Progenitor-derived hepatocyte-like (B-13/H) cells metabolise 1′-hydroxyestragole to a genotoxic species via a SULT2B1-dependent mechanism

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 1 mM) 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.