ToxSci Advance Access originally published online on March 16, 2006
Toxicological Sciences 2006 91(2):651-659; doi:10.1093/toxsci/kfj162
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Tienilic Acid Enhances Hyperbilirubinemia in Eisai Hyperbilirubinuria Rats through Hepatic Multidrug Resistance–Associated Protein 3 and Heme Oxygenase-1 Induction
Drug Safety Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo 134-8630, Japan
1 To whom correspondence should be addressed at Drug Safety Research Laboratory, Daiichi Pharmaceutical Co., Ltd., 16-13, Kita-Kasai 1-Chome, Edogawa-ku, Tokyo, Japan. Fax: +81-3-5696-8335. E-mail: nishiqm8{at}daiichipharm.co.jp.
Received January 31, 2006; accepted March 2, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
We demonstrated that tienilic acid, a diuretic drug withdrawn from the market because of hepatic failure, enhanced hyperbilirubinemia in Eisai hyperbilirubinuria rats (EHBR) with a defect of canalicular multidrug resistance–associated protein 2 (Mrp2). In contrast, no remarkable changes were noted in Sprague-Dawley (SD) rats, the parent strain for EHBR. To investigate a mechanism underlying this enhanced hyperbilirubinemia, we focused on comprehensive effects of tienilic acid on clinicopathological aspects and expression of hepatic transporters. Other than eventual hyperbilirubinemia with slightly increased biliary bilirubin, a single oral treatment of EHBR with tienilic acid at 300 mg/kg caused no changes in serum alanine aminotransferase and alkaline phosphatase, bile flow rate and biliary bile acid secretion, or hepatic morphology. In analyses of mRNA expression of the hepatic transporters, elevated Mrp3 expression in EHBR correlated with an increase in serum total bilirubin, suggesting increased bilirubin transport from the liver into the peripheral blood flow. Hepatic heme oxygenase-1 (Ho-1) mRNA, a stress-induced isoform of the rate-limiting enzyme in the catabolism of heme to bilirubin, was markedly upregulated in EHBR at the same dose at which increased serum bilirubin was seen. A time-course study revealed that marked induction of Ho-1 occurred earlier than that of Mrp3, followed by an increase in serum bilirubin. These results suggest that hepatic Mrp3 and Ho-1 may contribute to tienilic acid–enhanced hyperbilirubinemia in EHBR by inducing increased bilirubin transport from the liver into the blood stream, preceded by potentiation of bilirubin formation in the liver.
Key Words: Eisai hyperbilirubinuria rat; heme oxygenase-1; hyperbilirubinemia; tienilic acid; transporters.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Hyperbilirubinemia occurring under the cholestatic conditions has been simply interpreted as a result of decreased biliary excretion of conjugated bilirubin. According to recent in vitro studies (Jedlitschky et al., 1997
; Kamisako et al., 2000; Reichel et al., 1999), however, several functional proteins have been recognized as being actively involved in the efflux and influx of bilirubin across the hepatic plasma membrane, namely, multidrug resistance–associated proteins (Mrps) and organic anion–transporting polypeptides (Oatps). Their expression is cooperatively regulated upon biliary dysfunction to transfer excessive amounts of organic substances including bile salts and bilirubin through the hepatic excretions into blood (Donner and Keppler, 2001). In addition to endogenous substances, the hepatic transporters also play an important role in detoxification by eliminating exogenously administered parent compounds and associated toxic metabolites. For example, altered expression profiles of the transporters, as well as stress-responsive enzymes such as heme oxygenase-1 (Ho-1) after administration of the prototype hepatotoxicant acetaminophen or carbon tetrachloride, have been considered presumably as a consequence of protective responses against excessive exposure of the liver to the toxicants (Aleksunes et al., 2005). Based on the above information, the possibility is raised that certain hepatotoxic compounds would alter the expression of hepatic transporters and thereby induce abnormal bilirubin disposition and metabolism. Nevertheless, there is a dearth of information about the relationship between chemically induced hyperbilirubinemia and altered transporter expression in vivo.
