Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+
Identifieur interne : 001093 ( Istex/Corpus ); précédent : 001092; suivant : 001094Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+
Auteurs : Jalal Pourahmad ; Steve Ross ; Peter J. O RienSource :
- Free Radical Biology and Medicine [ 0891-5849 ] ; 2001.
English descriptors
- KwdEn :
- ANOVA, BAPTA, BSA, Copper, Cytotoxicity, DMSO, EGTA, Free radicals, HEPES, Hepatocyte, Lysosomes, Proteases, ROS, Redox cycling, SD, SOD, rpm.
- Teeft :
- Acridine, Aurothioglucose, Cadmium, Cell lysis, Cell viability, Chloroquine, Control hepatocytes, Copper chloride, Cysteine, Cytotoxic, Cytotoxic mechanism, Cytotoxicity, Dimethyl sulfoxide, Gentamicin, Hepatic, Hepatocyte, Hepatocyte cytotoxicity, Hepatocyte proteolysis, Hepatocytes, Inhibitor, Leupeptin, Lipid, Lipid peroxidation, Lysis, Lysosomal, Lysosomal enzymes, Lysosomal involvement, Lysosomal membrane damage, Lysosomal protease inhibitors, Lysosomal proteases, Lysosome, Methylamine, Mitochondrial membrane, Monensin, Oxidative, Oxidative stress, Pepstatin, Peroxidation, Pourahmad, Preincubated, Protease, Proteolysis, Redox, Redox cycling, Separate experiments, Superoxide dismutase, Toxicity, Trypan, Uorescence.
Abstract
Abstract: Previously we showed that the redox active Cu2+ was much more effective than Cd2+ at inducing reactive oxygen species (“ROS”) formation in hepatocytes and furthermore “ROS” scavengers prevented Cu2+-induced hepatocyte cytotoxicity (Pourahmad and O’Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu2+, but not Cd2+, was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, “ROS” formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu2+, but not Cd2+, as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu2+-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu2+-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu2+. Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu2+-induced hepatocyte cytotoxicity. It is proposed that Cu2+-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by “ROS” generated by lysosomal Cu2+ redox cycling.
Url:
DOI: 10.1016/S0891-5849(00)00450-0
Links to Exploration step
ISTEX:4A6B90703E79338718A7B90CB9F5489F8853F724Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+</title><author><name sortKey="Pourahmad, Jalal" sort="Pourahmad, Jalal" uniqKey="Pourahmad J" first="Jalal" last="Pourahmad">Jalal Pourahmad</name><affiliation><mods:affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</mods:affiliation></affiliation></author><author><name sortKey="Ross, Steve" sort="Ross, Steve" uniqKey="Ross S" first="Steve" last="Ross">Steve Ross</name><affiliation><mods:affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</mods:affiliation></affiliation></author><author><name sortKey="O Rien, Peter J" sort="O Rien, Peter J" uniqKey="O Rien P" first="Peter J" last="O Rien">Peter J. O Rien</name><affiliation><mods:affiliation>E-mail: peter.obrien@utoronto.ca</mods:affiliation></affiliation><affiliation><mods:affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:4A6B90703E79338718A7B90CB9F5489F8853F724</idno><date when="2001" year="2001">2001</date><idno type="doi">10.1016/S0891-5849(00)00450-0</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-0KW2SQ2Q-S/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001093</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001093</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+</title><author><name sortKey="Pourahmad, Jalal" sort="Pourahmad, Jalal" uniqKey="Pourahmad J" first="Jalal" last="Pourahmad">Jalal Pourahmad</name><affiliation><mods:affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</mods:affiliation></affiliation></author><author><name sortKey="Ross, Steve" sort="Ross, Steve" uniqKey="Ross S" first="Steve" last="Ross">Steve Ross</name><affiliation><mods:affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</mods:affiliation></affiliation></author><author><name sortKey="O Rien, Peter J" sort="O Rien, Peter J" uniqKey="O Rien P" first="Peter J" last="O Rien">Peter J. O Rien</name><affiliation><mods:affiliation>E-mail: peter.obrien@utoronto.ca</mods:affiliation></affiliation><affiliation><mods:affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Free Radical Biology and Medicine</title><title level="j" type="abbrev">FRB</title><idno type="ISSN">0891-5849</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001">2001</date><biblScope unit="volume">30</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="89">89</biblScope><biblScope unit="page" to="97">97</biblScope></imprint><idno