Many phase I and II microsomal enzyme inducers share common mechanisms of transcriptional activation and thus share a similar battery of genes that are coordinately regulated. Many phase II metabolites are thought to be transported out of cells by multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3). The purpose of this study was to determine the organ distribution of these three transporters in rat, and whether they are coordinately regulated with phase I and II drug-metabolizing enzymes. Therefore, Mrp1, 2, and 3 mRNAs were quantified using branched DNA signal amplification in multiple tissues and in tissues from rats that were treated with 18 chemicals thought to induce drug-metabolizing enzymes by six different transcription activation mechanisms [aryl-hydrocarbon receptor ligands, constitutive androstane receptor (CAR) activators, pregnane-X-receptor ligands, peroxisome proliferator activator receptor ligands, electrophile response element (EpRE) activators, and CYP2E1 inducers]. It was found that Mrp1 was expressed at a high level in kidney, lung, intestine, and brain, with low expression in liver. Mrp2 was highly expressed in liver and duodenum, and Mrp3 was highly expressed throughout the intestine but very low in liver. Microsomal enzyme inducers did not markedly increase the expression of Mrp1 or Mrp2. However, Mrp3 expression was significantly increased by each of the CAR activators and an EpRE activator in liver. Mrp3 was not similarly induced in kidney and large intestine, demonstrating that the coordinate inducibility of Mrp3 is specific to the liver. We conclude that rat hepatic Mrp3 is induced by CAR activators, thus enhancing the vectoral excretion of some phase II metabolites from the liver to the blood.
Members of the multidrug resistance protein (Mrp) family of xenobiotic transporters are important in the ATP-dependent transport of many organic anions including many phase II metabolites (Jedlitschky et al., 1997; Ito et al., 1998; Evers et al., 1998; van Aubel et al., 1998; Cui et al., 1999; Kamisako et al., 1999; Kawabe et al., 1999). Mrp1, 2, and 3 have all been shown to be conjugate export pumps and confer resistance to cytotoxic drugs (Stockel et al., 2000). Mrp1 and 3 are located on the basolateral membrane of polarized cells (Konig et al., 1999; Kool et al., 1999), whereas Mrp2 is localized to the apical membrane (canalicular in liver), which implies that in liver, Mrp2-mediated transport leads to increased excretion into bile, but Mrp1- and Mrp3-mediated transport into blood leads to increased excretion into urine.
Many structurally diverse chemicals have been shown to induce a variety of both phase I and phase II drug-metabolizing enzymes. These chemicals, termed microsomal enzyme inducers, can individually act through a common mechanism of transcriptional activation. Xenobiotics that activate the same transcriptional mechanism show a similar battery of coordinately regulated genes. A number of these mechanisms have been shown to regulate the expression of phase I and phase II genes that facilitate the metabolism and conjugation of both endogenous and exogenous compounds. These mechanisms include aryl-hydrocarbon receptor-mediated induction of CYP1A1, constitutive androstane receptor (CAR)-mediated induction of the CYP2B family, pregnane-X-receptor-mediated induction of the CYP3A family, peroxisome proliferator activator receptor-mediated induction of the CYP4A family, electrophile response element (EpRE)-mediated induction of NAD(P)H/quinone oxidoreductase, and CYP2E1 induction, which is not completely understood. Along with phase I and phase II enzyme induction, pretreatment with several microsomal enzyme inducers has been shown to alter the excretion of xenobiotics, which implies that phase III transport processes may also be similarly regulated (Klaassen and Plaa, 1968; Klaassen, 1970, 1974). Whether these microsomal enzyme inducers coordinately regulate the so-called phase III transport genes is currently unknown, but such information would add to our understanding of the disposition of xenobiotics.