Tienilic acid was launched in 1979 in the United States as a diuretic antihypertensive drug but has been withdrawn from the market because of fulminant hepatic failure. Tienilic acid is metabolized by the hepatic drug-metabolizing enzyme CYP2C9, and a reactive intermediate yielded covalently binds to macromolecules including the metabolizing enzyme itself (López-Garcia et al., 1994). Although immune-mediated mechanisms via this covalent binding have been believed to be associated with the idiosyncratic hepatic failure, direct toxic action by tienilic acid on primary cultured rat hepatocytes and the isolated perfused rat liver is also shown (Takagi et al., 1991; Zimmerman et al., 1982). Consistently, the hepatic injury in humans has the lack of peripheral eosinophilia and rash related to hypersensitivity in some cases (Zimmerman et al., 1984). In spite of extensive researches and informative findings relating to the hepatotoxicity, in normal experimental animals, tienilic acid has never induced the clinicopathological hallmarks such as marked elevations in serum transaminases and bilirubin level seen in humans (Zimmerman et al., 1984). Interestingly, according to our preliminary study, Eisai hyperbilirubinuria rats (EHBR), an animal model that exhibits a congenital defect of the canalicular ATP-binding cassette transporter Mrp2 (Ito et al., 1997; Kurisu et al., 1991), receiving tienilic acid showed a significant increase in serum bilirubin (Nishiya et al., 2004). In the present study, thus, the hyperbilirubinemic potential of tienilic acid was clinicopathologically assessed in both EHBR and Sprague-Dawley (SD) rats, the parent strain for EHBR. Afterward, the investigation was carried out to approach the initial mechanism underlying enhanced hyperbilirubinemia due to tienilic acid. Briefly, bile constituents were extensively determined in EHBR, and mRNA expressions of the hepatic transporters Mrp2, Mrp3, Na+/taurocholate co-transporting polypeptide (Ntcp), Oatp1, or Oatp2 involved in bilirubin and bile acid transport and Ho-1, a stress-induced isoform of the rate-limiting enzyme in heme catabolism to bilirubin, were measured by a real-time quantitative PCR method.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Test substance.
Tienilic acid was synthesized at Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan), and was suspended in 1% methylcellulose aqueous solution (Wako Pure Chemical Industries, Osaka, Japan). All other chemicals and reagents were the highest grade available from commercial sources.
Animals.
Male EHBR and SD rats aged 6–7 weeks were obtained from Japan SLC (Hamamatsu, Japan). They were housed in an air-conditioned facility (a temperature of 21–25°C, a relative humidity of 35–75%, and lighting at 8:00 A.M.–8:00 P.M. with a 12-h light cycle) and fed commercial rodent chow (F-2, Funabashi Farm, Chiba, Japan) and tap water ad libitum. All experimental procedures were performed in accordance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science (1987).
Laboratory tests and liver pathology.
To investigate the hyperbilirubinemic potential of tienilic acid, this agent was orally administered once at 300 mg/kg to EHBR and SD rats, and then all animals were euthanized 24 h after treatment. Rats of each strain given 1% methylcellulose solution alone in the same way served as the concurrent vehicle control. Blood samples were withdrawn from the jugular vein and carotid artery under ether anesthesia. Red blood cell counts and hemoglobin contents were analyzed by an ADVIA 120 (Bayer Corporation, Pittsburgh, PA), and serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bile acids, and total and conjugated bilirubin levels were determined with an H7315 automatic analyzer (Hitachi, Tokyo, Japan). Immediately after necropsy, the liver of each rat was excised, fixed in 10% neutral buffered formalin, trimmed, embedded in paraffin wax, cut at 3-µm thickness, stained with hematoxylin and eosin, and histopathologically examined. Next, to delineate a dose dependency for elevations in serum total bilirubin, tienilic acid was orally administered once at 10, 30, 100, and 300 mg/kg to EHBR, and serum total bilirubin was measured 24 h later. Furthermore, to assess the time course of serum total bilirubin, tienilic acid was orally administered once at 300 mg/kg to EHBR, and serum samples were collected 3, 6, 9, and 24 h later. For the interindividual variation of serum bilirubin level in naive EHBR, the difference from the predose value in each animal was utilized in the dose- and time-dependency studies.
Biliary excretion of bilirubin and bile acids.