type="ISSN">0891-5849</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0891-5849</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>ANOVA</term><term>BAPTA</term><term>BSA</term><term>Copper</term><term>Cytotoxicity</term><term>DMSO</term><term>EGTA</term><term>Free radicals</term><term>HEPES</term><term>Hepatocyte</term><term>Lysosomes</term><term>Proteases</term><term>ROS</term><term>Redox cycling</term><term>SD</term><term>SOD</term><term>rpm</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Acridine</term><term>Aurothioglucose</term><term>Cadmium</term><term>Cell lysis</term><term>Cell viability</term><term>Chloroquine</term><term>Control hepatocytes</term><term>Copper chloride</term><term>Cysteine</term><term>Cytotoxic</term><term>Cytotoxic mechanism</term><term>Cytotoxicity</term><term>Dimethyl sulfoxide</term><term>Gentamicin</term><term>Hepatic</term><term>Hepatocyte</term><term>Hepatocyte cytotoxicity</term><term>Hepatocyte proteolysis</term><term>Hepatocytes</term><term>Inhibitor</term><term>Leupeptin</term><term>Lipid</term><term>Lipid peroxidation</term><term>Lysis</term><term>Lysosomal</term><term>Lysosomal enzymes</term><term>Lysosomal involvement</term><term>Lysosomal membrane damage</term><term>Lysosomal protease inhibitors</term><term>Lysosomal proteases</term><term>Lysosome</term><term>Methylamine</term><term>Mitochondrial membrane</term><term>Monensin</term><term>Oxidative</term><term>Oxidative stress</term><term>Pepstatin</term><term>Peroxidation</term><term>Pourahmad</term><term>Preincubated</term><term>Protease</term><term>Proteolysis</term><term>Redox</term><term>Redox cycling</term><term>Separate experiments</term><term>Superoxide dismutase</term><term>Toxicity</term><term>Trypan</term><term>Uorescence</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Previously we showed that the redox active Cu2+ was much more effective than Cd2+ at inducing reactive oxygen species (“ROS”) formation in hepatocytes and furthermore “ROS” scavengers prevented Cu2+-induced hepatocyte cytotoxicity (Pourahmad and O’Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu2+, but not Cd2+, was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, “ROS” formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu2+, but not Cd2+, as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu2+-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu2+-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu2+. Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu2+-induced hepatocyte cytotoxicity. It is proposed that Cu2+-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by “ROS” generated by lysosomal Cu2+ redox cycling.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>lysosomal</json:string><json:string>hepatocytes</json:string><json:string>hepatocyte</json:string><json:string>cytotoxicity</json:string><json:string>acridine</json:string><json:string>lysosome</json:string><json:string>gentamicin</json:string><json:string>lipid</json:string><json:string>uorescence</json:string><json:string>aurothioglucose</json:string><json:string>peroxidation</json:string><json:string>leupeptin</json:string><json:string>pepstatin</json:string><json:string>methylamine</json:string><json:string>hepatocyte cytotoxicity</json:string><json:string>lipid 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Canada</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Original contribution</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Copper</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Redox cycling</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Lysosomes</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Proteases</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Hepatocyte</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Cytotoxicity</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Free radicals</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ANOVA : analysis of variance</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>BAPTA : 1 2-bis(2-Aminophenoxy)ethane-N N N′ N′-tetraacetic acid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>BSA : bovine serum albumin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DMSO : dimethyl sulfoxide DPPD N N′-Diphenyl-1 4-phenylenediamine</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>EGTA : ethyleneglycol-bis(P-aminoethylether)-N N N′ N′-tetraaceticacid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>HEPES : 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ROS : reactive oxygen species</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>rpm : rotations per minute</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SD : standard deviation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SOD : superoxide