Role and Regulation of Hepatobiliary ATP-Binding Cassette Transporters during Chemical-Induced Liver Injury.

Severity of drug-induced liver injury (DILI) ranges from mild, asymptomatic, and transient elevations in liver function tests to irreversible liver damage, often needing transplantation. Traditionally, DILI is classified mechanistically as high-frequency intrinsic DILI, commonly dose dependent or DILI that rarely occurs and is idiosyncratic in nature. This latter form is not dose dependent and has a pattern of histopathological manifestation that is not always uniform. Currently, a third type of DILI called indirect hepatotoxicity has been described that is associated with the pharmacological action of the drug. Historically, DILI was primarily linked to drug metabolism events; however, the impact of transporter-mediated rates of drug uptake and excretion has gained greater prominence in DILI research. This review provides a comprehensive view of the major findings from studies examining the contribution of hepatic ATP-binding cassette transporters as key contributors to DILI and how changes in their expression and function influence the development, severity, and overall toxicity outcome. SIGNIFICANCE STATEMENT: Drug-induced liver injury (DILI) continues to be a focal point in drug development research. ATP-binding cassette (ABC) transporters have emerged as important determinants of drug detoxification, disposition, and safety. This review article provides a comprehensive analysis of the literature addressing: (a) the role of hepatic ABC transporters in DILI, (b) the influence of genetic mutations in ABC transporters on DILI, and (c) new areas of research emphasis, such as the influence of the gut microbiota and epigenetic regulation, on ABC transporters.

P-glycoprotein activation by 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5) in rat distal ileum: ex vivo and in vivo studies.

In vitro studies showed that 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5) increases P-glycoprotein (P-gp) expression and activity in Caco-2 cells, preventing xenobiotic toxicity. The present study aimed at investigating TX5 effects on P-gp expression/activity using Wistar Han rats: a) in vivo, evaluating intestinal P-gp activity; b) ex vivo, evaluating P-gp expression in ileum brush border membranes (BBM) and P-gp activity in everted intestinal sacs; c) ex vivo, evaluating P-gp activity in everted intestinal sacs of the distal and proximal ileum. TX5 (30 mg/kg, b.w.), gavage, activated P-gp in vivo, given the significant decrease in the AUC of digoxin (0.25 mg/kg, b.w.). The efflux of rhodamine 123 (300 µM), a P-gp fluorescent substrate, significantly increased in TX5-treated everted sacs from the distal portion of the rat ileum, when P-gp activity was evaluated in the presence of TX5 (20 µM), an effect abolished by the P-gp inhibitor verapamil (100 µM). No increases on P-gp expression or activity were found in TX5-treated BBM of the distal ileum and everted distal sacs, respectively, 24 h after TX5 (10 mg/kg, b.w.) administration. In vivo, no differences were found on digoxin portal concentration between control (digoxin 0.025 mg/kg, b.w., intraduodenal) and TX5-treated (digoxin+TX5 20 µM, intraduodenal) rats. The observed discrepancies in digoxin results can be related to differences in TX5 dose administered and used methodologies. Thus, the results show that TX5 activates P-gp at the distal portion of the rat ileum, and, at the higher dose tested (30 mg/kg, b.w.), seems to modulate in vivo the AUC of P-gp substrates.

Newly Synthesized Oxygenated Xanthones as Potential P-Glycoprotein Activators: In Vitro, Ex Vivo, and In Silico Studies.

P-glycoprotein (P-gp) plays a crucial role in the protection of susceptible organs, by significantly decreasing the absorption/distribution of harmful xenobiotics and, consequently, their toxicity. Therefore, P-gp has been proposed as a potential antidotal pathway, when activated and/or induced. Knowing that xanthones are known to interact with P-gp, the main goal was to study P-gp induction or/and activation by six new oxygenated xanthones (OX 1-6). Furthermore, the potential protection of Caco-2 cells against paraquat cytotoxicity was also assessed. The most promising compound was further tested for its ability to increase P-gp activity ex vivo, using everted intestinal sacs from adult Wistar-Han rats. The oxygenated xanthones interacted with P-gp in vitro, increasing P-gp expression and/or activity 24 h after exposure. Additionally, after a short-incubation period, several xanthones were identified as P-gp activators, as they immediately increased P-gp activity. Moreover, some xanthones decreased PQ cytotoxicity towards Caco-2 cells, an effect prevented under P-gp inhibition. Ex vivo, a significant increase in P-gp activity was observed in the presence of OX6, which was selectively blocked by a model P-gp inhibitor, zosuquidar, confirming the in vitro results. Docking simulations between a validated P-gp model and the tested xanthones predicted these interactions, and these compounds also fitted onto previously described P-gp induction and activation pharmacophores. In conclusion, the in vitro, ex vivo, and in silico results suggest the potential of some of the oxygenated xanthones in the modulation of P-gp, disclosing new perspectives in the therapeutics of intoxications by P-gp substrates.

Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity.

Acetaminophen (APAP) is a well-known analgesic and antipyretic drug. It is considered to be safe when administered within its therapeutic range, but in cases of acute intoxication, hepatotoxicity can occur. APAP overdose is the leading cause of acute liver failure in the northern hemisphere. Historically, studies on APAP toxicity have been focused on liver, with alterations in brain function attributed to secondary effects of acute liver failure. However, in the last decade the pharmacological mechanism of APAP as a cannabinoid system modulator has been documented and some articles have reported "in situ" toxicity by APAP in brain tissue at high doses. Paradoxically, low doses of APAP have been reported to produce the opposite, neuroprotective effects. In this paper we present a comprehensive, up-to-date overview of hepatic toxicity as well as a thorough review of both toxic and beneficial effects of APAP in brain.

The effect of acetaminophen on the expression of BCRP in trophoblast cells impairs the placental barrier to bile acids during maternal cholestasis.

Acetaminophen is used as first-choice drug for pain relief during pregnancy. Here we have investigated the effect of acetaminophen at subtoxic doses on the expression of ABC export pumps in trophoblast cells and its functional repercussion on the placental barrier during maternal cholestasis. The incubation of human choriocarcinoma cells (JAr, JEG-3 and BeWo) with acetaminophen for 48h resulted in no significant changes in the expression and/or activity of MDR1 and MRPs. In contrast, in JEG-3 cells, BCRP mRNA, protein, and transport activity were reduced. In rat placenta, collected at term, acetaminophen administration for the last three days of pregnancy resulted in enhanced mRNA, but not protein, levels of Mrp1 and Bcrp. In fact, a decrease in Bcrp protein was found. Using in situ perfused rat placenta, a reduction in the Bcrp-dependent fetal-to-maternal bile acid transport after treating the dams with acetaminophen was found. Complete biliary obstruction in pregnant rats induced a significant bile acid accumulation in fetal serum and tissues, which was further enhanced when the mothers were treated with acetaminophen. This drug induced increased ROS production in JEG-3 cells and decreased the total glutathione content in rat placenta. Moreover, the NRF2 pathway was activated in JEG-3 cells as shown by an increase in nuclear NRF2 levels and an up-regulation of NRF2 target genes, NQO1 and HMOX-1, which was not observed in rat placenta. In conclusion, acetaminophen induces in placenta oxidative stress and a down-regulation of BCRP/Bcrp, which may impair the placental barrier to bile acids during maternal cholestasis.

The modulation of transcriptional expression and inhibition of multidrug resistance associated protein 4 (MRP4) by analgesics and their primary metabolites.

During the course of a toxic challenge, changes in gene expression can manifest such as induction of metabolizing enzymes as a compensatory detoxification response. We currently report that a single 400 mg/kg acetaminophen (APAP) dose to C57BL/6J mice led to an increase in multidrug resistance-associated (Mrp) 4 (Abcc4) mRNA 12 h after administration. Alanine aminotransferase, as a marker of liver injury, was also elevated indicating hepatotoxicity had occurred. Therefore, induction of Mrp4 mRNA was likely attributable to APAP-induced liver injury. Mrp4 has been shown to be upregulated during oxidative stress, and it is well-established that APAP overdose causes oxidative stress due to depletion of glutathione. Given the importance of Mrp4 upregulation as an adaptive response during cholestatic and oxidative liver injury, we next investigated the extent by which human MRP4 can be inhibited by the analgesics, APAP, diclofenac (DCF), and their metabolites. Using an in vitro assay with inside out human MRP4 vesicles, we determined that APAP-cysteine inhibited MRP4-mediated transport of leukotriene C4 with an apparent IC50 of 125 µM. APAP-glutathione also attenuated MRP4 activity though it achieved only 28% inhibition at 300 µM. Diclofenac acyl glucuronide (DCF-AG) inhibited MRP4 transport by 34% at 300 µM. The MRP4 in vitro inhibition occurs at APAP-cysteine and DCF-AG concentrations seen in vivo after toxic doses of APAP or DCF in mice, hence the findings are important given the role that Mrp4 serves as a compensatory response during oxidative stress following toxic challenge.

