HEPATIC SYNTHESIS AND URINARY ELIMINATION OF...

DMD #18127

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HEPATIC SYNTHESIS AND URINARY ELIMINATION OF ACETAMINOPHEN

GLUCURONIDE ARE EXACERBATED IN BILE DUCT-LIGATED RATS

Silvina S.M. Villanueva, María L. Ruiz, Carolina I. Ghanem, Marcelo G. Luquita, Viviana A. Catania,

and Aldo D. Mottino.

Instituto de Fisiología Experimental (CONICET) - Facultad de Ciencias Bioquímicas y Farmacéuticas

(UNR). Rosario. ARGENTINA (SSMV, MLR, MGL, VAC, ADM).

Instituto de Farmacología Experimental (CONICET) - Cátedra de Fisiopatología. Facultad de Farmacia y

Bioquímica (UBA). Buenos Aires. ARGENTINA (CIG).

DMD Fast Forward. Published on December 20, 2007 as doi:10.1124/dmd.107.018127

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 20, 2007 as DOI: 10.1124/dmd.107.018127

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Running title: Disposition of acetaminophen glucuronide in BDL rats.

Author for correspondence:

Aldo D. Mottino, PhD Instituto de Fisiología Experimental Facultad de Ciencias Bioquímicas y Farmacéuticas Universidad Nacional de Rosario Suipacha 570. (S2002LRL)-Rosario ARGENTINA TE: 54-341-4305799 FAX: 54-341-4399473 E-mail: [email protected]

Text pages: 16

Tables: 0

Figures: 2

References: 40

Words in Abstract: 253

Words in Introduction: 626

Words in Results and Discussion: 1773

Abbreviations:

APAP - acetaminophen, APAP-GLU - acetaminophen-glucuronide, Bsep - bile salt export pump, BDL -

bile duct ligation, GST - glutathione-S-transferase, GSH – glutathione, Mrp2 and Mrp3 - multidrug

resistance-associated proteins 2 and 3, NAPQI - N-acetyl-p-benzoquinone imine, UGT - UDP-

glucuronosyltransferase.


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ABSTRACT

Renal and intestinal disposition of acetaminophen glucuronide (APAP-GLU), a common substrate for

multidrug resistance-associated proteins 2 and 3 (Mrp2, 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 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 (Bcrp), which also transports non-toxic 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 upregulation of liver Mrp3

and renal Mrp2.


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INTRODUCTION

Biliary elimination of drugs is mainly mediated by members of the ABC (ATP binding cassette) family of

transporters such as multidrug resistance protein 1 (Mdr1, Abcb1), multidrug resistance-associated

protein 2 (Mrp2, Abcc2) and breast cancer resistance protein (Bcrp, Abcg2). Together with hepatic phase

I and phase II biotransformation reactions, they constitute a coordinated system to metabolize and excrete

into bile a wide variety of xenobiotics, including drugs of therapeutic application (Suzuki et al., 2003;

Catania et al., 2004; Aleksunes et al., 2005). Cholestasis, defined as decreased or ceased bile flow, is the

most frequent event related to liver disorders. Bile duct ligation (BDL) is an experimental model of

extrahepatic cholestasis widely used to resemble bile duct obstruction. Expression and function of major

canalicular transporters involved in bile flow formation, the bile salt export pump (Bsep) and Mrp2, are

impaired in BDL in rats (Trauner et al., 1997; Lee et al., 2000; Paulusma et al., 2000). We have

demonstrated recently that BDL rats, at 7 and 14 days post-surgery, present an impairment in the

glutathione-S-transferase (GST)-mediated conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) in vivo,

thus leading to decreased synthesis of the model Mrp2 substrate, dinitrophenyl-S-glutathione (DNP-SG)

(Villanueva et al., 2006). It is not known, however, whether conjugation reactions mediated by another

relevant phase II biotransformation pathway, UDP-glucuronosyltransferase (UGT), as well as subsequent

elimination of the corresponding derivatives via Mrp2 are similarly affected in hepatic vs. extrahepatic

tissues under BDL conditions.

Acetaminophen (APAP) is one of the analgesics most frequently used. At therapeutic doses, the

drug undergoes hepatic glucuronidation and to a lesser extent, sulphation (Thomas et al., 1993). Only a

small fraction of APAP results in the formation of the cytotoxic metabolite, N-acetyl-p-benzoquinone

imine (NAPQI), which reacts with cellular glutathione (GSH). Efficient glucuronic acid conjugation of

APAP is in consequence a crucial step in prevention of APAP toxicity. A previous report suggests that

biliary excretion of APAP glucuronide (APAP-GLU) is mediated by Mrp2 (Xiong et al., 2000).