Several studies have demonstrated the roles of Mrp1, 2, and 3 in drug resistance and their altered expression in several cancer cell lines. The regulation of Mrp2 and Mrp3 was also shown to be interdependent, because Mrp3 levels are increased to compensate for the absence of Mrp2 in a naturally occurring mutant rat strain (Keppler and Konig, 1997). However, the mechanisms for the regulation of the Mrp family are unresolved. Therefore, the aim of this study was to quantitatively assess the tissue distribution of Mrp1, 2, and 3 mRNA expression and to determine whether Mrp1, 2, and 3 are coordinately regulated through mechanisms similar to the well characterized phase I and II genes. In the present study, we have utilized Quantigene signal amplification technology to specifically and quantitatively monitor the mRNA levels of rat Mrp1, 2, and 3.
Materials and Methods
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was a gift from Dr. Karl Rozman (University of Kansas Medical Center, Kansas City, KS). Oltipraz was a gift of Dr. Ronald Lubet (National Cancer Institute, Bethesda, MD). 2,2′,4,4′,5-Pentachlorobiphenyl (PCB 99) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) were purchased from AccuStandard (New Haven, CT). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
To determine the tissue distribution of the three transporters, five male and female Sprague-Dawley rats (200–250 g; Sasco Inc., Wilmington, MA) were acclimated to the housing facility (2–3 rats/cage, 50% relative humidity, 12-h light/dark cycle) for 1 week and fed Teklad 8604 rodent chow (Harlan Teklad, Indianapolis, IN). Tissues were excised on day 5, snap-frozen in liquid nitrogen (intestinal epithelia were obtained by scraping prior to freezing), and stored at −80°C.
For the induction study, the following treatments were administered to five male Sprague-Dawley rats (200–250 g): TCDD (3.9 μg/kg, 1 day, i.p., in corn oil), indole-3-carbinol (I3C, 56 mg/kg, p.o., in corn oil), β-naphthoflavone (100 mg/kg, i.p., in corn oil), PCB 126 (40 μg/kg, i.p., in corn oil, 7 days), phenobarbital (80 mg/kg, i.p. in saline), PCB 99 (16 mg/kg, i.p., in corn oil, 7 day), diallyl sulfide (500 mg/kg, i.p., in corn oil), pregnenolone 16α-carbonitrile (PCN, 50 mg/kg, i.p., in corn oil), spironolactone (75 mg/kg, i.p., in corn oil), dexamethasone (40 mg/kg, i.p., in corn oil), clofibric acid (200 mg/kg, i.p., in saline), diethylhexylphthalate (1200 mg/kg, p.o., in corn oil), perfluorodecanoic acid (40 mg/kg, i.p., in corn oil, 1 day), ethoxyquin (50 mg/kg, p.o., in corn oil), oltipraz (150 mg/kg, p.o., in corn oil), isoniazid (200 mg/kg, i.p., in saline), acetylsalicylic acid (500 mg/kg, p.o., in corn oil), streptozotocin (STZ, 100 mg/kg, i.p., in 100 mM sodium citrate, 1 day), corn oil (i.p.), corn oil (p.o.), and saline (i.p.). All animals were treated for 4 days unless otherwise noted and injections were at a volume of 5 ml/kg. Tissues were excised as above.
Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) as per the manufacturer's protocol. RNA concentration was determined by UV spectrophotometry and its integrity was examined by ethidium bromide staining after agarose gel electrophoresis.
Development of Specific Oligonucleotide Probe Sets for bDNA Analysis.
The Mrp1, 2, and 3 gene sequences were accessed from GenBank. These target sequences were analyzed by ProbeDesigner Software Version 1.0 (Bayer Diagnostics, East Walpole, MA). The oligonucleotide probes designed were specific to a single mRNA transcript (i.e., Mrp1, 2, or 3; Table1). All oligonucleotide probes were designed with a Tm of approximately 63°C enabling hybridization conditions to be held constant (i.e., 53°C) during each hybridization step and for each oligonucleotide probe set. Every probe developed in ProbeDesigner was submitted to the National Center for Biotechnological Information for nucleotide comparison by the basic logarithmic alignment search tool (BLASTn; http://www.ncbi.nlm.nih.gov/BLAST/), to ensure minimal cross-reactivity with other known rat sequences and expressed sequence tags.
Branched DNA Assay.