Modulation of Hepatic MRP3/ABCC3 by Xenobiotics and Pathophysiological Conditions: Role in Drug Pharmacokinetics.

Liver transporters play an important role in the pharmacokinetics and disposition of pharmaceuticals, environmental contaminants, and endogenous compounds. Among them, the family of ATP-Binding Cassette (ABC) transporters is the most important due to its role in the transport of endo- and xenobiotics. The ABCC sub-family is the largest one, consisting of 13 members that include the cystic fibrosis conductance regulator (CFTR/ABCC7); the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9) and the multidrug resistanceassociated proteins (MRPs). The MRP-related proteins can collectively confer resistance to natural, synthetic drugs and their conjugated metabolites, including platinum-containing compounds, folate anti-metabolites, nucleoside and nucleotide analogs, among others. MRPs can be also catalogued into "long" (MRP1/ABCC1, -2/C2, -3/C3, -6/C6, and -7/C10) and "short" (MRP4/C4, -5/C5, -8/C11, -9/C12, and -10/C13) categories. While MRP2/ABCC2 is expressed in the canalicular pole of hepatocytes, all others are located in the basolateral membrane. In this review, we summarize information from studies examining the changes in expression and regulation of the basolateral hepatic transporter MPR3/ABCC3 by xenobiotics and during various pathophysiological conditions. We also focus, primarily, on the consequences of such changes in the pharmacokinetic, pharmacodynamic and/or toxicity of different drugs of clinical use transported by MRP3.

Acute acetaminophen intoxication induces direct neurotoxicity in rats manifested as astrogliosis and decreased dopaminergic markers in brain areas associated with locomotor regulation.

Acetaminophen (APAP) administration at therapeutic doses is safe, however overdosing produces hepatocellular injury via a multifactorial mechanism(s) that involves generation of reactive oxygen species (ROS), being the most common cause of acute liver failure (ALF) in the northern hemisphere. Brain alterations induced by APAP intoxication are usually considered secondary to hepatic encephalopathy development due to ALF. Although APAP is primarily metabolized in the liver, it is also distributed and metabolized homogeneously in the brain, affecting brain redox status. Nevertheless, comprehensive studies on the potential of APAP intoxication to produce brain toxicity are scarce. The aim of this study was to characterize the direct toxic effects of APAP in different regions of the brain and on behavior in rats where the magnitude of hepatotoxicity produced is not associated with ALF. The present work demonstrates that APAP intoxication producing hepatotoxicity, but not ALF in rats, is associated with marked hypolocomotion. Our studies also suggest that selective downregulation in dopamine levels in brain areas that regulate motor activity may be responsible, in part, for the decreased locomotion observed with APAP treatment. Furthermore, we observed that the brain histoarchitecture is conserved and that edema is not present. However, an increase in oxidative stress, reactive astrogliosis and a decrease in neuron processes are the main features observed in APAP-intoxicated animals. These effects might be partly due to direct toxic effects of APAP in brain, since the same reactive astrogliosis observed in rats was also observed in rat primary astrocyte cultures exposed to APAP.

Hepatic synthesis and urinary elimination of acetaminophen glucuronide are exacerbated in bile duct-ligated rats.

