Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture
Identifieur interne : 001357 ( Istex/Corpus ); précédent : 001356; suivant : 001358Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture
Auteurs : I. Leguen ; C. Carlsson ; E. Perdu-Durand ; P. Prunet ; P. P Rt ; J. P CravediSource :
- Aquatic Toxicology [ 0166-445X ] ; 2000.
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- KwdEn :
Abstract
The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10−6 to 10−12 M. After 24 h, TCDD showed a maximal induction at a concentration of 10−9 M while BNF showed a maximal induction at a concentration of 10−7 M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.
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DOI: 10.1016/S0166-445X(99)00043-0
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</title><author><name sortKey="Leguen, I" sort="Leguen, I" uniqKey="Leguen I" first="I" last="Leguen">I. Leguen</name><affiliation><mods:affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</mods:affiliation></affiliation></author><author><name sortKey="Carlsson, C" sort="Carlsson, C" uniqKey="Carlsson C" first="C" last="Carlsson">C. Carlsson</name><affiliation><mods:affiliation>Department of Environmental Toxicology, Norbyvdgen 18A, 752 36 Uppsala, Sweden</mods:affiliation></affiliation></author><author><name sortKey="Perdu Durand, E" sort="Perdu Durand, E" uniqKey="Perdu Durand E" first="E" last="Perdu-Durand">E. Perdu-Durand</name><affiliation><mods:affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Prunet, P" sort="Prunet, P" uniqKey="Prunet P" first="P" last="Prunet">P. Prunet</name><affiliation><mods:affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</mods:affiliation></affiliation></author><author><name sortKey="P Rt, P" sort="P Rt, P" uniqKey="P Rt P" first="P" last="P Rt">P. P Rt</name><affiliation><mods:affiliation>European Commission, C.C.R., Environment Institute, TP460, 21020 Ispra (VA), Italy</mods:affiliation></affiliation></author><author><name sortKey="Cravedi, J P" sort="Cravedi, J P" uniqKey="Cravedi J" first="J. P" last="Cravedi">J. P Cravedi</name><affiliation><mods:affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel.: +33-561-285-004; fax: +33-561-285-244</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: jean-pierre.cravedi@toulouse.inra.fr</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1016/S0166-445X(99)00043-0</idno><idno type="url">https://api.istex.fr/document/C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">001357</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001357</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</title><author><name sortKey="Leguen, I" sort="Leguen, I" uniqKey="Leguen I" first="I" last="Leguen">I. Leguen</name><affiliation><mods:affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</mods:affiliation></affiliation></author><author><name sortKey="Carlsson, C" sort="Carlsson, C" uniqKey="Carlsson C" first="C" last="Carlsson">C. Carlsson</name><affiliation><mods:affiliation>Department of Environmental Toxicology, Norbyvdgen 18A, 752 36 Uppsala, Sweden</mods:affiliation></affiliation></author><author><name sortKey="Perdu Durand, E" sort="Perdu Durand, E" uniqKey="Perdu Durand E" first="E" last="Perdu-Durand">E. Perdu-Durand</name><affiliation><mods:affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Prunet, P" sort="Prunet, P" uniqKey="Prunet P" first="P" last="Prunet">P. Prunet</name><affiliation><mods:affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</mods:affiliation></affiliation></author><author><name sortKey="P Rt, P" sort="P Rt, P" uniqKey="P Rt P" first="P" last="P Rt">P. P Rt</name><affiliation><mods:affiliation>European Commission, C.C.R., Environment Institute, TP460, 21020 Ispra (VA), Italy</mods:affiliation></affiliation></author><author><name sortKey="Cravedi, J P" sort="Cravedi, J P" uniqKey="Cravedi J" first="J. P" last="Cravedi">J. P Cravedi</name><affiliation><mods:affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel.: +33-561-285-004; fax: +33-561-285-244</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: jean-pierre.cravedi@toulouse.inra.fr</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Aquatic Toxicology</title><title level="j" type="abbrev">AQTOX</title><idno type="ISSN">0166-445X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">48</biblScope><biblScope unit="issue">2–3</biblScope><biblScope unit="page" from="165">165</biblScope><biblScope unit="page" to="176">176</biblScope></imprint><idno type="ISSN">0166-445X</idno></series><idno type="istex">C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4</idno><idno type="DOI">10.1016/S0166-445X(99)00043-0</idno><idno type="PII">S0166-445X(99)00043-0</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0166-445X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>CYPIA induction</term><term>Cytochrome P450</term><term>Gill cells</term><term>Hepatocytes</term><term>Metabolism</term><term>Rainbow trout</term><term>Transferases</term><term>Xenobiotics</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10−6 to 10−12 M. After 24 h, TCDD showed a maximal induction at a concentration of 10−9 M while BNF showed a