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

Philip M Probert,Ivonne M.C.M Rietjens,Wasma Alhusainy,Matthew C Wright,Aimen O Amer,Steven A White,David E Jones,Jeremy M Palmer

doi:10.1016/j.toxlet.2015.12.010

Philip M Probert, Ivonne M.C.M Rietjens + Show 6 more

Open Access

https://doi.org/10.1016/j.toxlet.2015.12.010

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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.

Highlights

Genotoxicity refers to the process by which chemicals or other agents damage DNA, leading to an alteration in DNA structure, information content or segregation (EFSA, 2011)
We demonstrate for the first time that B-13/H cells express functional SULT2B1 activity and that hepatocytes and cholangiocytes express this isoform in both rat and human liver
The data demonstrate that there was little evidence for genotoxicity when cells were exposed to estragole, likely because estragole is extensively metabolised to non-genotoxic metabolites as previously reported (Anthony et al, 1987; Luo and Guenthner, 1994; Luo and Guenthner, 1995; Punt et al, 2008); B-13/H cells express high levels of a number of cytochrome P450s (Marek et al, 2003; Probert et al, 2014a) and as reported the concentrations of 10-hydroxyestragole generated from estragole were low

Summary

Introduction

Genotoxicity refers to the process by which chemicals or other agents damage DNA, leading to an alteration in DNA structure, information content or segregation (EFSA, 2011). For drugs and chemicals knowledge of any potential genotoxic activity is essential in any assessment of safety. A variety of in vitro assays have been developed to screen for potential genotoxic chemicals. Since chemicals are very often indirect genotoxins and require metabolism to display their genotoxic activity, many assays (e.g. Ames tests, micronucleus tests) include the option of addition of liver extracts rich in enzymes that activate indirect genotoxins (e.g. S9 extracts). Hepatocytes provide an opportunity to examine potential chemical genotoxicity in cells that express a complex complement of xenobiotic metabolising enzymes (activating and de-activating) in an intact cell-based model. Primary hepatocytes rapidly dedifferentiate in culture (Wallace et al, 2010a)

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