Additionally, this metabolite can be excreted into blood through multidrug resistance-associated protein 3

(Mrp3, Abcc3), a basolateral Mrp family member, suggested to present a higher affinity for APAP-GLU

than Mrp2 (Xiong et al., 2000; Manautou et al., 2005). Mrp3 expression is low in human and rat liver

under normal conditions (König et al., 1999; Soroka et al., 2001). However, the expression of apical Mrp2

is down-regulated and, in contrast, that of sinusoidal Mrp3 is up-regulated in BDL rats (Trauner et al.,

1997; Paulusma et al., 2000; Donner et al., 2001; Soroka et al., 2001; Dietrich et al., 2004). More

importantly, biliary secretory function is severely affected in this model of obstructive cholestasis. Thus, a

vectorial change in the excretion of APAP-GLU from apical to basolateral transport is expected, causing

this metabolite to be excreted into blood instead of into bile. Interestingly, APAP liver toxicity was

attenuated in BDL rats, as judged by assessment of serum markers of liver damage and histology


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(Acevedo et al., 1995), thought the mechanism remains unknown. In view of the impossibility of biliary

excretion of APAP and its metabolites in this model of cholestasis, extrahepatic tissues could compensate

for impaired liver function. Whether derivation of non-toxic metabolites of APAP or APAP itself to

extrahepatic tissues, additionally contributes to explain higher tolerance to APAP toxicity at the liver, was

not explored. We here evaluated liver, renal and intestinal formation and intestinal and urinary

elimination of APAP-GLU under conditions of BDL in rats, either in response to i.v. administration of a

subtoxic test dose (150 mg/Kg b.w) or after i.p. administration of a toxic dose (1 g/Kg b.w.) of APAP.

The results indicate that irrespective of the dose of APAP, BDL rats produced more APAP-GLU than

controls, with a significant elimination through urine and a minor contribution of the intestine.

MATERIALS AND METHODS

Chemicals. Leupeptin, phenylmethylsulfonyl fluoride, pepstatin A, sucrose, APAP, APAP-GLU, UDP-

glucuronic acid (UDPGA), D-saccharic acid 1,4-lactone, Triton X-100, UDP-N-acetylglucosamine (UDP-

N-AG) and palmitoyl-lysophosphatidylcholine (PLPC) were obtained from Sigma Chemical Co. (St.

Louis, MO). All other chemicals and reagents were commercial products of analytical grade purity.

Animals and surgical procedures. Adult male Wistar rats weighing 300 to 350 g (National University of

Rosario, Argentina) were used. Animals had free access to food and water and received human care as

outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. BDL

was performed as described previously (Gartung et al., 1996) under ether anesthesia. Experiments were

performed 7 days after surgery. Controls underwent a sham operation that consisted of exposure, but no

ligation, of the common bile duct, and were studied 7 days later.

In vivo synthesis and excretion of APAP-GLU. Studies were performed using the in situ single-pass

intestinal perfusion technique (Gotoh et al., 2000) with simultaneous urine collection (Villanueva et al.,

2005). To evaluate the synthesis and disposition of APAP-GLU in response to administration with a

subtoxic dose of APAP, the bile duct (only in Shams), small intestine and urinary bladder were

cannulated under urethane anesthesia, as described (Villanueva et al., 2005). After a 30-min stabilization

period, a single dose of 150 mg/Kg b.w. of APAP was administered i.v. as described (Ghanem et al.,

2005). Bile, intestinal perfusate and urine were collected for 150 min at 10-, 15- and 30-min intervals,

respectively. Biliary, intestinal and urinary flows were determined gravimetrically. Additionally, a blood

sample was taken from the tail vein 5 min after administration of APAP. APAP-GLU content was

assessed in bile, intestinal perfusate, urine and serum by high-performance liquid chromatography

(HPLC) as described previously (Howie et al., 1997), with minor modifications (Ghanem et al., 2005).


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To evaluate the synthesis and disposition of APAP-GLU in response to administration with a

toxic dose of APAP, the animals were injected i.p. with 1 g/Kg b.w. of the drug. After 45 min, they were

anesthetized with urethane and the bile duct (only in Shams), small intestine and urinary bladder were

cannulated. One h after APAP was given; bile, intestinal perfusate and urine were collected for 150 min.