dismutase</value></json:item></subject><arkIstex>ark:/67375/6H6-0KW2SQ2Q-S</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Previously we showed that the redox active Cu2+ was much more effective than Cd2+ at inducing reactive oxygen species (“ROS”) formation in hepatocytes and furthermore “ROS” scavengers prevented Cu2+-induced hepatocyte cytotoxicity (Pourahmad and O’Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu2+, but not Cd2+, was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, “ROS” formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu2+, but not Cd2+, as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu2+-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu2+-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu2+. Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu2+-induced hepatocyte cytotoxicity. It is proposed that Cu2+-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by “ROS” generated by lysosomal Cu2+ redox cycling.</abstract><qualityIndicators><score>8.838</score><pdfWordCount>4486</pdfWordCount><pdfCharCount>29278</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>9</pdfPageCount><pdfPageSize>586 x 785 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>196</abstractWordCount><abstractCharCount>1459</abstractCharCount><keywordCount>18</keywordCount></qualityIndicators><title>Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+</title><pmid><json:string>11134899</json:string></pmid><pii><json:string>S0891-5849(00)00450-0</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Free Radical Biology and Medicine</title><language><json:string>unknown</json:string></language><publicationDate>2001</publicationDate><issn><json:string>0891-5849</json:string></issn><pii><json:string>S0891-5849(00)X0093-7</json:string></pii><volume>30</volume><issue>1</issue><pages><first>89</first><last>97</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1963</json:string><json:string>2001</json:string></date><geogName></geogName><orgName><json:string>University of Toronto</json:string><json:string>Ciba-Geigy Canada Ltd</json:string><json:string>Elsevier Science Inc</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Peter S. Pennefather</json:string></persName><placeName><json:string>Cytotoxicity</json:string><json:string>Germany</json:string><json:string>Montreal</json:string><json:string>Canada</json:string><json:string>Toronto</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>[10,11]</json:string><json:string>[12]</json:string><json:string>[26,27]</json:string><json:string>[33]</json:string><json:string>Pourahmad and O’Brien, 2000</json:string><json:string>[19, 20]</json:string><json:string>[32]</json:string><json:string>[16]</json:string><json:string>Moldeus et al. [17]</json:string><json:string>[31]</json:string><json:string>[37]</json:string><json:string>[28,29]</json:string><json:string>[36]</json:string><json:string>[35]</json:string><json:string>[38,39]</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-0KW2SQ2Q-S</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - endocrinology & metabolism</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Physiology (medical)</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2001</publicationDate><copyrightDate>2001</copyrightDate><doi><json:string>10.1016/S0891-5849(00)00450-0</json:string></doi><id>4A6B90703E79338718A7B90CB9F5489F8853F724</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-0KW2SQ2Q-S/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-0KW2SQ2Q-S/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-0KW2SQ2Q-S/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©2001 Elsevier Science Inc.</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>2001</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Section title: Original contribution</note><note type="content">Fig. 1: (A) Fluorescence micrograph of acridine orange loaded hepatocytes before addition of 50 μM Cu2+ demonstrated clear granular red fluorescence indicating accumulation of fluorescent dye inside the lysosomes. (B) Fluorescence micrograph of the same acridine orange loaded hepatocytes as in Fig. 1A demonstrated loss of granular red fluorescence and increase of nuclear and cytosolic fluorescence at about 20 min following the addition of 50 μM Cu2+.</note><note type="content">Fig. 2: Proposed cytotoxic mechanism for Cu2+ induced cell death. Copper ions released from Cu2+ storage proteins in the lysosomes may undergo redox cycling with lysosomal cysteine and react with H2O2 formed by damaged mitochondria. The “ROS” formed ruptures the lysosomal membrane and releases the lysosomal enzymes which target the cell membrane. • Storage protein-Cu2+: protein (metallothionein)-bound copper.