Specific Mrp or CYP450 (Hartley and Klaassen, 2000) oligonucleotide probes were diluted in lysis buffer supplied in the Quantigene bDNA Signal Amplification Kit (Bayer Diagnostics, East Walpole, MA). All reagents for analysis (i.e., lysis buffer, capture hybridization buffer, amplifier/label probe buffer, wash A and wash D, and substrate solution) were supplied in the Quantigene bDNA Signal Amplification Kit. Mrp1, 2, and 3 mRNAs were analyzed according to the method of Hartley and Klaassen (2000). Briefly, total RNA (1 μg/μl; 10 μl) was added to each well of a 96-well plate containing capture hybridization buffer and 100 μl of each diluted probe set. Total RNA was allowed to hybridize to each probe set overnight at 53°C. Subsequent hybridization steps were carried out as per the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 bDNA Luminometer interfaced with Quantiplex Data Management Software Version 5.02 for analysis of luminescence from 96-well plates.
Data were expressed as mean ± standard error. For multiple comparisons, analysis of variance was performed followed by Duncan's multiple range test. The level of significance was set at p < 0.05.
Tissue Distribution of Mrp1, 2, and 3 mRNA Expression.
In order to determine the tissues where Mrp1, 2, and 3 are expressed, total RNA from five male rats was individually isolated from liver, kidney, lung, stomach, duodenum, jejunem, ileum, large intestine, cerebellum and cerebral cortex and subjected to the Quantigene signal amplification assay (Fig. 1). To determine whether gender differences occur in Mrp mRNA expression, and to ensure the major tissues of expression were reported, single determinations were performed using pooled RNA from five male or female rats isolated from the above tissues as well as the following: heart, blood vessel, spleen, thymus, muscle, skin, adrenal, lymph node, thyroid, eye, pituitary, thalamus, brain stem, caudate, frontal cortex, hippocampus, olfactory bulb, spinal cord, bladder, testes, ventral prostate, dorsal prostate, ovary and uterus (data not shown). No significant differences were observed between male and female expression of Mrp1, 2, or 3.
Mrp1 mRNA was expressed at a relatively similar level in all tissues examined with the exception of liver. Large intestine expressed the highest level with the stomach expressing 83%, duodenum 40%, jejunum 55%, and ileum 38% as much. Kidney and lung each expressed 66%, whereas cerebellum and cerebral cortex expressed 79% and 68% as much as the large intestine. Liver expressed by far the lowest amount of Mrp1 mRNA with only 8% of that expressed in large intestine.
Mrp2 message was found highest in duodenum and sequentially decreasing distally (jejunum 60%, ileum 51%, and large intestine 30% in relation to duodenum). Liver, kidney, and brain expressed moderate levels of Mrp2 (65%, 38%, cerebral cortex 51% and cerebellum 26%), with lower expression in stomach (12%) and lung (11%).
Mrp3 was almost exclusively expressed in the intestines with levels increasing from proximal to distal (duodenum 33%, jejunum 24%, ileum 92% and large intestine 100%). Other tissues expressed much lower levels when compared with large intestine (stomach 13%, kidney 12%, cerebral cortex 11%, lung 8%, cerebellum, 4%, and liver 2%).
Because liver, kidney and large intestine are important organs for transport and excretion, and because they have been shown to be targets for chemical modulation of phase I and II gene expression, Mrp1, 2, and 3 expression was examined following administration of drug metabolizing enzyme inducers.
Validation of Chemical Treatment Regimen by Phase I Induction.