maximal induction at a concentration of 10−7 M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>I Leguen</name><affiliations><json:string>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</json:string></affiliations></json:item><json:item><name>C Carlsson</name><affiliations><json:string>Department of Environmental Toxicology, Norbyvdgen 18A, 752 36 Uppsala, Sweden</json:string></affiliations></json:item><json:item><name>E Perdu-Durand</name><affiliations><json:string>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</json:string></affiliations></json:item><json:item><name>P Prunet</name><affiliations><json:string>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</json:string></affiliations></json:item><json:item><name>P Pärt</name><affiliations><json:string>European Commission, C.C.R., Environment Institute, TP460, 21020 Ispra (VA), Italy</json:string></affiliations></json:item><json:item><name>J.P Cravedi</name><affiliations><json:string>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</json:string><json:string>Corresponding author. Tel.: +33-561-285-004; fax: +33-561-285-244</json:string><json:string>E-mail: jean-pierre.cravedi@toulouse.inra.fr</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Rainbow trout</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Gill cells</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Metabolism</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>CYPIA induction</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Cytochrome P450</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Transferases</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Xenobiotics</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Hepatocytes</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10−6 to 10−12 M. After 24 h, TCDD showed a maximal induction at a concentration of 10−9 M while BNF showed a maximal induction at a concentration of 10−7 M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.</abstract><qualityIndicators><score>8</score><pdfVersion>1.2</pdfVersion><pdfPageSize>536 x 674 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>8</keywordCount><abstractCharCount>1961</abstractCharCount><pdfWordCount>5085</pdfWordCount><pdfCharCount>33337</pdfCharCount><pdfPageCount>12</pdfPageCount><abstractWordCount>277</abstractWordCount></qualityIndicators><title>Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</title><pii><json:string>S0166-445X(99)00043-0</json:string></pii><genre><json:string>research-article</json:string></genre><host><volume>48</volume><pii><json:string>S0166-445X(00)X0054-9</json:string></pii><pages><last>176</last><first>165</first></pages><issn><json:string>0166-445X</json:string></issn><issue>2–3</issue><genre><json:string>journal</json:string></genre><language><json:string>unknown</json:string></language><title>Aquatic Toxicology</title><publicationDate>2000</publicationDate></host><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1016/S0166-445X(99)00043-0</json:string></doi><id>C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4</id><score>0.024557123</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©2000 Elsevier Science B.V.</p></availability><date>2000</date></publicationStmt><notesStmt><note type="content">Fig. 1: Induction of EROD activity in gill epithelial cells exposed to TCDD. The values are presented as the mean and S.D. for cells from five different fishes. Duplicate wells were measured for each concentration in each fish.</note><note type="content">Fig. 2: Induction of EROD activity in gill epithelial cells exposed to BNF. The values are presented as the mean and S.D. for five different wells for each concentration in one single fish.</note><note type="content">Fig. 3: Typical HPLC radio-chromatograms of culture medium after incubations of 2.5 μM [14C]7-EC with (a) gill epithelial cells, (b) gill filaments and (c) isolated hepatocytes. Incubation time was 2 h for (b) and (c) and 8 h for (a). Peaks 1, 2 and 3 corresponded to 7-EC-glucuronide, 7-hydroxycoumarin and 7-EC-sulfate respectively.</note><note type="content">Fig. 4: Typical HPLC radio-chromatograms of culture medium after 2-h incubations of IC-testosterone with (a) gill epithelial cells, (b) gill microsomes, (c) isolated hepatocytes and (d) liver microsomes. Substrate final concentration was 2.5 μM for (a) and (b) and 10 μM for (c) and (d). Peak identification: 6β-T, 6β-hydroxytestosterone; 16β-T, 16-hydroxytestosterone; Tgluc, testosterone glucuronide; Δ4, androstenedione; T, testosterone; 5α-DHT, 5α-dihydrotestosterone; A-diol, androstane-diol.</note><note type="content">Fig. 5: Typical HPLC profiles of culture medium after 2-h incubation of [14C]aniline (2.5 μM) with (a) gill epithelial cells and (b) hepatocytes. Peaks 1, 2 and 3 corresponded to 2-hydroxyaniline, aniline and acetanilide, respectively.</note><note type="content">Fig. 6: Biotransformation of 7-EC, testosterone and aniline in gill epithelial cells and isolated hepatocytes. Values are expressed as the mean±S.D. of three experiments and are calculated from 10 μM, 2-h incubations, except for 7-EC incubated with gill epithelial cells (10 μM, 8 h).