title: Early Alterations of Bile Canaliculi Dynamics and the Rho Kinase/Myosin Light Chain Kinase Pathway Are Characteristics of Drug-Induced Intrahepatic Cholestasis.
abstract: Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na+-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.
authors.affiliation: INSERM UMR991, Foie, Métabolismes et Cancer, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Université Rennes 1, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Biologie Servier, Gidy, France (M.G.B.); Institut de Recherches Internationales Servier, Suresnes, France (R.J.W.); and Biopredic International, St. Grégoire, Rennes, France (C.G.-G.).
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Burbank
authors.affiliation: INSERM UMR991, Foie, Métabolismes et Cancer, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Université Rennes 1, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Biologie Servier, Gidy, France (M.G.B.); Institut de Recherches Internationales Servier, Suresnes, France (R.J.W.); and Biopredic International, St. Grégoire, Rennes, France (C.G.-G.).
authors.forename: Audrey
authors.initials: A
authors.lastname: Burban
authors.affiliation: INSERM UMR991, Foie, Métabolismes et Cancer, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Université Rennes 1, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Biologie Servier, Gidy, France (M.G.B.); Institut de Recherches Internationales Servier, Suresnes, France (R.J.W.); and Biopredic International, St. Grégoire, Rennes, France (C.G.-G.).
authors.forename: Ahmad
authors.initials: A
authors.lastname: Sharanek
authors.affiliation: INSERM UMR991, Foie, Métabolismes et Cancer, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Université Rennes 1, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Biologie Servier, Gidy, France (M.G.B.); Institut de Recherches Internationales Servier, Suresnes, France (R.J.W.); and Biopredic International, St. Grégoire, Rennes, France (C.G.-G.).
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Weaver
authors.affiliation: INSERM UMR991, Foie, Métabolismes et Cancer, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Université Rennes 1, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Biologie Servier, Gidy, France (M.G.B.); Institut de Recherches Internationales Servier, Suresnes, France (R.J.W.); and Biopredic International, St. Grégoire, Rennes, France (C.G.-G.).
authors.forename: Christiane
authors.initials: C
authors.lastname: Guguen-Guillouzo
authors.affiliation: INSERM UMR991, Foie, Métabolismes et Cancer, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Université Rennes 1, Rennes, France (M.G.B., A.B., A.S., C.G.-G., A.G.); Biologie Servier, Gidy, France (M.G.B.); Institut de Recherches Internationales Servier, Suresnes, France (R.J.W.); and Biopredic International, St. Grégoire, Rennes, France (C.G.-G.) andre.guillouzo@univ-rennes1.fr.
authors.forename: André
authors.initials: A
authors.lastname: Guillouzo
mesh_terms: D000577:Amides / Q000494:pharmacology; D001647:Bile Acids and Salts / Q000378:metabolism; D001648:Bile Canaliculi / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002780:Cholestasis, Intrahepatic / Q000139:chemically induced / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013656:Taurocholic Acid / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000577:Amides; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009219:Myosin-Light-Chain Kinase; D011725:Pyridines; D013656:Taurocholic Acid; D054460:rho-Associated Kinases; C037643:bile acid binding proteins; C108830:Y 27632