{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T12:58:42Z","timestamp":1790945922993,"version":"4.1.0"},"reference-count":40,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001721","name":"Rijksuniversiteit Groningen","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001721","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Precision-cut liver slices (PCLSs) are an ex vivo liver model that allows isolation of extracellular vesicles (EVs), which are nano-sized particles involved in intercellular communication. However, EV recovery from PCLSs is hindered by adhesion to the extracellular matrix and macromolecules such as extracellular DNA. Enzymatic treatments, such as collagenase (COL) and the endonuclease benzonase (BEN), can aid their release, but potential effects on the tissue are unclear. Here, the effect of these enzymes separately, as well as combined, was analyzed to determine their effects on morphology, protein content, and the proteome of the PCLS. The enzymes showed minimal effects on cell viability and protein content after two different incubation times, whereas morphological analysis confirmed collagen degradation by COL and COL(BEN), along with decreased ribosomal and secretory secretory protein levels. BEN decreased the levels of proteins related to protein synthesis and degradation. Increased levels of proteins related to detoxification and energy production were observed upon addition of COL, BEN, and COL(BEN). This study emphasizes the potential and risks for the use of collagenase and benzonase for EV isolation from the PCLS.<\/jats:p>","DOI":"10.1021\/acs.jproteome.5c01232","type":"journal-article","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T05:02:06Z","timestamp":1789621326000},"page":"5049-5061","source":"Crossref","is-referenced-by-count":0,"title":["Toxicological\nExploration of Collagenase and Benzonase\non Rat Precision-Cut Liver Slices"],"prefix":"10.1021","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7157-3450","authenticated-orcid":true,"given":"Janine","family":"Stam","sequence":"first","affiliation":[{"name":"University of Groningen , , ,","place":["Groningen, The Netherlands, 9700 AD"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Olinga","sequence":"additional","affiliation":[{"name":"University of Groningen , , ,","place":["Groningen, The Netherlands, 9713 AV"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aswin","family":"Menke","sequence":"additional","affiliation":[{"name":"The Netherlands Organization for Applied Scientific Research (TNO) , , ,","place":["Leiden, The Netherlands, 2333 BE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3525-7351","authenticated-orcid":true,"given":"Pieter","family":"Annaert","sequence":"additional","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, B-3000"]},{"name":"BioNotus CommV , ,","place":["Niel, Belgium, 2845"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Klaas Nico","family":"Faber","sequence":"additional","affiliation":[{"name":"University of Groningen, University Medical Center Groningen , , ,","place":["Groningen, The Netherlands, 9713 GZ"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2218-1140","authenticated-orcid":true,"given":"Peter L.","family":"Horvatovich","sequence":"additional","affiliation":[{"name":"University of Groningen , , ,","place":["Groningen, The Netherlands, 9700 AD"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9849-0121","authenticated-orcid":true,"given":"Rainer","family":"Bischoff","sequence":"additional","affiliation":[{"name":"University of Groningen , , ,","place":["Groningen, The Netherlands, 9700 AD"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0066-3720","authenticated-orcid":true,"given":"Justina C.","family":"Wolters","sequence":"additional","affiliation":[{"name":"University of Groningen, University Medical Center Groningen , , ,","place":["Groningen, The Netherlands, 9713 AV"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,17]]},"reference":[{"issue":"1","key":"2026100208415109300_cit1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41392-024-02072-z","article-title":"Liver Diseases:\nEpidemiology, Causes, Trends and Predictions","volume":"10","author":"Gan","year":"2025","journal-title":"Signal Transduction Targeted Ther."},{"issue":"13","key":"2026100208415109300_cit2","doi-asserted-by":"publisher","first-page":"7137","DOI":"10.3390\/IJMS22137137\/S1","article-title":"Best Practices\nand Progress in Precision-Cut\nLiver Slice Cultures","volume":"22","author":"Dewyse","year":"2021","journal-title":"Int. J. Mol. Sci."