Reactive oxygen species-related induction of multidrug resistance-associated protein 2 expression in primary hepatocytes exposed to sulforaphane

Expression of multidrug resistance-associated protein 2 (MRP2), an efflux p ump contributing to biliary secretion of xenobiotics, was investigated in p rimary rat and human hepatocytes exposed to sulforaphane, a naturally-occur ring chemopreventive agent. Northern blot indicated that sulforaphane incre ased MRP2 mRNA levels in primary rat hepatocytes; it also induced expressio n of drug metabolizing enzymes such as glutathione S-transferase A1/2 isofo rms and NAD(P)H:quinone oxidoreductase in a dose-response and time-course m anner similar to that observed for the upregulation of MRP2 transcripts. Th is sulforaphane-related increase of MRP2 mRNAs paralleled increased express ion of 190 kD MRP2 protein as assessed by Western blotting; it was fully ab olished by the transcription inhibitor actinomycin D. MRP2 induction was as sociated with increased cellular production of reactive oxygen species (ROS ) and addition of dimethyl sulfoxide, that reduced sulforaphane-related for mation of ROS, and also decreased MRP2 mRNA levels in sulforaphane-treated primary rat hepatocytes; this suggests that sulforaphane-mediated productio n of ROS may contribute to MRP2 induction. This link between ROS and MRP2 r egulation was further supported by the increase of MRP2 expression occurrin g in response to t-butylhydroquinone, known to regulate drug metabolizing e nzymes through ROS formation. In addition to rat cells, primary human hepat ocytes exposed to sulforaphane also displayed induced MRP2 expression evide nced at both mRNA and protein levels. All these observations strongly suppo rt the conclusion that the export pump MRP2 can be classified among the det oxifying proteins that are regulated by sulforaphane and that are thought t o contribute, at least in part, to its anticarcinogenic properties. (C) 200 1 Academic Press.