Twenty-four hours after treatment of EHBR with tienilic acid at 300 mg/kg, bile was collected for 30 min via an indwelling polyethylene tube (PE-10, Becton Dickinson Co., Franklin Lakes, NJ) under pentobarbital anesthesia (30 mg/kg, iv). The bile volume was measured gravimetrically, and total bilirubin and total bile acid concentrations in the bile were determined with the automatic analyzer. The biliary secretions (bile flow rate x biliary concentration) were calculated.
Analyses of gene expression by real-time PCR.
Ntcp is a transporter located on the basolateral membrane in the liver, which is related to the sodium-dependent uptake of bile acids (Hagenbuch et al., 1991). The expression has been reported to be significantly downregulated in cholestasis (Geier et al., 2003). It is therefore essential to evaluate the cholestatic potential of tienilic acid on Ntcp mRNA expression. Initially, the basal expressions of the hepatic transporters Ntcp, Mrp2, Mrp3, Oatp1, and Oatp2 were measured in both naive EHBR and SD rats. The expression levels were determined 24 h after a single oral administration of tienilic acid at 300 mg/kg in both strains. As altered expressions of Mrp3, Oatp1, and Oatp2 were observed in EHBR after treatment, hepatic Mrp3, Oatp1, or Oatp2 mRNA expression was measured 3, 6, 9, and 24 h following a single treatment of EHBR with tienilic acid at 300 mg/kg. Next, hepatic Ho-1 mRNA expression was measured in the same time points in EHBR and SD rats. Finally, to ascertain a dose dependency and confirm its association with bilirubin elevation, Mrp3 and Ho-1 mRNA expressions in EHBR were measured 6 and 3 h, respectively, after a single administration of tienilic acid at 10, 30, 100, and 300 mg/kg. These quantitative analyses were performed in accordance with the following procedure. A portion (20–40 mg) of the left lobe in the liver was removed, cut into 5 mm in thickness, and immediately stored in commercially available RNA-stabilizing solution for tissues (RNAlater, QIAGEN GmbH, Hilden, Germany). Total RNA was isolated by an RNeasy Mini Kit (QIAGEN Inc., Valencia, CA) and reverse transcribed in a TaqMan reverse transcription (RT) buffer (Applied Biosystems, Foster City, CA) containing 5.5 mM MgCl2, 0.5 mM dNTP, 2.5 µM oligo dT16, 0.4 U/µl RNase inhibitor, and 1.25 U/µl MultiScribe Reverse Transcriptase at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min (Applied Biosystems). Target cDNA was amplified in an SYBR Green PCR buffer (Applied Biosystems) containing 50nM forward and reverse primers (Table 1), 3 mM MgCl2, 0.2 mM dATP/dCTP/dGTP, 0.4 mM dUTP, 0.01 U/microL AmpErase UNG, and 0.025 U/microL AmpliTaq Gold DNA polymerase (Applied Biosystems). The thermal cycling conditions for PCR were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. PCR primers were designed using a Primer Express (Applied Biosystems) to amplify the coding region of the gene. The primers were submitted to the National Center for Biotechnology Information for nucleotide comparison by the basic logarithmic alignment search tool (BLASTn) to minimize cross-reactivities with other known genes and expressed sequence tags. Each PCR product was proven as a target by a melting curve analysis and/or by an analysis using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). Quantification of the PCR product was conducted by measuring an increase in fluorescence with an ABI PRISM 7700 (Applied Biosystems). The number of cycles at which fluorescence intensity exceeded a threshold in the exponential growth of the PCR product was used to make a standard curve with a series of diluted samples, from which the relative amount of gene expression was determined. Each sample was assayed in duplicate, and all data were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA as a housekeeping gene. Primers for the housekeeping gene (GenBank accession no. M17701) are as follows: forward primer, 5'-AAACCTGCCAAGTATGATGACATC-3', and reverse primer, 5'-CTCGGCCGCCTGCTT-3'.
|
Statistical analysis.