},{"issue":"1","key":"2026100208415109300_cit3","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1007\/s12072-018-9913-7","article-title":"Precision-Cut Liver\nSlices: A Versatile\nTool to Advance Liver Research","volume":"13","author":"Palma","year":"2019","journal-title":"Hepatol. Int."},{"issue":"9","key":"2026100208415109300_cit4","doi-asserted-by":"publisher","first-page":"1540","DOI":"10.1038\/nprot.2010.111","article-title":"Preparation\nand Incubation of Precision-Cut Liver and Intestinal Slices for Application\nin Drug Metabolism and Toxicity Studies","volume":"5","author":"De Graaf","year":"2010","journal-title":"Nat.\nProtoc."},{"key":"2026100208415109300_cit5","doi-asserted-by":"publisher","first-page":"113795","DOI":"10.1016\/j.addr.2021.05.005","article-title":"An Overview of in Vitro,\nEx Vivo and in Vivo Models for Studying the Transport of Drugs across\nIntestinal Barriers","volume":"175","author":"Xu","year":"2021","journal-title":"Adv. Drug Delivery Rev."},{"issue":"2","key":"2026100208415109300_cit6","doi-asserted-by":"publisher","first-page":"G336","DOI":"10.1152\/ajpgi.00209.2019","article-title":"Investigating Fibrosis and Inflammation\nin an Ex Vivo\nNASH Murine Model","volume":"318","author":"Gore","year":"2020","journal-title":"Am. J. Physiol. Gastrointest.\nLiver Physiol."},{"key":"2026100208415109300_cit7","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1007\/978-1-4939-9420-5_23","article-title":"Ex Vivo Model in Cholestasis\nResearch","volume":"1981","author":"Karsten","year":"2019","journal-title":"Methods Mol. Biol."},{"issue":"3","key":"2026100208415109300_cit8","doi-asserted-by":"publisher","first-page":"507","DOI":"10.3390\/nu11030507","article-title":"A Pathophysiological\nModel of Non-Alcoholic Fatty Liver\nDisease Using Precision-Cut Liver Slices","volume":"11","author":"Prins","year":"2019","journal-title":"Nutrients"},{"issue":"5","key":"2026100208415109300_cit9","doi-asserted-by":"publisher","first-page":"626","DOI":"10.3390\/nu16050626","article-title":"Metabolic Dysfunction-Associated\nSteatotic Liver Disease in a Dish: Human Precision-Cut Liver Slices\nas a Platform for Drug Screening and Interventions","volume":"16","author":"Li","year":"2024","journal-title":"Nutrients"},{"issue":"3","key":"2026100208415109300_cit10","doi-asserted-by":"publisher","first-page":"495","DOI":"10.3390\/biomedicines14030495","article-title":"Extracellular\nVesicles: A Comprehensive Review of Their Origins, Functions, and\nTherapeutic Potential","volume":"14","author":"Schank","year":"2026","journal-title":"Biomedicines"},{"issue":"5","key":"2026100208415109300_cit11","doi-asserted-by":"publisher","DOI":"10.1002\/jex2.70043","article-title":"Characterization of Extracellular\nVesicles Derived From Human Precision-Cut Liver Slices in Metabolic\nDysfunction-Associated Steatotic Liver Disease","volume":"4","author":"Geng","year":"2025","journal-title":"J. Extracell. Biol."},{"issue":"8","key":"2026100208415109300_cit12","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1038\/nrgastro.2017.71","article-title":"Extracellular Vesicles in Liver Disease and Potential\nas Biomarkers and Therapeutic Targets","volume":"14","author":"Szabo","year":"2017","journal-title":"Nat.\nRev. Gastroenterol. Hepatol."},{"issue":"3","key":"2026100208415109300_cit13","doi-asserted-by":"publisher","first-page":"1548","DOI":"10.1038\/s41596-020-00466-1","article-title":"Isolation and Characterization of\nExtracellular Vesicle Subpopulations from Tissues","volume":"16","author":"Crescitelli","year":"2021","journal-title":"Nat. Protoc."},{"issue":"3","key":"2026100208415109300_cit14","doi-asserted-by":"publisher","first-page":"1674","DOI":"10.1007\/s12035-019-01797-1","article-title":"Extracellular\nVesicles in the Forebrain\nDisplay Reduced MiR-346 and MiR-331-3p in a Rat Model of Chronic Temporal\nLobe Epilepsy","volume":"57","author":"Gitai\u0301","year":"2020","journal-title":"Mol. Neurobiol."},{"issue":"5","key":"2026100208415109300_cit15","doi-asserted-by":"publisher","first-page":"4306","DOI":"10.3892\/mmr.2019.10107","article-title":"Identification of Potential Diagnostic Biomarkers for\nPneumonia Caused by Adenovirus Infection in Children by Screening\nSerum Exosomal MicroRNAs","volume":"49","author":"Huang","year":"2019","journal-title":"Mol. Med. Rep."},{"key":"2026100208415109300_cit16","doi-asserted-by":"publisher","first-page":"144","DOI":"10.3791\/59143","article-title":"Extraction of Extracellular Vesicles\nfrom Whole Tissue","volume":"7","author":"Hurwitz","year":"2019","journal-title":"J. Vis. Exp."