW, A. R ; HELD, G. A</creatorcontrib><description>C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development.</description><identifier>ISSN: 1096-6080</identifier><identifier>EISSN: 1096-0929</identifier><identifier>DOI: 10.1093/toxsci/47.1.76</identifier><identifier>PMID: 10048155</identifier><identifier>CODEN: TOSCF2</identifier><language>eng</language><publisher>Cary, NC: Oxford University Press</publisher><subject>AAh receptors ; Animals ; Aryl Hydrocarbon Receptor Nuclear Translocator ; Base Sequence ; Biological and medical sciences ; Blotting, Northern ; CYP1A1 protein ; Cytochrome P-450 CYP1A1 - genetics ; Cytochrome P-450 CYP1A1 - metabolism ; DNA-Binding Proteins ; Embryology: invertebrates and vertebrates. Teratology ; Environmental Pollutants - toxicity ; Female ; Fundamental and applied biological sciences. Psychology ; hydrocortisone ; Hydrocortisone - toxicity ; Maternal-Fetal Exchange ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; mRNA ; Palate - drug effects ; Palate - embryology ; Palate - metabolism ; Polychlorinated Dibenzodioxins - toxicity ; Pregnancy ; Receptors, Aryl Hydrocarbon - genetics ; Receptors, Aryl Hydrocarbon - metabolism ; Receptors, Glucocorticoid - genetics ; Receptors, Glucocorticoid - metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger - metabolism ; Teratogens - toxicity ; Teratology. Teratogens ; Transcription Factors - genetics ; Transcription Factors - metabolism</subject><ispartof>Toxicological sciences, 1999, Vol.47 (1), p.76-85</ispartof><rights>1999 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c349t-d6361ccd9f53cdd478245bd608ef4897015e1aac079916fa1d846a3f70e42a833</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,4024,27923,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=2004228$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10048155$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>ABBOTT, B. D</creatorcontrib><creatorcontrib>SCHMID, J. E</creatorcontrib><creatorcontrib>BROWN, J. G</creatorcontrib><creatorcontrib>WOOD, C. R</creatorcontrib><creatorcontrib>WHITE, R. D</creatorcontrib><creatorcontrib>BUCKALEW, A. R</creatorcontrib><creatorcontrib>HELD, G. A</creatorcontrib><title>RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone</title><title>Toxicological sciences</title><addtitle>Toxicol Sci</addtitle><description>C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development.</description><subject>AAh receptors</subject><subject>Animals</subject><subject>Aryl Hydrocarbon Receptor Nuclear Translocator</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Blotting, Northern</subject><subject>CYP1A1 protein</subject><subject>Cytochrome P-450 CYP1A1 - genetics</subject><subject>Cytochrome P-450 CYP1A1 - metabolism</subject><subject>DNA-Binding Proteins</subject><subject>Embryology: invertebrates and vertebrates. Teratology</subject><subject>Environmental Pollutants - toxicity</subject><subject>Female</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>hydrocortisone</subject><subject>Hydrocortisone - toxicity</subject><subject>Maternal-Fetal Exchange</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Molecular Sequence Data</subject><subject>mRNA</subject><subject>Palate - drug effects</subject><subject>Palate - embryology</subject><subject>Palate - metabolism</subject><subject>Polychlorinated Dibenzodioxins - toxicity</subject><subject>Pregnancy</subject><subject>Receptors, Aryl Hydrocarbon - genetics</subject><subject>Receptors, Aryl Hydrocarbon - metabolism</subject><subject>Receptors, Glucocorticoid - genetics</subject><subject>Receptors, Glucocorticoid - metabolism</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA, Messenger - metabolism</subject><subject>Teratogens - toxicity</subject><subject>Teratology. Teratogens</subject><subject>Transcription Factors - genetics</subject><subject>Transcription Factors - metabolism</subject><issn>1096-6080</issn><issn>1096-0929</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkUtrGzEUhUVpaV7ddlm0KF3NONKMRhotB5NHISRhcBddCVkPojIjOZIMdv9O_mhkbEpX93L57rmcewD4itECI95e57BLyl0TtsALRj-A8zKlNeIN_3jqKerRGbhI6Q9CGFPEP4MzjBDpcdedg7dxVT8vR_i6lT4765TMLngYLBzuxwoO4-Oqgnelk17D5e9nPGA4j48DdB6qKL0LVionJ5hdSluTDptmXsd98E7B2SkDzW4TktEwB9hUbcWqvs4mR6lephCDdmvj_4Z6U2sXdkX1cOhlr2NQIRbR4M0V-GTllMyXU70Ev25vVsv7-uHp7udyeKhVS3iuNW0pVkpz27VKa8L6hnRrXfwbS3rOEO4MllIhxjmmVmLdEypby5Ahjezb9hL8OOpuYngtXrKYXVJmmqQ3YZsEZgRxhA7g4giqGFKKxopNdLOMe4GROMQijrEIwgQWjJaFbyfl7Xo2-j_8mEMBvp8AmZScbPmscukf1xSuafr2HTn5lgc</recordid><startdate>1999</startdate><enddate>1999</enddate><creator>ABBOTT, B. D</creator><creator>SCHMID, J. E</creator><creator>BROWN, J. G</creator><creator>WOOD, C. R</creator><creator>WHITE, R. D</creator><creator>BUCKALEW, A. R</creator><creator>HELD, G. A</creator><general>Oxford University Press</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7U7</scope><scope>C1K</scope></search><sort><creationdate>1999</creationdate><title>RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone</title><author>ABBOTT, B. D ; SCHMID, J. E ; BROWN, J. G ; WOOD, C. R ; WHITE, R. D ; BUCKALEW, A. R ; HELD, G. 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Psychology</topic><topic>hydrocortisone</topic><topic>Hydrocortisone - toxicity</topic><topic>Maternal-Fetal Exchange</topic><topic>Mice</topic><topic>Mice, Inbred C57BL</topic><topic>Molecular Sequence Data</topic><topic>mRNA</topic><topic>Palate - drug effects</topic><topic>Palate - embryology</topic><topic>Palate - metabolism</topic><topic>Polychlorinated Dibenzodioxins - toxicity</topic><topic>Pregnancy</topic><topic>Receptors, Aryl Hydrocarbon - genetics</topic><topic>Receptors, Aryl Hydrocarbon - metabolism</topic><topic>Receptors, Glucocorticoid - genetics</topic><topic>Receptors, Glucocorticoid - metabolism</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>RNA, Messenger - metabolism</topic><topic>Teratogens - toxicity</topic><topic>Teratology. Teratogens</topic><topic>Transcription Factors - genetics</topic><topic>Transcription Factors - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>ABBOTT, B. D</creatorcontrib><creatorcontrib>SCHMID, J. E</creatorcontrib><creatorcontrib>BROWN, J. G</creatorcontrib><creatorcontrib>WOOD, C. R</creatorcontrib><creatorcontrib>WHITE, R. D</creatorcontrib><creatorcontrib>BUCKALEW, A. R</creatorcontrib><creatorcontrib>HELD, G. 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A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone</atitle><jtitle>Toxicological sciences</jtitle><addtitle>Toxicol Sci</addtitle><date>1999</date><risdate>1999</risdate><volume>47</volume><issue>1</issue><spage>76</spage><epage>85</epage><pages>76-85</pages><issn>1096-6080</issn><eissn>1096-0929</eissn><coden>TOSCF2</coden><abstract>C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development.</abstract><cop>Cary, NC</cop><pub>Oxford University Press</pub><pmid>10048155</pmid><doi>10.1093/toxsci/47.1.76</doi><tpages>10</tpages></addata></record> |
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subjects | AAh receptors Animals Aryl Hydrocarbon Receptor Nuclear Translocator Base Sequence Biological and medical sciences Blotting, Northern CYP1A1 protein Cytochrome P-450 CYP1A1 - genetics Cytochrome P-450 CYP1A1 - metabolism DNA-Binding Proteins Embryology: invertebrates and vertebrates. Teratology Environmental Pollutants - toxicity Female Fundamental and applied biological sciences. Psychology hydrocortisone Hydrocortisone - toxicity Maternal-Fetal Exchange Mice Mice, Inbred C57BL Molecular Sequence Data mRNA Palate - drug effects Palate - embryology Palate - metabolism Polychlorinated Dibenzodioxins - toxicity Pregnancy Receptors, Aryl Hydrocarbon - genetics Receptors, Aryl Hydrocarbon - metabolism Receptors, Glucocorticoid - genetics Receptors, Glucocorticoid - metabolism Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger - metabolism Teratogens - toxicity Teratology. Teratogens Transcription Factors - genetics Transcription Factors - metabolism |
title | RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone |
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