</note><note type="content">Table 1: Preventing Cu2+-Induced Hepatocyte Lysosomal Membrane Damage by Inhibitors of Oxidative Stress or Endocytosislegend legend legend</note><note type="content">Table 2: Preventing Cu2+- but not Cd2+-Induced Hepatocyte Oxidative Stress and Cell Lysis with Lysosomotropic/Endocytosis Agents or Lysosomal Protease Inhibitorslegend legend legend legend</note><note type="content">Table 3: Preventing Cu2+-Induced Hepatocyte Proteolysis with Inhibitors of Oxidative Stress Lysosomotropic/Endocytosis Agents or Lysosomal Protease Inhibitorslegend legend legend</note><note type="content">Table 4: Lysosomal Toxins Modulate Cu2+- but Not Cd2+-Induced Hepatocyte Necrosislegend legend legend</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+</title><author xml:id="author-0000"><persName><forename type="first">Jalal</forename><surname>Pourahmad</surname></persName><affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Steve</forename><surname>Ross</surname></persName><affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Peter J</forename><surname>O’Brien</surname></persName><email>peter.obrien@utoronto.ca</email><affiliation>Address correspondence to: Peter J. O’Brien, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, ON M5S 2S2, Canada; Tel: (416) 978-2716; Fax: (416) 978-8511</affiliation><affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</affiliation></author><idno type="istex">4A6B90703E79338718A7B90CB9F5489F8853F724</idno><idno type="ark">ark:/67375/6H6-0KW2SQ2Q-S</idno><idno type="DOI">10.1016/S0891-5849(00)00450-0</idno><idno type="PII">S0891-5849(00)00450-0</idno></analytic><monogr><title level="j">Free Radical Biology and Medicine</title><title level="j" type="abbrev">FRB</title><idno type="pISSN">0891-5849</idno><idno type="PII">S0891-5849(00)X0093-7</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001"></date><biblScope unit="volume">30</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="89">89</biblScope><biblScope unit="page" to="97">97</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2001</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Previously we showed that the redox active Cu2+ was much more effective than Cd2+ at inducing reactive oxygen species (“ROS”) formation in hepatocytes and furthermore “ROS” scavengers prevented Cu2+-induced hepatocyte cytotoxicity (Pourahmad and O’Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu2+, but not Cd2+, was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, “ROS” formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu2+, but not Cd2+, as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu2+-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu2+-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu2+. Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu2+-induced hepatocyte cytotoxicity. It is proposed that Cu2+-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by “ROS” generated by lysosomal Cu2+ redox cycling.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Original contribution</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Copper</term></item><item><term>Redox cycling</term></item><item><term>Lysosomes</term></item><item><term>Proteases</term></item><item><term>Hepatocyte</term></item><item><term>Cytotoxicity</term></item><item><term>Free radicals</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>ANOVA</term><term>analysis of variance</term></item><item><term>BAPTA</term><term>1 2-bis(2-Aminophenoxy)ethane-N N N′ N′-tetraacetic acid</term></item><item><term>BSA</term><term>bovine serum albumin</term></item><item><term>DMSO</term><term>dimethyl sulfoxide DPPD N N′-Diphenyl-1 4-phenylenediamine</term></item><item><term>EGTA</term><term>ethyleneglycol-bis(P-aminoethylether)-N N N′ N′-tetraaceticacid</term></item><item><term>HEPES</term><term>4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid</term></item><item><term>ROS</term><term>reactive oxygen species</term></item><item><term>rpm</term><term>rotations per minute</term></item><item><term>SD</term><term>standard deviation</term></item><item><term>SOD</term><term>superoxide dismutase</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000-09-19">Modified</change><change when="2001">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-0KW2SQ2Q-S/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:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>FRB</jid><aid>6293</aid><ce:pii>S0891-5849(00)00450-0</ce:pii><ce:doi>10.1016/S0891-5849(00)00450-0</ce:doi><ce:copyright type="full-transfer" year="2001">Elsevier Science Inc.