At the end of this period, the animals were sacrificed by exsanguination. APAP-GLU content was

assessed in all the samples by HPLC. Whereas the experiments on i.v. administration of the test dose of

APAP were performed to study in detail its glucuronidation and subsequent elimination of APAP-GLU

under non-saturating conditions (Ghanem et al., 2005), administration of the 1g/Kg dose, in the way of an

i.p. single injection, was used to study APAP-GLU synthesis and disposition under toxic conditions.

Because of the potential role of the kidney in elimination of APAP and its major metabolite

APAP-GLU in experimental obstructive cholestasis, it was also of interest to evaluate urinary excretion of

intact APAP as well as its tissue content and that of APAP-GLU in renal cortex. The tissue content was

evaluated at the end of the 150 min urinary collection period in rats receiving 150 mg/Kg b.w. of APAP.

Homogenate preparation and subsequent HPLC analysis were performed as described (Ruiz et al., 2006).

Western blotting studies. Because of the different localizations of Mrp2 and Bcrp with respect to Mrp3

(apical vs. basolateral), and to perform a comparative analysis of their expression in response to BDL, we

prepared homogenates from liver, jejunum and renal cortex, which were solubilized with Triton X-100 as

described (Cao et al., 2002). The final preparations were used immediately in western blot studies using a

monoclonal antibody to human MRP2 (M2 III-6, Alexis Biochemicals, Carlsbad, CA), a rabbit polyclonal

antibody to mouse Bcrp (M-70, Santa Cruz Biotechnology, Santa Cruz, CA) and a rabbit polyclonal

antibody to rat Mrp3 (Ogawa et al., 2000), respectively. Expression of UGT1A6, demonstrated to be a

major isoform involved in APAP glucuronidation (Kessler et al., 2002), was evaluated by western

blotting of liver, jejunum and renal cortex microsomal preparations (Catania et al., 1998) using a specific

rabbit polyclonal anti-rat antibody (Ikushiro et al., 1995). The immunoreactive bands were quantified

using the Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD) software.

UGT activity. Microsomal APAP glucuronidating activity was measured as described previously

(Kessler et al., 2002), except that PLPC (0.15 mg/mg protein) was used to fully activate the microsomal

suspension and D-saccharic acid 1,4-lactone (2 mM) was routinely included in the incubation media to

inhibit enzymatic hydrolysis of APAP-GLU. Hepatic UGT activity was alternatively assessed in the

presence of UDP-N-AG, the physiologic activator of UGTs, instead of PLPC. UDP-N-AG was

incorporated to the incubation mixtures at a 2 mM final concentration. The APAP-GLU formed was

detected by HPLC.


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Statistical Analysis. Data are presented as mean ± SD. Statistical analysis was performed using the

Student’s t test. Values of p < 0.05 were considered to be statistically significant.

RESULTS AND DISCUSSION

BDL induced a significant increase in the liver weight to body weight ratio by 68% over Shams (0.052 ±

0.004 vs. 0.031 ± 0.003 for BDL and Sham rats, respectively, p < 0.05, N = 3) and in the mass of both

kidneys relative to body mass (+33%, 0.008 ± 0.001 vs. 0.006 ± 0.001, p < 0.05, N = 3). In contrast, the

relative mass of the fragment of small intestine perfused in vivo for transport studies was not affected by

BDL (0.013 ± 0.003 vs. 0.015 ± 0.002, N = 3). This portion of the small intestine (~50 cm long)

corresponds mainly to jejunum, where the highest expression and activity of Mrp2 were reported (Gotoh

et al., 2000; Mottino et al., 2000), whereas expression of Mrp3 and Bcrp were the lowest (Rost et al.,

2002; Tanaka et al., 2005). Excretion rate of APAP-GLU by these three organs is shown in Fig 1. Biliary

elimination of APAP-GLU after administration of a subtoxic, test dose, of APAP (150 mg/Kg b.w.) to

Sham rats showed its maximum value at 40 min, and thereafter decreased with time (Fig 1A). Although

statistically significant differences in intestinal excretion of APAP-GLU were observed in some periods

in response to BDL (Fig 1B), these differences had little impact on the cumulative measure (inset), which

did not differ between groups. In contrast, urinary excretion of APAP-GLU was increased significantly by

BDL from 60 min onwards (Fig 1C). Cumulative renal excretion of the glucuronide was increased by

200% in BDL rats respect to Shams (inset in Fig 1C). Total excretion of APAP-GLU was also calculated

for each organ and expressed as percentage of the dose of APAP. The data are shown in Fig 1D (left).

