Beneficial Effect of Spironolactone Administration on Ethynylestradiol-Induced Cholestasis in the Rat: Involvement of Up-Regulation of Multidrug Resistance-Associated Protein 2

Abstract

ABSTRACT:The effect of spironolactone (SL) administration on 17␣-ethynylestradiol (EE)-induced cholestasis was studied, with emphasis on expression and activity of Mrps. Adult male Wistar rats were divided into the following groups: EE (5 mg/kg daily for 5 days, s.c.), SL (200 mol/kg daily for 3 days, i.p.), EE؉SL (same doses, SL administered the last 3 days of EE treatment), and controls. SL prevented the decrease in bile salt-independent fraction of bile flow induced by EE, in association with normalization of biliary excretion of glutathione. Western blot studies indicate that EE decreased the expression of multidrug resistance-associated protein 2 (Mrp2) by 41% and increased that of Mrp3 by 200%, whereas SL only affected Mrp2 expression (؉60%) with respect to controls. The EE؉SL group showed increased levels of Mrp2 and Mrp3 to the same extent as that registered for the individual treatments.Real-time polymerase chain reaction studies indicated that upregulation of Mrp2 and Mrp3 by SL and EE, respectively, was at the transcriptional level. To estimate Mrp2 and Mrp3 activities, apical and basolateral excretion of acetaminophen glucuronide (APAPglu), a common substrate for both transporters, was measured in the recirculating isolated perfused liver model. Biliary/perfusate excretion ratio was decreased in EE (؊88%) and increased in SL (؉36%) with respect to controls. Coadministration of rats with SL partially prevented (؊53%) impairment induced by EE in this ratio. In conclusion, SL administration to EE-induced cholestatic rats counteracted the decrease in bile flow and biliary excretion of glutathione and APAP-glu, a model Mrp substrate, findings associated with up-regulation of Mrp2 expression.

Regulation of Expression and Activity of Rat Intestinal Multidrug Resistance-Associated Protein 2 by Cholestatic Estrogens

“…In view of these results, we further evaluated the possibility of reversing EE effects by coadministration of the therapeutic agent spironolactone (SL). SL, an Mrp2 inducer, has been shown to prevent the impairment in bile flow formation and biliary excretion of glutathione associated with down-regulation of canalicular Mrp2 induced by EE (Ruiz et al, 2007). The data confirmed such a protective effect.…”

“…Bile flow rate and the serum markers of cholestasis alkaline phosphatase (ALP) and bile salts were assessed using commercial kits as described previously (Ruiz et al, 2007). A different set of normal rats was used to test the acute effect of E2-17G on intestinal localization and activity of Mrp2 as described below.…”

“…Basal bile and serum were collected in all of these groups as described (Ruiz et al, 2007) to confirm induction of cholestasis by EE and its prevention by SL. Bile flow rate and the serum markers of cholestasis alkaline phosphatase (ALP) and bile salts were assessed using commercial kits as described previously (Ruiz et al, 2007).…”

“…Real-time quantitative PCR was performed on cDNA samples using the MiniOpticom System (Bio-Rad, Hercules, CA). Sequences of primer pairs and conditions for Mrp2 and 18S were designed to optimally detect the respective mRNAs (Ruiz et al, 2007). Quantification of the target cDNAs in all samples was normalized to 18S ribosomal RNA (Ct target Ϫ Ct 18S ϭ ⌬Ct), and the difference in expression for each target cDNA in the treated groups was referred to the amount in the control group (⌬Ct treated Ϫ ⌬Ct control ϭ ⌬⌬Ct).…”

Estrogen receptor-α mediates human multidrug resistance associated protein 3 induction by 17α-ethynylestradiol. Role of activator protein-1

“…In previous work we demonstrated that 17α-ethynylestradiol (EE), a synthetic estrogen widely used in contraceptive formulations and in estrogen replacement therapies, up-regulates hepatic Mrp3 in rats (12,13). We later demonstrated that induction of Mrp3 by EE in rat liver is independent of cholestasis and occurs via activation of ER (14), though the underlying mechanism downstream ER remains unknown.…”