Renal and intestinal disposition of acetaminophen glucuronide (APAP-GLU), a common substrate for multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), was assessed in bile duct-ligated rats (BDL) 7 days after surgery using an in vivo perfused jejunum model with simultaneous urine collection. Doses of 150 mg/kg b.w. (i.v.) or 1 g/kg b.w. (i.p.) of acetaminophen (APAP) were administered, and its glucuronide was determined in bile (only Shams), urine, and intestinal perfusate throughout a 150-min period. Intestinal excretion of APAP-GLU was unchanged or decreased (-58%) by BDL for the 150 mg and 1 g/kg b.w. doses of APAP, respectively. In contrast, renal excretion was increased by 200 and 320%, respectively. Western studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, whereas Mrp3 was substantially increased in liver and not affected in kidney or intestine. The global synthesis of APAP-GLU, determined as the sum of cumulative excretions, was higher in BDL rats (+51 and +110%) for these same doses of APAP as a consequence of a significant increase in functional liver mass, with no changes in specific glucuronidating activity. Expression of apical breast cancer resistance protein, which also transports nontoxic metabolites of APAP, was decreased by BDL in liver and renal cortex, suggesting a minor participation of this route. We demonstrate a more efficient hepatic synthesis and basolateral excretion of APAP-GLU followed by its urinary elimination in BDL group, the latter two processes consistent with up-regulation of liver Mrp3 and renal Mrp2.

Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice.

Changes in expression of liver ABC transporters have been described during acute APAP intoxication. However, the effect of APAP on brain ABC transporters is poorly understood. The aim of this study was to evaluate the effect of APAP on brain ABC transporters expression and the role of the oxidative stress sensor Nrf2. Male C57BL/6J mice were administered APAP (400mg/kg) for analysis of brain mRNA and protein expression of Mrp1-6, Bcrp and P-gp. The results show induction of P-gp, Mrp2 and Mrp4 proteins, with no changes in Bcrp, Mrp1 or Mrp5-6. The protein values were accompanied by corresponding changes in mRNA levels. Additionally, brain Nrf2 nuclear translocation and expression of two Nrf2 target genes, NAD(P)H: quinone oxidoreductase 1 (Nqo1) and Hemoxygenase 1 (Ho-1), was evaluated at 6, 12 and 24h after APAP treatment. Nrf2 nuclear content increased by 58% at 12h after APAP along with significant increments in mRNA and protein expression of Nqo1 and Ho-1. Furthermore, APAP treated Nrf2 knockout mice did not increase mRNA or protein expression of Mrp2 and Mrp4 as observed in wildtypes. In contrast, P-gp induction by APAP was observed in both genotypes. In conclusion, acute APAP intoxication induces protein expression of brain P-gp, Mrp2 and Mrp4. This study also suggests that brain changes in Mrp2 and Mrp4 expression may be due to in situ Nrf2 activation by APAP, while P-gp induction is independent of Nrf2 function. The functional consequences of these changes in brain ABC transporters by APAP deserve further attention.

Differential effects of environment-induced changes in body temperature on modafinil's actions against methamphetamine-induced striatal toxicity in mice.

Methamphetamine (METH) exposure can produce hyperthermia that might lead to toxicity and death. Modafinil is a wake-promoting compound that is also been prescribed off-label to treat METH dependence. Modafinil has shown neuroprotective properties against METH harmful effects in animal models. The goal of the present study was to test if the prevention of hyperthermia might play a role on the neuroprotective actions of modafinil against METH toxicity using various ambient temperatures. METH was administered to female C57BL/6 mice in a binge regimen: 4 × 5 mg/kg, 2 h apart; modafinil (90 mg/kg) was injected twice, 1 h before first and fourth METH injections. Drugs were given at cold ambient temperature (14 °C) or hot ambient temperature (29 °C). Body temperature was measured during treatments. Brains were dissected out 6 days after treatments and processed for tyrosine hydroxylase (TH), dopamine transporter (DAT), GFAP and c-Fos immunohistochemistry. Exposure to hot ambient temperature exacerbated METH toxicity evidenced by striatal reductions in TH and DAT and increased GFAP immmunoreactivity. Modafinil counteracted reductions in TH and DAT, but failed to block astroglial activation. At both ambient temperatures tested modafinil did induce increments in GFAP, but the magnitude was significantly lower than the one induced by METH. Both drugs induced increases in c-Fos positive nuclei; modafinil did not block this effect. Our results suggest that protective effects of modafinil against METH-induced neurotoxicity may be dependent, in part, to its hypothermic effects. Nevertheless, modafinil maintained some protective properties on METH-induced alterations in the striatum at different ambient temperatures.