Under normal conditions, biliary and urinary excretion of APAP-GLU accounted for elimination of 13

and 9% of the administered APAP, respectively, with a minor contribution for the intestinal excretion (~

1%). The data also indicate that normal contribution of biliary excretion of APAP-GLU, absent in BDL

rats, was overrode by its renal excretion, which exhibited an increase of 264% with respect to Shams.

Intestinal elimination was not affected in BDL group. The total amount of APAP-GLU eliminated by

hepatic and extrahepatic tissues after administration of the test dose was significantly increased (+51%) in

BDL group, as seen in the same figure, clearly indicating exacerbated production of the glucuronide

derivative in this group when compared to Shams. We additionally evaluated serum APAP-GLU levels 5

min after administration with this same dose of APAP. The data indicate higher values for BDL rats

(1194 ± 247 µM) than for Shams (496 ± 64 µM), p < 0.05, N = 3, suggesting more efficient basolateral

secretion into blood. Urinary excretion of intact APAP was also increased in BDL rats (18.1 ± 0.3% of

the dose) when compared to Shams (11.9 ± 2.4% of the dose), p < 0.05, N = 3, likely reflecting

impossibility of excretion through bile. Renal tissue content of APAP-GLU by the end of the experiment

was higher in BDL (3.4 ± 0.2% of the dose) vs. Sham (2.3 ± 0.2% of the dose) rats, p < 0.01, N = 3, as it


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is the content of intact APAP (1.3 ± 0.1% and 0.9 ± 0.1% of the dose for BDL and Sham rats,

respectively, p < 0.05, N = 3). These data are consistent with increased exposition of the kidney to APAP

and its glucuronide, as a consequence of a failure in their biliary elimination.

Cumulative elimination of APAP-GLU in response to administration of the 1 g/Kg b.w. dose of

APAP is shown in Fig 1D (right) as percentage of the total dose of APAP. Intestinal contribution in

Shams was minor, as already seen for the test dose, and was further decreased by BDL surgery (-55%). In

contrast, a substantial increase in APAP-GLU urinary excretion was observed in BDL rats when

compared to Shams (+397%), which again, overrode normal contribution of biliary elimination. Fig 1D

shows that the amount of APAP-GLU excreted by all tissues, was exacerbated in BDL rats when

compared to Shams (+110%), clearly indicating overproduction of the glucuronide derivative, as reported

above for the test dose.

APAP-GLU is a model substrate for both Mrp2 and Mrp3 (Xiong et al., 2000; Manautou et al.,

2005). Though Mrp2 has been considered a crucial step in liver elimination of endogenous and exogenous

glucuronides, more recently, Mrp3 was found to play also a significant role, as clearly demonstrated using

the Mrp3 null mice model (Belinsky et al., 2005; Manautou et al., 2005; Zelcer et al., 2006). To establish

a potential association between disposition of this metabolite and its plasma membrane transport in the

three tissues, we estimated the expression of Mrp2 and Mrp3 by western blotting. Fig 2A shows a

significant decrease in the content of hepatic and intestinal Mrp2 protein in BDL rats by 42% and 47%,

respectively, with respect to controls, whereas it was increased by 57% in renal cortex. These data on

tissue-dependent regulation of this transporter under conditions of obstructive cholestasis agree well with

previous reports (Trauner et al., 1997; Paulusma et al., 2000; Lee et al., 2001; Tanaka et al., 2002;

Dietrich et al., 2004; Villanueva et al., 2006). BDL treatment also resulted in a marked induction of

hepatic Mrp3 (+202%), in agreement with previous reports (Donner et al., 2001; Soroka et al., 2001). The

current western blot studies further demonstrate that expression of Mrp3 in jejunum and renal cortex was

not affected by BDL surgery (Fig 2A). Because of its high affinity for APAP-GLU, the remarkable

induction of hepatic Mrp3 could have explained an increase in blood accumulation of this metabolite

under BDL conditions, even in absence of increased glucuronidation capacity. Up-regulation of renal

Mrp2 may account for subsequent tubular secretion and thereby urinary elimination of this metabolite,

which was found to be significantly increased in response to administration of either a test or a toxic dose

of APAP. It should be considered, however, the possibility of a proportion of this metabolite to be

eliminated in urine by glomerular filtration. This needs further examination. In contrast to renal findings,

contribution of jejunum to elimination of APAP-GLU was of much less relevance. Moreover, decreased

expression of Mrp2 detected in intestine from BDL animals correlates well with impaired excretion of


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APAP-GLU, as detected in vivo under saturating conditions resulting from administration with the 1 g/Kg

b.w. dose of APAP.

