Consequences of Mrp2 deficiency for diclofenac-induced toxicity in rat intestine in vitro

Diclofenac (DCF), a widely used non-steroidal anti-inflammatory drug (NSAID), is associated with high prevalence of severe intestinal side-effects. The reactive metabolite diclofenac acylglucuronide (DAG) formed in the liver, and transported by bile into the intestine was reported to be involved in the intestinal injury, based on the observation that Mrp2 deficient (TR-) rat intestine was less sensitive to DCF toxicity due to the reduced biliary transport and intestinal exposure to DAG. However, it is not clear what are the direct consequences of Mrp2 deficiency in the intestine itself. Previously we reported that DCF was toxic in the rat intestine in vitro without the presence of liver metabolites. Therefore, using precision cut intestinal slices (PCIS), we compared wild type (WT) and Mrp2 deficient (TR-) rat intestine in vitro, by studying direct toxicity, DCF disposition and intracellular glutathione concentration. PCIS from WT and TR- rats were incubated with a concentration range of DCF. DCF induced similar dose-dependent toxicity and 200 μM DCF caused a significant decrease of ATP in both strains of rats indicating that the intestine from TRrat is not intrinsically less sensitive to DCF toxicity. As glutathione is a substrate of Mrp2, Mrp2 deficiency may influence its accumulation and thereby the DCF induced toxicity. Intestinal GSH level in the TR-rats was significantly lower than in WT rats but did not make the TR- rat intestine more vulnerable. In both strains, hydroxyl DCF as well as DAG were detected as the main intestinal metabolites after 5 hours incubation, less amount was excreted into the medium by PCLS of the TR- rats. The study of DCF disposition is ongoing with ussing chamber. In conclusion, the TR- rat intestine is not intrinsically less sensitive to DCF toxicity and the lower GSH level does not make it more vulnerable. Less intestinal metabolites are excreted by the PCLS of TR- rats, but whether this is due to lower production or lower excretion ability needs to be further validated.