Quantitative data are expressed as the mean and SD of the group. In dose-dependency studies for increases in bilirubin and Mrp3 and Ho-1 mRNA expressions, linear regression analysis was performed with 5% of the significant level. Differences between treatment and vehicle control groups and EHBR and SD rats (species control) were statistically analyzed by Williams test (among three groups or more) or Student's t-test (between two groups) with 5% of the significant level. These analyses were performed by using LATOX (FUJITSU LIMITED, Tokyo, Japan) and EXSAS (Arm Co., Ltd., Osaka, Japan).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Laboratory Tests, Liver Pathology, and Biliary Excretion of Bilirubin and Bile Acids
A single oral administration of tienilic acid at 300 mg/kg to EHBR significantly increased serum total and conjugated bilirubin (5.12 and 3.71 mg/dl, p < 0.05) as compared with the concurrent vehicle control (1.46 and 1.13 mg/dl) 24 h after treatment, although there were trivial increases in serum total bilirubin in SD rats (Table 2). No change in serum ALT, ALP, and total bile acids (Table 2); red cell counts, hemoglobin contents; or liver morphology was noted in either strain (data not shown). Serum total bilirubin in EHBR was elevated in a dose-dependent manner over 30 mg/kg with statistical significance (Fig. 1A). Linear regression analysis indicated a close relationship between serum bilirubin concentrations and dosage levels of tienilic acid (Fig. 1A, p < 0.001). In a time-course study after the administration of tienilic acid at 300 mg/kg to EHBR, serum total bilirubin started to increase at 6 h postdose (p < 0.05, Fig. 1B). In analyses of biliary constituents, biliary concentrations of total bilirubin increased with statistical significance (p < 0.05), although the biliary secretion showed no statistical significance (Fig. 2). Change in neither the bile flow rate nor biliary secretion and concentration of total bile acids was noted even at 300 mg/kg (Fig. 2).
|
|
|
Analyses of mRNA Expression of Hepatic Transporters and Ho-1
Initially, the basal levels of the mRNA expressions of hepatic transporters Ntcp, Mrp2, Mrp3, Oatp1, and Oatp2 were investigated in both naive EHBR and SD rats (Table 3). Mrp2 mRNA was about 20-fold lower and Mrp3 mRNA was about 4-fold higher in EHBR than in SD rats, whereas Ntcp, Oatp1, and Oatp2 mRNA levels in EHBR were essentially the same as those in SD rats. Next, effects of tienilic acid on these transporters were quantitatively examined in both strains. Following a single oral administration of tienilic acid at 300 mg/kg to EHBR, a marked increase in Mrp3 mRNA and decreases in Mrp2, Oatp1, and Oatp2 mRNAs were noted (Fig. 3), but no significant difference in Ntcp mRNA was seen between the vehicle control and treatment groups (data not shown). In SD rats at the same dose, Mrp3 mRNA was slightly increased, but its extent was much smaller than that in EHBR. Decreases in Mrp2 and Oatp2 mRNAs were observed, but no significant difference in Ntcp or Oatp1 mRNA was seen between the vehicle control and treatment groups.
|
|
For the changes in transporters Mrp3, Oatp1 and Oatp2 mRNA expression in EHBR, the time-course study was performed (Fig. 4). Mrp3 mRNA level began to increase at 3 h postdose (191% of the initial value, p < 0.05), reached 826% of the initial value at 6 h postdose, and then plateaued at 9 and 24 h postdose with statistical significance (p < 0.05). The Oatp1 mRNA level was reduced to 46 and 48% of the initial value (p < 0.05) at 9 and 24 h postdose, respectively. The Oatp2 mRNA level decreased to 65 and 55% of the initial value (p < 0.05) at 9 and 24 h postdose, respectively. For hepatic Ho-1 mRNA expression, the time-course study was performed in both EHBR and SD rats (Fig. 5). In EHBR, the Ho-1 mRNA level increased to 4811% of the initial vehicle control at 3 h postdose and reached a maximum (p < 0.05), being 7249% of the vehicle control, at 6 h postdose with statistical significance (p < 0.05). The level was relatively high at 9 and 24 h post-dose, being 682% and 190% of the initial vehicle control (p < 0.05), respectively. In SD rats, the Ho-1 mRNA level increased to 748% of the initial vehicle control at 3 h postdose (p < 0.05) and reached a maximum, being 1469% of the vehicle control, at 6 h postdose with statistical significance (p < 0.05). The level was relatively high at 9 h postdose, being 786% of the (p < 0.05). However, it was lower than that of the vehicle control at 24 h (p < 0.05), being 40% of the initial vehicle control. Overall, the extent of Ho-1 mRNA induction was much larger in EHBR than in SD rats. Finally, the dose dependency for the hepatic Mrp3 and Ho-1 mRNA expressions in EHBR was investigated (Fig. 6). Treatment with tienilic acid significantly increased Mrp3 to 185, 362, 611, and 775% at 10, 30, 100, and 300 mg/kg and Ho-1 to 312, 1119, and 4907% at 30, 100, and 300 mg/kg, respectively, compared with the vehicle control (p < 0.05), whereas a marginal change (126%) was observed at 10 mg/kg. Linear regression analysis showed dose dependency between the Mrp3 or Ho-1 expression and dosage levels of tienilic acid (p < 0.001).