Bcrp is involved in apical transport of sulfate conjugate of several drugs, whereas glucuronides

are transported to a lesser extent (Suzuki et al., 2003; Zamek-Gliszczynski et al., 2005; Zamek-

Gliszczynski et al., 2006). Because some overlap exists in substrate specificity between Bcrp and Mrps, it

was also of interest to explore the effect of BDL on expression of Bcrp in homogenate from the different

tissues. The data in panel A from Fig 2 clearly show downregulation in liver and renal cortex. Intestinal

content was below the limits of detection with the current methodology. Decreased levels of Bcrp in

kidney strongly suggests a minor role for this pathway as a compensatory mechanism to eliminate the

non-toxic metabolites of APAP, APAP-GLU and APAP sulfate, under conditions of experimental

obstructive cholestasis.

Because of the findings on increased production of APAP-GLU in vivo, it was of interest to

explore the origin of overproduction of this metabolite. UGT activity towards APAP was first examined

in vitro in fully activated microsomes. The data (nmol/min/mg protein) indicate that neither liver (4.1 ±

0.4 vs. 4.9 ± 0.9, N = 3), nor jejunum (1.1 ± 0.3 vs. 1.4 ± 0.3, N = 3) or renal cortex (2.5 ± 0.2 vs. 2.1 ±

0.1, N = 3) exhibited any change in UGT activity in BDL when compared to Sham rats. These data

correlate well with the data on detection of a major UGT isoform involved in APAP conjugation by

western blotting. Fig 2B shows that only intestinal UGT1A6 was affected by BDL, with an increase of

27% over Shams. This likely had no consequences on APAP glucuronidating activity as reported above.

Because UGT activity detected in fully activated microsomes likely correlates with the number of

catalytic units or enzyme molecules (and thus with western blot studies) but not necessarily with the in

situ activity as conditioned by the lipid environment, we performed an additional assay using liver

microsomes in the presence of the physiologic activator UDP-N-AG, which does not produce membrane

perturbation (Zakim et al., 1977; Hauser et al., 1988). The study indicates that UGT activity did not differ

between BDL (1.5 ± 0.1 nmol/min/mg protein) and Sham (1.7 ± 0.8 nmol/min/mg protein) rats (N = 3).

Microsomal protein yield per liver mass unit was neither affected (36 ± 5 and 41 ± 6 mg per g of liver

weight in DBL vs. Sham rats, respectively). In consequence, UGT activity per liver mass unit is also

expected to be preserved in BDL rats. Because a net increase of the liver weight was observed in BDL vs.

Sham rats (16.5 ± 1.3 vs. 10.4 ± 0.3 g, p < 0.01, N = 3), it is likely that the higher amount of APAP-GLU

produced in cholestatic rats and expressed as percentage of the dose of administered APAP, resulted from

a net increase in functional liver mass. The possibility that the renal cortex or the intestine also

contributed to production of the excess of APAP-GLU in BDL group is less likely, since their UGT

activities were lower than that of the liver, and the increase in kidney mass in response to BDL was less

relevant when compared with the liver one.


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Liver glutathione conjugation of CDNB was severely impaired in BDL rats, whereas renal cortex

constituted the main place for its metabolism and subsequent elimination through urine (Villanueva et al.,

2006). It is known that Mrp3 has preference for glucuronides rather than for glutathione conjugates as

substrates (Hirohashi et al., 1999; Zelcer et al., 2001). Taken together, the evidence clearly demonstrates

tissue-specific differences in conjugation of different phase II substrates, as well as in re-distribution and

disposition of the corresponding conjugates, in this model of obstructive cholestasis.

In summary, we demonstrate that cholestasis by BDL led to a marked increase in liver synthesis

of APAP-GLU followed by its efficient urinary excretion, whereas intestinal elimination played a minor

role. This was consistent with upregulation of basolateral Mrp3 in liver and apical Mrp2 in renal cortex.