},{"issue":"1","key":"2026100208415109300_cit17","doi-asserted-by":"publisher","first-page":"1635420","DOI":"10.1080\/20013078.2019.1635420","article-title":"Mitochondrial\nProtein Enriched Extracellular Vesicles Discovered in Human Melanoma\nTissues Can Be Detected in Patient Plasma","volume":"8","author":"Jang","year":"2019","journal-title":"J.\nExtracell. Vesicles"},{"key":"2026100208415109300_cit18","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1016\/j.neurobiolaging.2019.07.016","article-title":"A Pleiotropic Role\nfor Exosomes Loaded with the Amyloid \u03b2 Precursor Protein Carboxyl-Terminal\nFragments in the Brain of Down Syndrome Patients","volume":"84","author":"Pe\u0301rez-Gonza\u0301lez","year":"2019","journal-title":"Neurobiol. Aging"},{"key":"2026100208415109300_cit19","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1016\/j.abb.2019.04.003","article-title":"Quantitative\nAtomic Force Microscopy Provides New Insight\ninto Matrix Vesicle Mineralization","volume":"667","author":"Plaut","year":"2019","journal-title":"Arch. Biochem.\nBiophys."},{"issue":"10","key":"2026100208415109300_cit20","doi-asserted-by":"publisher","first-page":"3744","DOI":"10.1074\/jbc.RA118.004825","article-title":"CNS-Derived Extracellular Vesicles from Superoxide\nDismutase 1 (SOD1)G93A ALS Mice Originate from Astrocytes\nand Neurons and Carry Misfolded SOD1","volume":"294","author":"Silverman","year":"2019","journal-title":"J. Biol.\nChem."},{"issue":"1","key":"2026100208415109300_cit21","doi-asserted-by":"publisher","first-page":"1348885","DOI":"10.1080\/20013078.2017.1348885","article-title":"A Rigorous Method to Enrich for Exosomes from Brain\nTissue","volume":"6","author":"Vella","year":"2017","journal-title":"J. Extracell. Vesicles"},{"key":"2026100208415109300_cit22","doi-asserted-by":"publisher","first-page":"100069","DOI":"10.1016\/j.vesic.2025.100069","article-title":"A Method for Isolating\nSmall Extracellular Vesicles from Parenchymal Tissues","volume":"5","author":"Sun","year":"2025","journal-title":"Extracell. Vesicle"},{"issue":"3","key":"2026100208415109300_cit23","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1006\/jmbi.1999.2694","article-title":"The DNA\/RNA Non-Specific Serratia\nNuclease Prefers Double-Stranded A-Form Nucleic Acids as Substrates","volume":"288","author":"Meiss","year":"1999","journal-title":"J. Mol. Biol."},{"issue":"1","key":"2026100208415109300_cit24","doi-asserted-by":"publisher","first-page":"288","DOI":"10.1016\/j.tiv.2015.10.008","article-title":"Viability,\nFunction and Morphological Integrity of\nPrecision-Cut Liver Slices during Prolonged Incubation: Effects of\nCulture Medium","volume":"30","author":"Starokozhko","year":"2015","journal-title":"Toxicol. In Vitro"},{"issue":"10","key":"2026100208415109300_cit25","doi-asserted-by":"publisher","first-page":"3403","DOI":"10.1007\/S00204-017-1960-7","article-title":"Rat\nPrecision-Cut Liver Slices Predict\nDrug-Induced Cholestatic Injury","volume":"91","author":"Starokozhko","year":"2017","journal-title":"Arch. Toxicol."},{"issue":"9","key":"2026100208415109300_cit26","doi-asserted-by":"publisher","first-page":"1100","DOI":"10.1016\/S0090-9556(24)15193-8","article-title":"Increased post-thaw viability and phase I and II biotransformation\nactivity in cryopreserved rat liver slices after improvement of a\nfast-freezing method","volume":"28","author":"De Graaf","year":"2000","journal-title":"Drug Metab. Dispos."},{"issue":"9","key":"2026100208415109300_cit27","doi-asserted-by":"publisher","first-page":"3204","DOI":"10.1021\/acs.jproteome.6b00419","article-title":"Translational\nTargeted Proteomics Profiling of Mitochondrial Energy Metabolic Pathways\nin Mouse and Human Samples","volume":"15","author":"Wolters","year":"2016","journal-title":"J. Proteome Res."},{"key":"2026100208415109300_cit28","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1007\/978-3-662-66786-6","article-title":"Repeated-Measures Analysis of Variance (ANOVA)","volume":"2023","author":"Janczyk","year":"2023","journal-title":"Understanding Inferential Statistics"},{"key":"2026100208415109300_cit29","unstructured":"Oliveros, J. C. V.\n          \n          An\nInteractive\nTool for Comparing Lists with Venn\u2019s Diagrams; 2007, https:\/\/bioinfogp.cnb.csic.es\/tools\/venny\/."