Effect of acetaminophen on expression and activity of rat liver multidrug resistance-associated protein 2 and P-glycoprotein.

We evaluated the effect of acetaminophen (APAP), given as a single, 1g/kg body weight dose, on expression and activity of rat liver multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp), two major canalicular drug transporters. The studies were performed 24h after administration of the drug. APAP induced an increase in plasma membrane content of Mrp2 detected by western blotting, consistent with increased detection of the protein at the canalicular level by immunoflourescence microscopy. In vivo biliary excretion of dinitrophenyl-S-glutathione, a well known Mrp2 substrate, was slightly but significantly increased by APAP, agreeing well with upregulation of the transporter. Basal biliary excretion of oxidized glutathione, an endogenous Mrp2 substrate, was also increased by APAP, likely indicating increased hepatic synthesis as a result of APAP-induced oxidative stress followed by accelerated canalicular secretion mediated by Mrp2. APAP also increased the expression of P-gp detected by western blotting and immunofluorescence microscopy as well as the in vivo biliary secretory rate of digoxin, a model P-gp substrate. Because specific APAP-conjugated metabolites are Mrp2 substrates, we postulate that induction of Mrp2 by APAP may represent an adaptive mechanism to accelerate liver disposition of the drug. In addition, increased Mrp2-mediated elimination of oxidized glutathione may be essential in maintaining the redox equilibrium in the hepatocyte under conditions of APAP-induced oxidative stress.

Acetaminophen inhibits intestinal p-glycoprotein transport activity.

Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 µM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP.

Acetaminophen-induced stimulation of MDR1 expression and activity in rat intestine and in LS 174T human intestinal cell line.

The well-known analgesic and antipyretic drug N-acetyl-p-aminophenol (acetaminophen; APAP) has been previously reported to affect MDR1 expression in rat liver. In this study, we have investigated the effect of subtoxic doses of APAP on MDR1 expression and activity in rat intestine and human intestinal cells. Administration of APAP at increasing doses of 0.2, 0.3, and 0.6g/kg b.w., i.p. over three consecutive days, induced MDR1 expression in rat duodenum (+240%) and ileum (+160%) as detected by western blotting. This was accompanied by preserved localization of the protein at the surface of the villus, as detected by confocal immunofluorescence microscopy. MDR1 activity was increased by 50% in APAP treated rats, as evaluated by serosal to mucosal secretion of rhodamine 123 in everted intestinal sacs. Treatment with APAP also decreased by 65% the portal vein concentrations of digoxin found in anesthetized rats after intraduodenal administration of this drug, which is consistent with an APAP-induced increased efficacy of intestinal barrier for digoxin net absorption. Exposure of LS 174T human colon adenocarcinoma cells to subtoxic APAP concentration (5mM) induced an increase in MDR1 mRNA expression (+46%), which was accompanied with an enhanced ability (+78%) to reduce intracellular content of rhodamine 123. Taken together these data suggest the existence of APAP-induced stimulation of MDR1 transcription in the intestinal epithelium. These findings are of clinical relevance, as co-administration of APAP with other MDR1 substrates could indirectly inhibit the net intestinal absorption of these drugs, leading to changes in their pharmacokinetics and therapeutic efficacy.

Effect of repeated administration with subtoxic doses of acetaminophen to rats on enterohepatic recirculation of a subsequent toxic dose.