This may contribute to explain reduced sensitivity of BDL rats to APAP toxicity, as previously reported

(Acevedo et al., 1995). In contrast, dowregulation of renal Bcrp would indicate a minor role for this

protein in exacerbated urinary elimination of non-toxic metabolites of APAP.

ACKNOWLEDGEMENTS

We want to express our gratitude to Dr. J. Elena Ochoa for her valuable technical assistance. We also

thank Drs. Y. Sugiyama and H. Suzuki (University of Tokyo, Japan) for kindly providing the anti-rat

Mrp3 antibody and Dr. S. Ikushiro for kindly providing the anti-UGT1A6 antibody.


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REFERENCES

Acevedo C, Bengochea L, Tchercansky DM, Ouviña G, Perazzo JC, Lago N, Lemberg A, and Rubio MC (1995)

Cholestasis as a liver protective factor in paracetamol acute overdose. Gen Pharmacol 26:1619-24.

Aleksunes LM, Slitt AM, Cherrington NJ, Thibodeau MS, Klaassen CD, and Manautou JE (2005) Differential

expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride. Toxicol Sci

3:44-52.

Belinsky MG, Dawson PA, Shchaveleva I, Bain LJ, Wang R, Ling V, Chen ZS, Grinberg A, Westphal H, Klein-

Szanto A, Lerro A, and Kruh GD. (2005) Analysis of the in vivo functions of Mrp3. Mol Pharmacol 68:160-168.

Cao J, Stieger B, Meier PJ, and Vore M (2002) Expression of rat hepatic multidrug resistance-associated proteins

and organic anion transporters in pregnancy. Am J Physiol Gastrointest Liver Physiol 283:757-766.

Catania VA, Luquita MG, Sánchez Pozzi EJ, and Mottino AD (1998) Enhancement of intestinal UDP-

glucuronosyltranferase activity in partially hepatectomized rats. Biochim Biophys Acta 1380:345-353.

Catania VA, Sánchez Pozzi EJ, Luquita MG, Ruiz ML, Villanueva SSM, Jones B, and Mottino AD (2004) Co-

regulation of expression of phase II metabolizing enzymes and multidrug resistance-associated protein 2. Ann

Hepatol 3:11–17.

Dietrich C, Geier A, Salein N, Lammert F, Roeb E, Oude Elferink RP, Matern S, and Gartung C (2004)

Consequences of bile duct obstruction on intestinal expression and function of multidrug resistance-associated

protein 2. Gastroenterology 126:1044–1053.

Donner MG, and Keppler D (2001) Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic

rat liver. Hepatology 34:351–359.

Gartung C, Ananthanarayanan M, Rahman MA, Schuele S, Nundy S, Soroka CJ, Stolz A, Suchy FL, and Boyer JL

(1996) Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic

cholestasis. Gastroenterology 110:199–209.

Ghanem CI, Ruiz ML, Villanueva SS, Luquita MG, Catania VA, Jones B, Bengochea LA, Vore M, and Mottino AD

(2005) Shift from biliary to urinary elimination of acetaminophen-glucuronide in acetaminophen-pretreated rats. J

Pharmacol Exp Ther 315:987-995.

Goon D, and Klaassen CD (1990) Dose-dependent intestinal glucuronidation and sulfation of acetaminophen in the

rat in situ. J Pharmacol Exp Ther 252:201-207.

Gotoh Y, Suzuki H, Kinoshita S, Hirohashi T, Kato Y, and Sugiyama Y (2000) Involvement of an organic anion

transporter (canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2 in

gastrointestinal secretion of glutathione conjugates in rats. J Pharmacol Exp Ther 292:433–439.


at ASPE

T Journals on A

ugust 19, 2019dm


ownloaded from


DMD #18127

12

Hauser SC, Ransil BJ, Ziurys JC, and Gollan JL (1988) Interaction of uridine 5'-diphosphoglucuronic acid with

microsomal UDP-glucuronosyltransferase in primate liver: the facilitating role of uridine 5'-diphospho-N-

acetylglucosamine. Biochim Biophys Acta 967:141-148.