|
|
|
DISCUSSION
The present study was performed to clarify the initial mechanism underlying tienilic acid–induced hyperbilirubinemia through active transporters and enzymes in coordinated regulation and contribution to its pathogenesis. A single oral treatment with tienilic acid dose dependently increased serum total bilirubin in EHBR, whereas no abnormality was seen in SD rats. No change in either serum hepatic enzymes (ALT and ALP), markers for cholestatic liver damage (total bile acids), or hepatic morphology was noted in either strain (Table 2). In addition, tienilic acid did not affect the bile flow rate or biliary excretion of bile acids (Fig. 2), suggesting that enhanced hyperbilirubinemia in EHBR was not caused by preventing biliary secretion. Rather, the biliary bilirubin concentration was increased, probably by the direct action of tienilic acid on hepatic bilirubin synthesis as mentioned below. Meanwhile, the enhanced hyperbilirubinemia is not associated with the inhibition of bilirubin glucuronidation since tienilic acid increased serum conjugated bilirubin (Table 2), which is in contrast to high level of serum unconjugated bilirubin in Gunn rats with congenital defect of the bilirubin glucuronidation enzyme UDP-glucuronosyltransferase (UGT) 1A1 (Sato et al., 1991
). Furthermore, although certain drugs have been known to provoke hyperbilirubinemia through their hemolytic actions, tienilic acid showed no anemia, such as decreases in red blood cell counts and hemoglobin contents, in EHBR even at high doses where serum bilirubin was increased.
Our results clearly demonstrated that basal expression of Mrp3 mRNA in the liver was exceedingly higher in EHBR than in SD rats, as previously reported (Hirohashi et al., 1998), although those of Ntcp, Oatp1, and Oatp2 were comparable in both strains (Table 3). In analyses of the hepatic transporter mRNAs following treatment with tienilic acid, marked induction of Mrp3 mRNA was observed in EHBR (Fig. 3). Mrp3/MRP3 is localized on the basolateral membrane and transports bilirubin glucuronide into the blood stream (Lee et al., 2004). Thus, tienilic acid would facilitate the excretion of conjugated bilirubin from the liver into the blood stream via an Mrp3 induction, thereby leading to elevated serum total bilirubin. This notion was supported by the fact that the change in Mrp3 mRNA was prominent in EHBR with tienilic acid, whereas it was subtle in SD rats (Fig. 3); the dose dependency for Mrp3 induction was very similar to that for elevation in serum total bilirubin concentration (Figs. 1 and 6), and the increase in Mrp3 mRNA was followed by the increase in the serum bilirubin (Fig. 4). Extensive investigations have indicated that Mrp3 may compensate for impaired canalicular functions in cholestasis, based on its ability to transport organic anions into the sinusoid and its upregulation coordinately with the downregulation of Mrp2 (Donner and Keppler, 2001; Soroka et al., 2001). Furthermore, it has been reported that Mrp3 null mice have lower serum bilirubin glucuronide than do the wild-type mice after bile duct ligation, although the difference is not seen between the respective sham-operated mice (Belinsky et al., 2005). Accordingly, Mrp3 would be a causal determinant for serum bilirubin level, especially in EHBR that exhibit the limited biliary secretion of bilirubin.