</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</title><author xml:id="author-1"><persName><forename type="first">I</forename><surname>Leguen</surname></persName><affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</affiliation></author><author xml:id="author-2"><persName><forename type="first">C</forename><surname>Carlsson</surname></persName><affiliation>Department of Environmental Toxicology, Norbyvdgen 18A, 752 36 Uppsala, Sweden</affiliation></author><author xml:id="author-3"><persName><forename type="first">E</forename><surname>Perdu-Durand</surname></persName><affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</affiliation></author><author xml:id="author-4"><persName><forename type="first">P</forename><surname>Prunet</surname></persName><affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</affiliation></author><author xml:id="author-5"><persName><forename type="first">P</forename><surname>Pärt</surname></persName><affiliation>European Commission, C.C.R., Environment Institute, TP460, 21020 Ispra (VA), Italy</affiliation></author><author xml:id="author-6"><persName><forename type="first">J.P</forename><surname>Cravedi</surname></persName><email>jean-pierre.cravedi@toulouse.inra.fr</email><affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</affiliation><affiliation>Corresponding author. Tel.: +33-561-285-004; fax: +33-561-285-244</affiliation></author></analytic><monogr><title level="j">Aquatic Toxicology</title><title level="j" type="abbrev">AQTOX</title><idno type="pISSN">0166-445X</idno><idno type="PII">S0166-445X(00)X0054-9</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">48</biblScope><biblScope unit="issue">2–3</biblScope><biblScope unit="page" from="165">165</biblScope><biblScope unit="page" to="176">176</biblScope></imprint></monogr><idno type="istex">C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4</idno><idno type="DOI">10.1016/S0166-445X(99)00043-0</idno><idno type="PII">S0166-445X(99)00043-0</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10−6 to 10−12 M. After 24 h, TCDD showed a maximal induction at a concentration of 10−9 M while BNF showed a maximal induction at a concentration of 10−7 M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Rainbow trout</term></item><item><term>Gill cells</term></item><item><term>Metabolism</term></item><item><term>CYPIA induction</term></item><item><term>Cytochrome P450</term></item><item><term>Transferases</term></item><item><term>Xenobiotics</term></item><item><term>Hepatocytes</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1999-04-15">Modified</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4ab" NDATA="IMAGE" name="gr4ab"></istex:entity><istex:entity SYSTEM="gr4cd" NDATA="IMAGE" name="gr4cd"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>AQTOX</jid><aid>1079</aid><ce:pii>S0166-445X(99)00043-0</ce:pii><ce:doi>10.1016/S0166-445X(99)00043-0</ce:doi><ce:copyright type="full-transfer" year="2000">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</ce:title><ce:author-group><ce:author><ce:given-name>I</ce:given-name><ce:surname>Leguen</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>C</ce:given-name><ce:surname>Carlsson</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>E</ce:given-name><ce:surname>Perdu-Durand</ce:surname><ce:cross-ref refid="AFF3"><ce:sup>c</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>P</ce:given-name><ce:surname>Prunet</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>P</ce:given-name><ce:surname>Pärt</ce:surname><ce:cross-ref refid="AFF4"><ce:sup>d</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>J.P</ce:given-name><ce:surname>Cravedi</ce:surname><ce:cross-ref refid="AFF3"><ce:sup>c</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>jean-pierre.cravedi@toulouse.inra.fr</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Department of Environmental Toxicology, Norbyvdgen 18A, 752 36 Uppsala, Sweden</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>c</ce:label><ce:textfn>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</ce:textfn></ce:affiliation><ce:affiliation id="AFF4"><ce:label>d</ce:label><ce:textfn>European Commission, C.C.R., Environment Institute, TP460, 21020 Ispra (VA), Italy</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +33-561-285-004; fax: +33-561-285-244</ce:text></ce:correspondence></ce:author-group><ce:date-received day="12" month="2" year="1999"></ce:date-received><ce:date-revised day="15" month="4" year="1999"></ce:date-revised><ce:date-accepted day="12" month="5" year="1999"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (<ce:italic>Oncorhynchus mykiss</ce:italic>) gills. As a first approach, ethoxyresorufin-<ce:italic>O</ce:italic>-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-<ce:italic>p</ce:italic>-dioxin (TCDD) in concentrations ranging from 10<ce:sup>−6</ce:sup> to 10<ce:sup>−12</ce:sup> M. After 24 h, TCDD showed a maximal induction at a concentration of 10<ce:sup>−9</ce:sup> M while BNF showed a maximal induction at a concentration of 10<ce:sup>−7</ce:sup> M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Rainbow trout</ce:text></ce:keyword><ce:keyword><ce:text>Gill