Hirohashi T, Suzuki H, and Sugiyama Y (1999) Characterization of the transport properties of cloned rat multidrug

resistance-associated protein 3 (Mrp3). J Biol Chem 274:15181-15185

Howie D, Adriaenssens PI, and Prescott LF (1997) Paracetamol metabolism following overdosage: application of

high performance liquid chromatography. J Pharm Pharmacol 9:235–237.

Ikushiro S, Emi Y, and Iyanagi T (1995) Identification and analysis of drug responsive expression of UDP-

glucuronosyltransferase family 1 (UGT1) isozyme in rat hepatic microsomes using anti-peptide antibodies. Arch

Biochem Biophys 324:267-272.

Kessler FK, Kessler MR, Auyeung DJ, and Ritter JK (2002) Glucuronidation of acetaminophen catalyzed by

multiple rat phenol UDP-glucuronosyltransferases. Drug Metab Dispos 30:324-330.

König J, Rost D, Cui Y, and Keppler D (1999) Characterization of the human multidrug resistance protein isoform

MRP3 localized to the basolateral hepatocyte membrane. Hepatology 29:1156-1163.

Lee JM, Trauner M, Soroka CJ, Stieger B, Meier PJ, and Boyer JL (2000) Expression of the bile salt export pump is

maintained after chronic cholestasis in the rat. Gastroenterology 118:163-172.

Lee J, Azzaroli F, Wang L, Soroka CJ, Gigliozzi A, Setchell KDR, Kramer W, and Boyer JL (2001) Adaptive

regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology

121:1473–1484.

Manautou JE, de Waart DR, Kunne C, Zelcer N, Goedken M, Borst P, and Elferink O (2005) Altered disposition of

acetaminophen in mice with a disruption of the Mrp3 gene. Hepatology 42:1091-1098.

Mottino AD, Hoffman T, Jennes L, and Vore M (2000) Expression and localization of multidrug resistant protein

mrp2 in rat small intestine. J Pharm Exp Ther 293:717–723.

Ogawa K, Suzuki H, Hirohashi T, Ishikawa T, Meier PJ, Hirose K, Akizawa T, Yoshioka M, and Sugiyama Y

(2000) Characterization of inducible nature of MRP3 in rat liver. Am J Physiol Gastrointest Liver Physiol 278:438–

446.

Paulusma CC, Kothe MJ, Bakker CT, Bosma PJ, van Bokhoven I, van Marle J, Bolder U, Tytgat GN, and Oude

Elferink RP (2000) Zonal down-regulation and redistribution of the multidrug resistance protein 2 during bile duct

ligation in rat liver. Hepatology 31:684-693.

Rost D, Mahner S, Sugiyama Y, and Stremmel W (2002) Expression and localization of the multidrug resistance-

associated protein 3 in rat small and large intestine. Am J Physiol Gastrointest Liver Physiol 282:720-726.

Ruiz ML, Villanueva SS, Luquita MG, Vore M, Mottino AD, and Catania VA (2006) Ethynylestradiol increases

expression and activity of rat liver MRP3. Drug Metab Dispos 34:1030-1034.


at ASPE

T Journals on A

ugust 19, 2019dm


ownloaded from


DMD #18127

13

Soroka CJ, Lee JM, Azzaroli F, and Boyer JL (2001) Cellular localization and up-regulation of multidrug resistance-

associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver. Hepatology

33:783–791.

Suzuki M, Suzuki H, Sugimoto Y, and Sugiyama Y (2003) ABCG2 transports sulfated conjugates of steroids and

xenobiotics. J Biol Chem 278:22644-22649.

Tanaka Y, Kobayashi Y, Garbazza E, Higuchi K, Kamisako T, Kuroda M, Takeuchi K, Iwasa K, Kaito M, and

Adachi Y (2002) Increased renal expression of bilirubin glucuronide transporters in a rat model of obstructive

jaundice. Am J Physiol Gastrointest Liver Physiol 282:656–662.

Tanaka Y, Slitt AL, Leazer TM, Maher JM, and Klaassen CD (2005) Tissue distribution and hormonal regulation of

the breast cancer resistance protein (Bcrp/Abcg2) in rats and mice. Biochem Biophys Res Commun 326:181-187.

Thomas SH (1993) Paracetamol (acetaminophen) poisoning. Pharmacol Ther 60:91–120.

Trauner M, Arrese M, Soroka CJ, Ananthanarayanan M, Koeppel TA, Schlosser SF, Suchy FJ, Keppler D, and

Boyer JL (1997) The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive

cholestasis. Gastroenterology 113:255-264.