</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Original contribution</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Original contribution</ce:textfn></ce:dochead><ce:title>Lysosomal involvement in hepatocyte cytotoxicity induced by Cu<ce:sup>2+</ce:sup> but not Cd<ce:sup>2+</ce:sup></ce:title><ce:author-group><ce:author><ce:given-name>Jalal</ce:given-name><ce:surname>Pourahmad</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Steve</ce:given-name><ce:surname>Ross</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Peter J</ce:given-name><ce:surname>O’Brien</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>peter.obrien@utoronto.ca</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Address correspondence to: Peter J. O’Brien, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, ON M5S 2S2, Canada; Tel: (416) 978-2716; Fax: (416) 978-8511</ce:text></ce:correspondence></ce:author-group><ce:date-received day="22" month="6" year="2000"></ce:date-received><ce:date-revised day="19" month="9" year="2000"></ce:date-revised><ce:date-accepted day="28" month="9" year="2000"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Previously we showed that the redox active Cu<ce:sup>2+</ce:sup> was much more effective than Cd<ce:sup>2+</ce:sup> at inducing reactive oxygen species (“ROS”) formation in hepatocytes and furthermore “ROS” scavengers prevented Cu<ce:sup>2+</ce:sup>-induced hepatocyte cytotoxicity (Pourahmad and O’Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu<ce:sup>2+</ce:sup>, but not Cd<ce:sup>2+</ce:sup>, was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, “ROS” formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu<ce:sup>2+</ce:sup>, but not Cd<ce:sup>2+</ce:sup>, as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu<ce:sup>2+</ce:sup>-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu<ce:sup>2+</ce:sup>-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu<ce:sup>2+</ce:sup>. Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu<ce:sup>2+</ce:sup>-induced hepatocyte cytotoxicity. It is proposed that Cu<ce:sup>2+</ce:sup>-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by “ROS” generated by lysosomal Cu<ce:sup>2+</ce:sup> redox cycling.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Copper</ce:text></ce:keyword><ce:keyword><ce:text>Redox cycling</ce:text></ce:keyword><ce:keyword><ce:text>Lysosomes</ce:text></ce:keyword><ce:keyword><ce:text>Proteases</ce:text></ce:keyword><ce:keyword><ce:text>Hepatocyte</ce:text></ce:keyword><ce:keyword><ce:text>Cytotoxicity</ce:text></ce:keyword><ce:keyword><ce:text>Free radicals</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>ANOVA</ce:text><ce:keyword><ce:text>analysis of variance</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>BAPTA</ce:text><ce:keyword><ce:text>1 2-bis(2-Aminophenoxy)ethane-N N N′ N′-tetraacetic acid</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>BSA</ce:text><ce:keyword><ce:text>bovine serum albumin</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>DMSO</ce:text><ce:keyword><ce:text>dimethyl sulfoxide DPPD N N′-Diphenyl-1 4-phenylenediamine</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>EGTA</ce:text><ce:keyword><ce:text>ethyleneglycol-bis(P-aminoethylether)-N N N′ N′-tetraaceticacid</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>HEPES</ce:text><ce:keyword><ce:text>4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ROS</ce:text><ce:keyword><ce:text>reactive oxygen species</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>rpm</ce:text><ce:keyword><ce:text>rotations per minute</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SD</ce:text><ce:keyword><ce:text>standard deviation</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SOD</ce:text><ce:keyword><ce:text>superoxide dismutase</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Lysosomal involvement in hepatocyte cytotoxicity induced by Cu</title></titleInfo><name type="personal"><namePart type="given">Jalal</namePart><namePart type="family">Pourahmad</namePart><affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Steve</namePart><namePart type="family">Ross</namePart><affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Peter J</namePart><namePart type="family">O’Brien</namePart><affiliation>E-mail: peter.obrien@utoronto.ca</affiliation><affiliation>Faculty of Pharmacy, University of Toronto, Toronto ON, Canada</affiliation><description>Address correspondence to: Peter J. O’Brien, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, ON M5S 2S2, Canada; Tel: (416) 978-2716; Fax: (416) 978-8511</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2001</dateIssued><dateModified encoding="w3cdtf">2000-09-19</dateModified><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Previously we showed that the redox active Cu2+ was much more effective than Cd2+ at inducing reactive oxygen species (“ROS”) formation in hepatocytes and furthermore “ROS” scavengers prevented Cu2+-induced hepatocyte cytotoxicity (Pourahmad and O’Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu2+, but not Cd2+, was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, “ROS” formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu2+, but not Cd2+, as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu2+-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu2+-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu2+. Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu2+-induced hepatocyte cytotoxicity. It is proposed that Cu2+-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by “ROS” generated by lysosomal Cu2+ redox cycling.</abstract><note type="content">Section title: Original contribution</note><note type="content">Fig. 1: (A) Fluorescence micrograph of acridine orange loaded hepatocytes before addition of 50 μM Cu2+ demonstrated clear granular red fluorescence indicating accumulation of fluorescent dye inside the lysosomes. (B) Fluorescence micrograph of the same acridine orange loaded hepatocytes as in Fig. 1A demonstrated loss of granular red fluorescence and increase of nuclear and cytosolic fluorescence at about 20 min following the addition of 50 μM Cu2+.</note><note type="content">Fig. 2: Proposed cytotoxic mechanism for Cu2+ induced cell death. Copper ions released from Cu2+ storage proteins in the lysosomes may undergo redox cycling with lysosomal cysteine and react with H2O2 formed by damaged mitochondria. The “ROS” formed ruptures the lysosomal membrane and releases the lysosomal enzymes which target the cell membrane. • Storage protein-Cu2+: protein (metallothionein)-bound copper.</note><note type="content">Table 1: Preventing Cu2+-Induced Hepatocyte Lysosomal Membrane Damage by Inhibitors of Oxidative Stress or Endocytosislegend legend legend</note><note type="content">Table 2: Preventing Cu2+- but not Cd2+-Induced Hepatocyte Oxidative Stress and Cell Lysis with Lysosomotropic/Endocytosis Agents or Lysosomal Protease Inhibitorslegend legend legend legend</note><note type="content">Table 3: Preventing Cu2+-Induced Hepatocyte Proteolysis with Inhibitors of Oxidative Stress Lysosomotropic/Endocytosis Agents or Lysosomal Protease Inhibitorslegend legend legend</note><note type="content">Table 4: Lysosomal Toxins Modulate Cu2+- but Not Cd2+-Induced Hepatocyte Necrosislegend legend legend</note><subject><genre>article-category</genre><topic>Original contribution</topic></subject><subject lang="en"><genre>Keywords</genre><topic>Copper</topic><topic>Redox cycling</topic><topic>Lysosomes</topic><topic>Proteases</topic><topic>Hepatocyte</topic><topic>Cytotoxicity</topic><topic>Free radicals</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>ANOVA : analysis of variance</topic><topic>BAPTA : 1 2-bis(2-Aminophenoxy)ethane-N N N′ N′-tetraacetic acid</topic><topic>BSA : bovine serum albumin</topic><topic>DMSO : dimethyl sulfoxide DPPD N N′-Diphenyl-1 4-phenylenediamine</topic><topic>EGTA : ethyleneglycol-bis(P-aminoethylether)-N N N′ N′-tetraaceticacid</topic><topic>HEPES : 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid</topic><topic>ROS : reactive oxygen species</topic><topic>rpm : rotations per minute</topic><topic>SD : standard deviation</topic><topic>SOD : superoxide dismutase</topic></subject><relatedItem type="host"><titleInfo><title>Free Radical Biology and Medicine</title></titleInfo><titleInfo type="abbreviated"><title>FRB</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2001</dateIssued></originInfo><identifier type="ISSN">0891-5849</identifier><identifier type="PII">S0891-5849(00)X0093-7</identifier><part><date>2001</date><detail type="volume"><number>30</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>140</end></extent><extent unit="pages"><start>89</start><end>97</end></extent></part></relatedItem><identifier type="istex">4A6B90703E79338718A7B90CB9F5489F8853F724</identifier><identifier type="ark">ark:/67375/6H6-0KW2SQ2Q-S</identifier><identifier type="DOI">10.1016/S0891-5849(00)00450-0</identifier><identifier type="PII">S0891-5849(00)00450-0</identifier><accessCondition type="use and reproduction" contentType="copyright">©2001 Elsevier Science Inc.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science Inc., ©2001</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-0KW2SQ2Q-S/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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