},{"issue":"D1","key":"2026100208415109300_cit30","doi-asserted-by":"publisher","first-page":"D638","DOI":"10.1093\/nar\/gkac1000","article-title":"The STRING\nDatabase in 2023: Protein-Protein Association Networks and Functional\nEnrichment Analyses for Any Sequenced Genome of Interest","volume":"51","author":"Szklarczyk","year":"2023","journal-title":"Nucleic Acids Res."},{"issue":"D1","key":"2026100208415109300_cit31","doi-asserted-by":"publisher","first-page":"D543","DOI":"10.1093\/nar\/gkae1011","article-title":"The PRIDE\nDatabase at 20 Years: 2025 Update","volume":"53","author":"Perez-Riverol","year":"2025","journal-title":"Nucleic Acids\nRes."},{"issue":"11","key":"2026100208415109300_cit32","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2021.e08318","article-title":"High Concentration of Extracellular\nNucleotides Suppresses Cell Growth via Delayed Cell Cycle Progression\nin Cancer and Noncancer Cell Lines","volume":"7","author":"Sawa","year":"2021","journal-title":"Heliyon"},{"issue":"9","key":"2026100208415109300_cit33","doi-asserted-by":"publisher","first-page":"100273","DOI":"10.1016\/j.mcpro.2022.100273","article-title":"Quantitative\nProteomics Identifies Proteins Enriched in Large and Small Extracellular\nVesicles","volume":"21","author":"Lischnig","year":"2022","journal-title":"Mol. Cell. Proteomics"},{"issue":"22","key":"2026100208415109300_cit34","doi-asserted-by":"publisher","first-page":"12874","DOI":"10.1093\/nar\/gkaa674","article-title":"Fragmentation\nof Extracellular Ribosomes and TRNAs Shapes the Extracellular RNAome","volume":"48","author":"Tosar","year":"2020","journal-title":"Nucleic Acids Res."},{"issue":"5","key":"2026100208415109300_cit35","doi-asserted-by":"publisher","first-page":"1232","DOI":"10.1053\/jhep.1996.v23.pm0008621158","article-title":"Binding of\nApolipoprotein A-I and Acetaldehyde-Modified Apolipoprotein A-I to\nLiver Extracellular Matrix","volume":"23","author":"Paradis","year":"1996","journal-title":"Hepatology"},{"issue":"1","key":"2026100208415109300_cit36","doi-asserted-by":"publisher","first-page":"11","DOI":"10.3390\/cells8010011","article-title":"A Rise in ATP, ROS, and Mitochondrial Content upon\nGlucose Withdrawal Correlates with a Dysregulated Mitochondria Turnover\nMediated by the Activation of the Protein Deacetylase SIRT1","volume":"8","author":"Song","year":"2019","journal-title":"Cells"},{"issue":"4","key":"2026100208415109300_cit37","doi-asserted-by":"publisher","first-page":"1205","DOI":"10.1152\/physrev.00037.2014","article-title":"Membrane Repair: Mechanisms and Pathophysiology","volume":"95","author":"Cooper","year":"2015","journal-title":"Physiol. Rev."},{"issue":"1","key":"2026100208415109300_cit38","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41467-019-12640-5","article-title":"DNA Damage\nand Transcription Stress\nCause ATP-Mediated Redesign of Metabolism and Potentiation of Anti-Oxidant\nBuffering","volume":"10","author":"Milanese","year":"2019","journal-title":"Nat. Commun."},{"key":"2026100208415109300_cit39","doi-asserted-by":"publisher","first-page":"1502","DOI":"10.3389\/fphar.2019.01502","article-title":"Intracellular ATP Delivery\nCauses Rapid Tissue Regeneration via Upregulation of Cytokines, Chemokines,\nand Stem Cells","volume":"10","author":"Mo","year":"2020","journal-title":"Front. Pharmacol."},{"issue":"3","key":"2026100208415109300_cit40","doi-asserted-by":"publisher","DOI":"10.1038\/cddis.2013.78","article-title":"Regulation of Cytochrome\nP450 2e1 Expression by Ethanol: Role of Oxidative Stress-Mediated\nPkc\/Jnk\/Sp1 Pathway","volume":"4","author":"Jin","year":"2013","journal-title":"Cell Death Dis."}],"container-title":["Journal of Proteome Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jproteome.5c01232","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5049\/68539427\/acs.jproteome.5c01232.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5049\/68539427\/acs.jproteome.5c01232.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T12:51:41Z","timestamp":1790945501000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jprobs\/article\/25\/10\/5049\/5431757\/Toxicological-Exploration-of-Collagenase-and"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,17]]},"references-count":40,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,9,17]]},"published-print":{"date-parts":[[2026,10,2]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jproteome.5c01232","relation":{},"ISSN":["1535-3893","1535-3907"],"issn-type":[{"value":"1535-3893","type":"print"},{"value":"1535-3907","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,17]]}}}