Villanueva SS, Ruiz ML, Luquita MG, Sánchez Pozzi EJ, Catania VA, and Mottino AD (2005) Involvement of

Mrp2 in hepatic and intestinal disposition of dinitrophenyl-S-glutathione in partially hepatectomized rats. Toxicol

Sci 84:4-11.

Villanueva SS, Ruiz ML, Soroka CJ, Cai SY, Luquita MG, Torres AM, Sánchez Pozzi EJ, Pellegrino JM, Boyer JL,

Catania VA, and Mottino AD (2006) Hepatic and extrahepatic synthesis and disposition of dinitrophenyl-S-

glutathione in bile duct-ligated rats. Drug Metab Dispos 34:1301-1309.

Xiong H, Turner KC, Ward ES, Jansen PL, and Brouwer KL (2000) Altered hepatobiliary disposition of

acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(-

) rats. J Pharmacol Exp Ther 295:512–518.

Zakim D, and Vessey DA (1977) Regulation of microsomal UDP-glucoronyltransferase. Mechanism of activation

by UDP-N-acetylglucosamine. Biochem Pharmacol 26:129-131.

Zamek-Gliszczynski MJ, Hoffmaster KA, Tian X, Zhao R, Polli JW, Humphreys JE, Webster LO, Bridges AS,

Kalvass JC, and Brouwer KL (2005) Multiple mechanisms are involved in the biliary excretion of acetaminophen

sulfate in the rat: role of Mrp2 and Bcrp1. Drug Metab Dispos 33:1158-1165.

Zamek-Gliszczynski MJ, Nezasa K, Tian X, Kalvass JC, Patel NJ, Raub TJ, and Brouwer KL (2006) The important

role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-

methylumbelliferone, and harmol in mice. Mol Pharmacol 70:127-133.

Zelcer N, Saeki T, Reid G, Beijnen JH, and Borst P (2001) Characterization of drug transport by the human

multidrug resistance protein 3 (ABCC3). J Biol Chem 276:46400-46407.


at ASPE

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Zelcer N, van de Wetering K, de Waart R, Scheffer GL, Marschall HU, Wielinga PR, Kuil A, Kunne C, Smith A,

van der Valk M, Wijnholds J, Elferink RO, and Borst P (2006) Mice lacking Mrp3 (Abcc3) have normal bile salt

transport, but altered hepatic transport of endogenous glucuronides. J Hepatol 44:768-775.


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Footnotes:

This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica,

Consejo Nacional de Investigaciones Científicas y Técnicas, and Universidad Nacional de Rosario,

Argentina.


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FIGURE LEGENDS

Fig 1. Biliary, intestinal and urinary excretion of APAP-GLU.

Excretion of APAP-GLU, a common substrate for both Mrp2 and Mrp3, in bile (A), intestinal perfusate

(B), and urine (C) was evaluated at 10, 15 or 30 min periods, respectively, after administration of a 150

mg/Kg b.w. dose of APAP. Insets depict cumulative excretion of APAP-GLU by 150 min. The total

amount of APAP-GLU excreted by the whole organs, expressed as percentage of the amount of APAP

administered, is depicted in Fig 1D for the dose of 150 mg/Kg b.w. (left) or for the dose of 1 g/Kg b.w.

(right). Data are means ± SD of 3 animals per group.

* significantly different from Sham group (p < 0.05).

** significantly different from Sham group (p < 0.01).

Fig 2. Expression of Mrp2, Mrp3, Bcrp (panel A) and UGT1A6 (panel B) in liver, intestine and

kidney.

Twenty µg of protein from hepatic homogenates and 40 µg from jejunum and renal cortex homogenates

were loaded in the gels for simultaneous detection of Mrp2 and Mrp3. 60 µg of protein from liver and

renal cortex homogenates were loaded in the gels for detection of Bcrp. Fifteen µg of protein from hepatic

and renal cortex microsomes and 30 µg from jejunum microsomes were loaded in the gels for detection of

UGT1A6. These amounts of protein gave a densitometric signal in the linear range of the response curve

for the different antibodies. Uniformity of loading and transfer from gel to nitrocellulose membrane was

controlled with Ponceau S. Data on densitometry are means ± SD of 3 animals per group. Bcrp was not

detected in jejunum in spite that 100 µg of protein were loaded in the gel.

* significantly different from Sham group (p < 0.05).


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