A decrease in Oatp1 mRNA was noted only in EHBR given tienilic acid, whereas Oatp2 mRNA was reduced in both strains (Fig. 3). Oatp1 and Oatp2 related to the uptake of organic anions are located on the sinusoidal membrane in the liver (Bergwerk et al., 1996; Reichel et al., 1999). Regarding bilirubin transport, Oatp1 has been reported to deliver bilirubin monoglucuronide (Reichel et al., 1999) and has been postulated to contribute to the bilirubin clearance from the systemic circulation by the uptake into hepatocytes. The reduction in Oatp1 mRNA due to tienilic acid, however, was unlikely to be a main cause for the enhanced hyperbilirubinemia because it was preceded by an increase in serum bilirubin (Fig. 4).
In addition to Oatp1, Oatp4, a rat orthologue of human OATP2 that has shown to mediate bilirubin transport into hepatocytes (Cui et al., 2001), is expressed in the sinusoidal membrane of hepatocytes (Cattori et al., 2000; Li et al., 2002). Unfortunately, as little information is available for involvement of Oatp4 in bilirubin transports and its regulation by bilirubin, the relevance of Oatp4 to tienilic acid–enhanced hyperbilirubinemia remains to be investigated.
Ho is a rate-limiting enzyme in the degradation of heme to bilirubin (Tenhunen et al., 1968). Ho-1, an isoform of Ho, is known as a heat-shock protein (HSP32) and significantly evoked by oxidative stresses and hepatic injury (Llesuy and Tomaro, 1994; Yamaguchi et al., 1996). Hepatic Ho-1 mRNA was markedly increased in EHBR treated with tienilic acid, and its high expression remained up to 24 h after treatment (Fig. 5), suggesting that tienilic acid facilitated bilirubin formation in the liver of EHBR. As with Mrp3, high Ho-1 expression was seen at the same dose at which the increase in serum bilirubin was observed (Figs. 1 and 6). These indicate that hepatic Ho-1 induction may participate in enhanced hyperbilirubinemia in EHBR. The present data (Fig. 5) also demonstrated that Ho-1 expression was increased in SD rats, but its extent was smaller than that in EHBR. This may be explained by a difference in increased serum bilirubin level. Mrp2/MRP2 is involved in the transport of endogenous organic anions as well as exogenous substances including glucuronide and glutathione conjugates (Oude Elferink and Jansen, 1994; Sasabe et al., 1998). Indeed, EHBR displayed a decrease in biliary excretion of drugs and their metabolites (Ishizuka et al., 1997; Sasabe et al., 1998). In addition to Mrp2 deficiency, hepatic activities and protein expression of CYP isoforms have been reported to be different between EHBR and SD rats (Newton et al., 2005; Ohmori et al., 1991). An experiment using the isolated perfused rat liver indicates that metabolite(s) of tienilic acid produced in the cytochrome P450 system may contribute to the onset of hepatotoxicity (Zimmerman et al., 1982). Thus, marked Ho-1 and Mrp3 inductions by tienilic acid would be derived from impaired disposition of tienilic acid and/or its metabolites due to the Mrp2 defect and/or abnormal biotransformation by CYPs. The kinetics of tienilic acid in both strains may be informative to understand the mechanisms underlying high susceptibility of EHBR to tienilic acid.
Mrp3 mRNA expression has been reported to increase when bilirubin was exogenously administered (Ogawa et al., 2000). Based on the finding, the presence of newly biosynthesized bilirubin in the liver by Ho-1 induction seems to facilitate Mrp3 mRNA expression. This phenomenon coincided closely with evidence that a marked increase in hepatic Ho-1 mRNA was seen as early as 3 h, whereas Mrp3 mRNA began to elevate at 3 h postdose and reached a maximum level at 6 h postdose (Figs. 4 and 5). Hence, our data may provide an additional insight into hepatic Mrp3 induction that is provoked by increased hepatic bilirubin derived from the Ho-1 induction.