The chemically induced expression of six phase I genes was used to verify that the treatments selected were adequate to activate the appropriate mechanisms to result in transcriptional activation. Table2 lists the phase I genes used as indicators of transcriptional activation as well as the fold induction of that gene by chemical treatment. These microsomal enzyme inducers are all thought to induce phase I genes by activating one of six different transcriptional activation pathways. The inducers can be separated into six categories: aryl-hydrocarbon receptor ligands, constitutive androstane receptor activators, pregnane-X-receptor ligands, peroxisome proliferator activator receptor ligands, electrophile response element activators, and CYP2E1 inducers. CYP1A1 induction by the aryl-hydrocarbon receptor ligands ranged between 456- and 693-fold with the exception of indole-3-carbinol (6.7-fold). CYP2B1/2 induction by constitutive androstane receptor activators ranged between 54- and 96-fold. Pregnane-X-receptor ligands induced CYP3A1/23 from 14- to 33-fold, and peroxisome proliferator activator receptor ligands induced CYP4A2/3 between 4- and 9-fold. Electrophile response element activators increased quinone reductase mRNA expression by 3- to 5-fold, whereas the treatments designed to increase CYP2E1 mRNA expression levels were only effective up to 2-fold.
Expression of Mrp1, 2, and 3 in Xenobiotic-Treated Rats.
The mRNA expression levels of Mrp1, 2, and 3 after treatment with microsomal enzyme inducers were examined. Mrp1 tended to be increased slightly over control by several treatments in liver including TCDD (110%), indole-3-carbinol (90%), phenobarbital (120%), diallyl sulfide (60%), spironolactone (60%), dexamethasone (130%), diethylhexylphthalate (90%), ethoxyquin (60%), oltipraz (80%), and acetylsalicylic acid (60%) (Fig. 2). However, only PCN significantly increased Mrp1 mRNA (140%). In kidney, only β-naphthoflavone significantly increased Mrp1 mRNA expression (70% over control), although indole-3-carbinol tended to increase expression 50% over control. In large intestine, TCDD, indole-3-carbinol, and isoniazid significantly increased Mrp1 expression by 70 to 80% over control.
Mrp2 mRNA expression in liver was not significantly altered by any treatment, although PCN, spironolactone, clofibric acid, and diethylhexylphthalate all increased expression 50, 90, 110, and 90%, respectively, over control, whereas perfluorodecanoic acid and isoniazid decreased expression to 40 and 49% of control (Fig.3). In kidney, indole-3-carbinol (130%), β-naphthoflavone (200%), diethylhexylphthalate (120%), and streptozotocin (110%) treatment significantly increased Mrp2 mRNA expression over control. In large intestine, Mrp2 mRNA expression was not significantly increased by any treatment, although β-naphthoflavone and diallyl sulfide both tended to decrease Mrp2 expression to 42% of control.
Mrp3 mRNA expression in liver was significantly increased by each of the CAR activators (phenobarbital, 390%; PCB 99, 580%; and diallyl sulfide, 540% over control) and by the EpRE activator oltipraz (670% over control) (Fig. 4). Although ethoxyquin increased Mrp3 mRNA expression 130% over control, it was not statistically significant. Incidentally, the ethoxyquin treatment regimen increased quinone reductase mRNA but was not as effective as oltipraz (Table 2). In contrast, only β-naphthoflavone treatment caused a significant increase of Mrp3 mRNA (160% over control) in kidney. None of the treatments in large intestine had significant effects on Mrp3 expression.
In the present study, the organ distribution of rat Mrp1, 2, and 3 mRNAs was determined. Also, multiple chemical activators of six transcriptional activation pathways were used in an effort to determine whether the expression of MRP1, 2, and 3 is coordinately regulated along with phase I and II drug-metabolizing enzymes. The method employed in this study (Quantigene signal amplification) has certain advantages over more commonly used methods such as the use of multiple short oligos as components of a larger probe set that retains the specificity to discriminate between closely related members of the same gene family without sacrificing sensitivity (Hartley and Klaassen, 2000). The end result (luminescence) is also directly measured and expressed as a numeric value. Most previous reports only examined a limited number of varying tissues and relied upon quantitation of autoradiographs from Northern blots that used large sections of cDNA as probes. The tissue distribution data presented herein were the result of an initial screen using pooled RNA to acquire single determinations of Mrp1, 2, and 3 mRNA expression levels in 34 tissues from both male and female rats. It was determined that there were no gender differences and that the 10 tissues reported herein represent the major organs of expression for Mrp2 and 3 and demonstrate the widespread expression of Mrp1.
