title: Progenitor-derived hepatocyte-like (B-13/H) cells metabolise 1'-hydroxyestragole to a genotoxic species via a SULT2B1-dependent mechanism.
abstract: Rat B-13 progenitor cells are readily converted into functional hepatocyte-like B-13/H cells capable of phase I cytochrome P450-dependent activation of pro-carcinogens and induction of DNA damage. The aim of the present study was to investigate whether the cells are also capable of Phase II sulphotransferase (SULT)-dependent activation of a pro-carcinogen to an ultimate carcinogen. To this end we therefore examined the bioactivation of the model hepatic (hepato- and cholangio-) carcinogen estragole and its proximate SULT1A1-activated genotoxic metabolite 1'-hydroxyestragole. Exposing B-13 or B-13/H cells to estragole (at concentrations up to 1mM) resulted in the production of low levels of 1'-hydroxyestragole, but did not result in detectable DNA damage. Exposing B-13/H cells - but not B-13 cells - to 1'-hydroxyestragole resulted in a dose-dependent increase in DNA damage in comet assays, confirmed by detection of N(2)-(trans-isoestragol-3'-yl)-2'-deoxyguanosine adducts. Genotoxicity was inhibited by general SULT inhibitors, supporting a role for SULTS in the activation of 1-hydroxyestragole in B-13/H cells. However, B-13 and B-13/H cells did not express biologically significant levels of SULT1A1 as determined by qRT-PCR, Western blotting and its associated 7-hydroxycoumarin sulphation activity. B-13 and B-13/H cells expressed - relative to intact rat liver - high levels of SULT2B1 (primarily the b isoform) and SULT4A1 mRNAs and proteins. B-13 and B-13/H cells also expressed the 3'-phosphoadenosine 5'-phosphosulphate synthase 1 required for the generation of activated sulphate cofactor 3'-phosphoadenosine 5'-phosphosulphate. However, only B-13/H cells expressed functional SULT activities towards SULT2B1 substrates DHEA, pregnenolone and 4 methylumbelliferone. Since liver progenitor cells are bi-potential and also form cholangiocytes, we therefore hypothesised that B-13 cells express a cholangiocyte-like SULT profile. To test this hypothesis, the expression of SULTs was examined in liver by RT-PCR and immunohistochemistry. SULT2B1 - but not SULT1A1 - was determined to be expressed in both rat and human cholangiocytes. Since 1'-hydroxyestragole exposure readily produced DNA injury in B-13/H cells, these data suggest that cholangiocarcinomas generated in rats fed estragole may be dependent, in part, on SULT2B1 activation of the 1'-hydroxyestragole metabolite.
authors.affiliation: Institute Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Probert
authors.affiliation: Institute Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom.
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Palmer
authors.affiliation: Division of Toxicology, Wageningen University, Tuinlaan 5, 6703HE Wageningen, The Netherlands.
authors.forename: Wasma
authors.initials: W
authors.lastname: Alhusainy
authors.affiliation: Institute Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom.
authors.forename: Aimen O
authors.initials: AO
authors.lastname: Amer
authors.affiliation: Division of Toxicology, Wageningen University, Tuinlaan 5, 6703HE Wageningen, The Netherlands.
authors.forename: Ivonne M C M
authors.initials: IM
authors.lastname: Rietjens
authors.affiliation: Institute Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom.
authors.forename: Steven A
authors.initials: SA
authors.lastname: White
authors.affiliation: Institute Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom.
authors.forename: David E
authors.initials: DE
authors.lastname: Jones
authors.affiliation: Institute Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom. Electronic address: M.C.Wright@ncl.ac.uk.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Wright
mesh_terms: D000081248:Allylbenzene Derivatives; D000818:Animals; D000840:Anisoles / Q000008:administration & dosage / Q000633:toxicity*; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002273:Carcinogens / Q000008:administration & dosage / Q000633:toxicity; D002460:Cell Line; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects*; D003687:Dehydroepiandrosterone / Q000494:pharmacology; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000187:drug effects*; D006801:Humans; D006923:Hymecromone / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011284:Pregnenolone / Q000494:pharmacology; D051381:Rats; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D000081248:Allylbenzene Derivatives; D000840:Anisoles; D002273:Carcinogens; D006923:Hymecromone; D003687:Dehydroepiandrosterone; C014037:1'-hydroxyestragole; D011284:Pregnenolone; C007633:estragole; D015238:Sulfotransferases; D015239:Arylsulfotransferase; C117544:SULT1A1 protein, human; C433248:SULT2B1 protein, human
keywords: Comet; Estragole; Genotoxicity; Liver; Sulfotransferase