It has been reported that Mrp3 is also upregulated by drug-metabolizing enzyme inducers that produce transcriptional activation via the constitutive androstane receptor (CAR) (Cherrington et al., 2002). The increase in Mrp3 expression by tienilic acid, however, may be independent of drug-metabolizing enzyme induction, inasmuch as tienilic acid had no effects on hepatic cytochrome P450 contents or activities in SD rats by a 14-day repeated oral administration (Sellman and Parkki, 1983) or on CYP2B1/2 mRNA, which was shown to increase through a CAR-mediated activation, in EHBR (unpublished data, Nishiya T). Alternatively, Mrp3 was induced by electrophile response element activators via probably nuclear transcription factor-E2 p45-related factor (Nrf2) that regulated the expression of stress-induced genes including Ho-1 (Cherrington et al., 2002). In fact, since coinduction of Mrp3 and Ho-1 was observed during liver injuries by acetaminophen to protect against further hepatic injury (Aleksunes et al., 2005) and tienilic acid reduced glutathione in the primary cultured hepatocytes (Takagi et al., 1991), it is plausible that induction of Mrp3 and Ho-1 due to tienilic acid may require Nrf2. Expression analyses of genes as indicators of transcriptional activation may provide clues to this regulation.
Hyperbilirubinemia is the most common feature of the tienilic acid–associated hepatotoxicity (Zimmerman et al., 1984) as well as elevation of transaminases. Although relevance of the pathological features in the present model to hepatic injury in humans are still obscure, in addition to enhanced hyperbilirubinemia, marked Mrp3 and/or Ho-1 inductions in the liver were seen as with other hepatotoxicants including acetaminophen, carbon tetrachloride, and diclofenac (Aleksunes et al., 2005; Cantoni et al., 2003). Therefore, the present data add support to the likelihood that tienilic acid may cause or aggravate, in part, jaundice in humans by potentiation of bilirubin biosynthesis and its sinusoidal transport as a consequence of the stress response in the liver.
In conclusion, tienilic acid enhances hyperbilirubinemia in EHBR, and hepatic Mrp3 and Ho-1 may contribute greatly to this event.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Aleksunes, L. M., Slitt, A. M., Cherrington, N. J., Thibodeau, M. S., Klaassen, C. D., and Manautou, J. E. (2005). Differential expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride. Toxicol. Sci. 83, 44–52.
Belinsky, M. G., Dawson, P. A., Shchaveleva, I., Bain, L. J., Wang, R., Ling, V., Chen, Z. S., Grinberg, A., Westphal, H., Klein-Szanto, A., et al. (2005). Analysis of the in vivo functions of Mrp3. Mol. Pharmacol. 68, 160–168.
Bergwerk, A. J., Shi, X., Ford, A. C., Kanai, N., Jacquemin, E., Burk, R. D., Bai, S., Novikoff, P. M., Stieger, B., Meier, P. J., et al. (1996). Immunologic distribution of an organic anion transport protein in rat liver and kidney. Am. J. Physiol. 271, G231–G238.[Medline]
Cantoni, L., Valaperta, R., Ponsoda, X., Castell, J. V., Barelli, D., Rizzardini, M., Mangolini, A., Hauri, L., and Villa, P. (2003). Induction of hepatic heme oxygenase-1 by diclofenac in rodents: Role of oxidative stress and cytochrome P-450 activity. J. Hepatol. 38, 776–783.[CrossRef][Medline]
Cattori, V., Hagenbuch, B., Hagenbuch, N., Stieger, B., Ha, R., Winterhalter, K. E., and Meier, P. J. (2000). Identification of organic anion transporting polypeptide 4 (Oatp4) as a major full-length isoform of the liver-specific transporter-1 (rlst-1) in rat liver. FEBS Lett. 474, 242–245.[CrossRef][ISI][Medline]
Cherrington, N. J., Hartley, D. P., Li, N., Johnson, D. R., and Klaassen, C. D. (2002). Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats. J. Pharmacol. Exp. Ther. 300, 97–104.
Cui, Y., König, J., Leier, I., Buchholz, U., and Keppler, D. (2001). Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6. J. Biol. Chem. 276, 9626–9630.