The tissue distribution of Mrp1 has not previously been studied extensively in rats. In the present study, rat Mrp1 is expressed at a similar level in all tissues examined with the exception of liver, where expression was very low. MRP1 mRNA in humans is also expressed at similar levels in a number of tissues and is lower in liver, but is also lower in brain in relation to other tissues (Zaman et al., 1993;Kruh et al., 1995). In humans, MRP1 has been shown to be expressed on the basolateral membrane of human hepatocytes and bile duct epithelial cells (Roelofsen et al., 1997). It has recently been suggested that Mrp1 may play an important role in the blood-inner ear barrier in the rat (Saito et al., 2001).
Mrp2 mRNA expression in intestine gradually decreased distally along the intestinal tract from highest expression in duodenum to low Mrp2 expression in large intestine. Previous studies in rat intestine showed Mrp2 expression to be higher in duodenum and jejunum than in ileum and colon (Gotoh et al., 2000), and higher in the villus tip with lower expression in the lower villus and crypt cells (Mottino et al., 2000). Mrp2 expression in organs such as liver, kidney, and intestine suggests the importance of Mrp2 in the excretion of xenobiotics, as well as protection against dietary xenobiotics. The function of Mrp2 expression in brain has yet to be determined, although the absence of Mrp2 in bovine brain microvessel endothelial cells suggests that Mrp2 is not important for the blood-brain barrier (Zhang et al., 2000). The differential expression of Mrp1 and Mrp2 in intestine suggests distinct functions for these transporters despite their similar substrate specificities (Keppler et al., 1997, 1998).
Similar to previous reports (Hirohashi et al., 1998; Ortiz et al., 1999), Mrp3 mRNA expression increases along the intestinal tract (large intestine > duodenum). Mrp3 mRNA expression is low in kidney and lung. Very little Mrp3 was expressed in liver under normal physiological conditions, and previous studies showed that expression of Mrp3 in rat liver is only detected under various cholestatic conditions (Ogawa et al., 2000). Because of its high expression in ileum as well as its ability to transport various bile acids (Hirohashi et al., 2000), Mrp3 may serve the important function of cooperatively reabsorbing bile acids from the intestine back into the bloodstream. The ileal sodium-dependent bile acid transporter has been shown to be important in the recirculation of bile acids by facilitating the uptake of bile acids from the lumen of the ileum into enterocytes (Wong et al., 1994). Mrp3 has been shown to be localized to the basolateral membrane in the hepatocyte (Konig et al., 1999; Soroka et al., 2001) and to accept various bile acids such as taurocholate and taurolithocholate sulfate as substrates (Hirohashi et al., 2000). If Mrp3 is also on the basolateral membrane in intestine, Mrp3 could facilitate the sodium-independent transport (Weinberg et al., 1986) of bile acids from the enterocytes to blood.
With regard to relative isoform expression within organ systems, each of these three transporters is expressed in kidney, whereas Mrp2 is expressed much more than Mrp1 and Mrp3 in liver, where it plays an important physiological role in biliary excretion (Morikawa et al., 2000; Nishino et al., 2001). The predominant Mrp expression appears to be different in various sections of the intestine. Mrp2 expression appears to be more important proximally, whereas Mrp3 expression is dramatically higher distally, and Mrp1 expression appears to be consistent throughout.
Coordinate regulation of phase I and II drug-metabolizing genes plays an important part in the elimination of xenobiotics. To extend the implications of coordinate regulation of phase I and II drug-metabolizing enzymes to transporter genes, 18 different treatments were selected based upon six major proposed mechanisms of drug-metabolizing enzyme induction (aryl-hydrocarbon receptor ligands, CAR activators, pregnane-X-receptor ligands, peroxisome proliferator activator receptor ligands, EpRE activators, and CYP2E1 inducers). Each treatment was intended to activate a corresponding transcriptional activation pathway and thereby increase transcription of the associated gene battery (e.g., TCDD induction of CYP1A1). The effectiveness of each treatment was demonstrated by assessing the induction of appropriate phase I drug-metabolizing genes. The use of multiple, structurally dissimilar activators of these transcriptional activation pathways can implicate with some confidence the mechanism of induction in coordination with the phase I drug-metabolizing enzymes (Hartley and Klaassen, 2000).