cells</ce:text></ce:keyword><ce:keyword><ce:text>Metabolism</ce:text></ce:keyword><ce:keyword><ce:text>CYPIA induction</ce:text></ce:keyword><ce:keyword><ce:text>Cytochrome P450</ce:text></ce:keyword><ce:keyword><ce:text>Transferases</ce:text></ce:keyword><ce:keyword><ce:text>Xenobiotics</ce:text></ce:keyword><ce:keyword><ce:text>Hepatocytes</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture</title></titleInfo><name type="personal"><namePart type="given">I</namePart><namePart type="family">Leguen</namePart><affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">C</namePart><namePart type="family">Carlsson</namePart><affiliation>Department of Environmental Toxicology, Norbyvdgen 18A, 752 36 Uppsala, Sweden</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">E</namePart><namePart type="family">Perdu-Durand</namePart><affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">P</namePart><namePart type="family">Prunet</namePart><affiliation>Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">P</namePart><namePart type="family">Pärt</namePart><affiliation>European Commission, C.C.R., Environment Institute, TP460, 21020 Ispra (VA), Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">J.P</namePart><namePart type="family">Cravedi</namePart><affiliation>Laboratoire des Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France</affiliation><affiliation>Corresponding author. Tel.: +33-561-285-004; fax: +33-561-285-244</affiliation><affiliation>E-mail: jean-pierre.cravedi@toulouse.inra.fr</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateModified encoding="w3cdtf">1999-04-15</dateModified><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10−6 to 10−12 M. After 24 h, TCDD showed a maximal induction at a concentration of 10−9 M while BNF showed a maximal induction at a concentration of 10−7 M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.</abstract><note type="content">Fig. 1: Induction of EROD activity in gill epithelial cells exposed to TCDD. The values are presented as the mean and S.D. for cells from five different fishes. Duplicate wells were measured for each concentration in each fish.</note><note type="content">Fig. 2: Induction of EROD activity in gill epithelial cells exposed to BNF. The values are presented as the mean and S.D. for five different wells for each concentration in one single fish.</note><note type="content">Fig. 3: Typical HPLC radio-chromatograms of culture medium after incubations of 2.5 μM [14C]7-EC with (a) gill epithelial cells, (b) gill filaments and (c) isolated hepatocytes. Incubation time was 2 h for (b) and (c) and 8 h for (a). Peaks 1, 2 and 3 corresponded to 7-EC-glucuronide, 7-hydroxycoumarin and 7-EC-sulfate respectively.</note><note type="content">Fig. 4: Typical HPLC radio-chromatograms of culture medium after 2-h incubations of IC-testosterone with (a) gill epithelial cells, (b) gill microsomes, (c) isolated hepatocytes and (d) liver microsomes. Substrate final concentration was 2.5 μM for (a) and (b) and 10 μM for (c) and (d). Peak identification: 6β-T, 6β-hydroxytestosterone; 16β-T, 16-hydroxytestosterone; Tgluc, testosterone glucuronide; Δ4, androstenedione; T, testosterone; 5α-DHT, 5α-dihydrotestosterone; A-diol, androstane-diol.</note><note type="content">Fig. 5: Typical HPLC profiles of culture medium after 2-h incubation of [14C]aniline (2.5 μM) with (a) gill epithelial cells and (b) hepatocytes. Peaks 1, 2 and 3 corresponded to 2-hydroxyaniline, aniline and acetanilide, respectively.</note><note type="content">Fig. 6: Biotransformation of 7-EC, testosterone and aniline in gill epithelial cells and isolated hepatocytes. Values are expressed as the mean±S.D. of three experiments and are calculated from 10 μM, 2-h incubations, except for 7-EC incubated with gill epithelial cells (10 μM, 8 h).</note><subject lang="en"><genre>Keywords</genre><topic>Rainbow trout</topic><topic>Gill cells</topic><topic>Metabolism</topic><topic>CYPIA induction</topic><topic>Cytochrome P450</topic><topic>Transferases</topic><topic>Xenobiotics</topic><topic>Hepatocytes</topic></subject><relatedItem type="host"><titleInfo><title>Aquatic Toxicology</title></titleInfo><titleInfo type="abbreviated"><title>AQTOX</title></titleInfo><genre type="journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">200003</dateIssued></originInfo><identifier type="ISSN">0166-445X</identifier><identifier type="PII">S0166-445X(00)X0054-9</identifier><part><date>200003</date><detail type="volume"><number>48</number><caption>vol.</caption></detail><detail type="issue"><number>2–3</number><caption>no.</caption></detail><extent unit="issue pages"><start>75</start><end>356</end></extent><extent unit="pages"><start>165</start><end>176</end></extent></part></relatedItem><identifier type="istex">C66907179B6CDE0AAE03621EBC04F14BD6B4B5F4</identifier><identifier type="DOI">10.1016/S0166-445X(99)00043-0</identifier><identifier type="PII">S0166-445X(99)00043-0</identifier><accessCondition type="use and reproduction" contentType="copyright">©2000 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>Elsevier Science B.V., ©2000</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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