Development of resistance to toxic effects of acetaminophen (APAP) was reported in rodents and humans, though the mechanism is only partially understood. We examined in rats the effect of administration with subtoxic daily doses (0.2, 0.3, and 0.6g/kg, i.p.) of APAP on enterohepatic recirculation and liver toxicity of a subsequent i.p. toxic dose of 1g/kg, given 24h after APAP pre-treatment. APAP and its major metabolite APAP-glucuronide (APAP-Glu) were determined in bile, urine, serum and liver homogenate. APAP pre-treatment was not toxic, as determined by serum markers of liver damage and neither induced oxidative stress as demonstrated by assessment of ROS generation in liver or glutathione species in liver and bile. APAP pre-treatment induced a partial shift from biliary to urinary elimination of APAP-Glu after administration with the toxic dose, and decreased hepatic content and increased serum content of this conjugate, consistent with a marked up-regulation of its basolateral transporter Mrp3 relative to apical Mrp2. Preferential secretion of APAP-glu into blood decreased enterohepatic recirculation of APAP, thus attenuating liver exposition to the intact drug, as demonstrated 6h after administration with the toxic dose. The beneficial effect of interfering the enterohepatic recirculation was alternatively tested in animals receiving activated charcoal by gavage to adsorb APAP of biliary origin. The data indicated decreased liver APAP content and glutathione consumption. We conclude that selective up-regulation of Mrp3 expression by APAP pre-treatment may contribute to development of resistance to APAP hepatotoxicity, at least in part by decreasing its enterohepatic recirculation.

Induction of rat intestinal P-glycoprotein by spironolactone and its effect on absorption of orally administered digoxin.

The effect of the diuretic spironolactone (SL) on expression and function of intestinal P-glycoprotein (P-gp), as well as its impact on intestinal absorption of digoxin, was explored. Rats were treated with daily doses of 200 micromol/kg b.wt. of SL intraperitoneally for 3 consecutive days. The small intestine was divided into four equal segments of approximately 25 cm, with segment I being the most proximal. Brush-border membranes were isolated and used in analysis of P-gp expression by Western blot analysis. P-gp content increased in the SL group by 526, 292, 210, and 622% over controls for segments I, II, III, and IV, respectively. Up-regulation of apical P-gp was confirmed by immunofluorescence microscopy. P-gp transport activity was explored in intestinal sacs prepared from segment IV using two different model substrates. Serosal to mucosal transport (efflux) of rhodamine 123 was 140% higher, and mucosal to serosal transport (absorption) of digoxin was 40% lower in the SL group, both indicating increased P-gp function. In vivo experiments showed that intestinal absorption of a single dose of digoxin administered p.o. was attenuated by SL pretreatment. Thus, concentration of digoxin in portal and peripheral blood was lower in SL versus control groups, as well as its accumulation in kidney and liver. Urinary excretion of digoxin was significantly decreased in the SL group, probably reflecting decreased systemic availability of digoxin for subsequent urinary elimination. We conclude that SL induces P-gp expression with potential impact on intestinal absorption of substrates with therapeutic application.

Shift from biliary to urinary elimination of acetaminophen-glucuronide in acetaminophen-pretreated rats.

Despite its toxicity, acetaminophen (APAP) is used increasingly as an analgesic, antipyretic, and anti-inflammatory agent. We examined the effect of prior exposure to APAP on its biliary and urinary elimination. The biliary and urinary elimination of a test dose of APAP (150 mg/kg i.v.) was determined in male Wistar rats 24 h after pretreatment with vehicle, a single dose (1.0 g/kg i.p.), or increasing daily doses (0.2, 0.3, 0.6, and 1.0 g/kg/day i.p.) of APAP. Although elimination of the parent APAP was minimally affected, biliary excretion of APAP glucuronide was significantly decreased 70 and 80%, whereas urinary excretion was significantly increased 90 and 100% in the groups pretreated with single and repeated doses of APAP, respectively, relative to vehicle controls. Western analysis and confocal immunofluorescent microscopy indicated a marked increase in hepatic expression of multidrug resistance-associated protein 3 (Mrp3) in both groups pretreated with APAP, relative to expression of Mrp2. ATP-dependent transport of [3H]taurocholate, an Mrp3 substrate, was significantly increased in basolateral liver plasma membrane vesicles from rats pretreated with repeated doses of APAP relative to controls. Enterohepatic recirculation of APAP glucuronide after administration of the same test dose of the drug was significantly decreased in rats pretreated with repeated doses of APAP. These data indicate that APAP pretreatment induced a shift from biliary to urinary elimination of APAP glucuronide, consistent with the increased expression of Mrp3 in the basolateral domain of the hepatocyte. We postulate that decreased enterohepatic recirculation contributes to decreased APAP hepatotoxicity by reducing liver exposure.