Phenobarbital, PCB 99, and diallyl sulfide all markedly increased hepatic Mrp3 mRNA levels. These chemicals are quite structurally diverse but share a common transcriptional activation of CYP2B by activation of the nuclear receptor CAR (Honkakoski et al., 1998;Kawamoto et al., 1999; Sueyoshi et al., 1999). Additional evidence that Mrp3 is under the transcriptional regulation of CAR is the liver-specific induction of Mrp3 by CAR activators, because CAR is expressed almost exclusively in liver (Baes et al., 1994; Choi et al., 1997). Similarly, oltipraz and, to a lesser extent, ethoxyquin increase hepatic Mrp3 mRNA levels and are structurally quite dissimilar but share a common transcriptional activation of quinone oxidoreductase through activation of the EpRE (Buetler et al., 1995). However, since ethoxyquin failed to increase Mrp3 levels significantly, involvement of the EpRE is questionable.
CAR has been described as a cellular sensor that is capable of responding to chemical toxicity and mediating CYP2B family induction (Honkakoski et al., 1998; Kawamoto et al., 1999; Sueyoshi et al., 1999). Activation of this cellular sensor leads to an increase of Mrp3 mRNA, which may lead to an enhanced ability of the liver to eliminate organic anions into the sinusoidal blood, thereby reducing hepatic toxicity. The importance of the coordinate regulation of both the phase III transport (export) by Mrp3 and phase I and II metabolism in liver has not been studied and warrants further investigation. However, the addition of phase III transport regulation by CAR lends greater understanding to the role of CAR as a biological sensor, but more as an important means of managing hepatoprotection during chemical exposure.
Studies conducted in our laboratory before the discovery of CAR showed that several CAR activators, including phenobarbital, significantly decreased biliary excretion of the acetaminophen (APAP) metabolites, APAP-glucuronide and APAP-sulfate, by up to 89 and 57%, respectively (Gregus et al., 1990), whereas the CAR activatortrans-stilbene oxide increased APAP-glucuronide blood levels up to 11-fold and urinary excretion up to 3.6-fold. This alteration in vectoral excretion from bile to blood may be explained by the induction of hepatic Mrp3 levels by CAR activators. Substrates, such as APAP metabolites, which are predominantly excreted into bile (potentially by Mrp2 on the canalicular membrane), could be transported more rapidly back into the blood because of increased levels of Mrp3.
In conclusion, these results show that Mrp2 mRNA expression in the liver is higher than that of Mrp1 or 3, whereas in intestine, the predominant Mrp expression appears to change from Mrp2 proximally to Mrp3 distally. Because of the distribution of these transporters, distinct endogenous functions are likely, despite the similarities in substrate specificities. These results also show that hepatic Mrp3 mRNA levels are increased by CAR activators and an EpRE activator, thereby potentially enhancing the excretion of organic anions from the liver to the blood. The extent of this induction is less than the induction of many phase I and II drug-metabolizing genes, in particular, the coordinately regulated CYP2B family. Presumably, chemical insult resulting in CAR activation leads to an increase in Mrp3 mRNA, thereby providing an important mechanism for hepatoprotection from the toxicity of organic anions.
↵1 Current address: Department of Drug Metabolism, Merck Research Laboratories, Mail Stop RY80D-100, Rahway, NJ 07065.
↵2 Current address: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655.
This work was supported by National Institutes of Health Grants ES-09716 and ES-03192, and by National Institutes of Health Training Grant ES-07079 (to N.J.C., D.P.H., and D.R.J.) and Grant ES-05883 (to N.J.C.).
- multidrug resistance protein
- constitutive androstane receptor
- electrophile response element
- PCB 99
- PCB 126
- pregnenolone 16α-carbonitrile
- branched DNA
- Received June 8, 2001.
- Accepted September 21, 2001.
- The American Society for Pharmacology and Experimental Therapeutics