Donner, M. G., and Keppler, D. (2001). Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 34, 351–359.[CrossRef][ISI][Medline]
Geier, A., Dietrich, C. G., Gerloff, T., Haendly, J., Kullak-Ublick, G. A., Stieger, B., Meier, P. J., Matern, S., and Gartung, C. (2003). Regulation of basolateral organic anion transporters in ethinylestradiol-induced cholestasis in the rat. Biochim. Biophys. Acta 1609, 87–94.[Medline]
Hagenbuch, B., Stieger, B., Foguet, M., Lübbert, H., and Meier, P. J. (1991). Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system. Proc. Natl. Acad. Sci. U.S.A. 88, 10629–10633.
Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998). Hepatic expression of multidrug resistance-associated protein-like proteins maintained in Eisai hyperbilirubinemic rats. Mol. Pharmacol. 53, 1068–1075.
Ishizuka, H., Konno, K., Naganuma, H., Sasahara, K., Kawahara, Y., Niinuma, K., Suzuki, H., and Sugiyama, Y. (1997). Temocaprilat, a novel angiotensin-converting enzyme inhibitor, is excreted in bile via an ATP-dependent active transporter (cMOAT) that is deficient in Eisai hyperbilirubinemic mutant rats (EHBR). J. Pharmacol. Exp. Ther. 280, 1304–1311.
Ito, K., Suzuki, H., Hirohashi, T., Kume, K., Shimizu, T., and Sugiyama, Y. (1997). Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR. Am. J. Physiol. 272, G16–G22.[Medline]
Japanese Association for Laboratory Animal Science (1987). Guidelines for animal experimentation. Exp. Anim. 3, 285–288.
Jedlitschky, G., Leier, I., Buchholz, U., Hummel-Eisenbeiss, J., Burchell, B., and Keppler, D (1997). ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2. Biochem. J. 327, 305–310.[Medline]
Kamisako, T., Kobayashi, Y., Takeuchi, K., Ishihara, T., Higuchi, K., Tanaka, Y., Gabazza, E. C., and Adachi, Y. (2000). Recent advances in bilirubin metabolism research: The molecular mechanism of hepatocyte bilirubin transport and its clinical relevance. J. Gastroenterol. 35, 659–664.[CrossRef][Medline]
Kurisu, H., Kamisaka, K., Koyo, T., Yamasuge, S., Igarashi, H., Maezawa, H., Uesugi, T., and Tagaya, O. (1991). Organic anion transport study in mutant rats with autosomal recessive conjugated hyperbilirubinemia. Life Sci. 49, 1003–1011.[CrossRef][ISI][Medline]
Lee, Y. M., Cui, Y., König, J., Risch, A., Jäger, B., Drings, P., Bartsch, H., Keppler, D., and Nies, A. T. (2004). Identification and functional characterization of the natural variant MRP3-Arg1297His of human multidrug resistance protein 3 (MRP3/ABCC3). Pharmacogenetics 14, 213–223.[CrossRef][Medline]
Li, N., Hartley, D. P., Cherrington, N. J., and Klaassen, C. D. (2002). Tissue expression, ontogeny, and inducibility of rat organic anion transporting polypeptide 4. J. Pharmacol. Exp. Ther. 301, 551–560.
Llesuy, S. F., and Tomaro, M. L. (1994). Heme oxygenase and oxidative stress. Evidence of involvement of bilirubin as physiological protector against oxidative damage. Biochim. Biophys. Acta 1223, 9–14.[Medline]
López-Garcia, M. P., Dansette, P. M., and Mansuy, D. (1994). Thiophene derivatives as new mechanism-based inhibitors of cytochromes P-450: Inactivation of yeast-expressed human liver cytochrome P-450 2C9 by tienilic acid. Biochemistry 33, 166–175.[CrossRef][Medline]
Newton, D. J., Wang, R. W., and Evans, D. C. (2005). Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR(–) and EHBR rats. Life Sci. 77, 1106–1115.[CrossRef][Medline]
Nishiya, T., Kataoka, H., Mori, K., Sugawara, T., and Furuhama, K. (2004). Proceedings of the 31st Japanese Society of Toxicology Annual Meeting (P6-01). J. Toxicol. Sci. 29, 411.
Ogawa, K., Suzuki, H., Hirohashi, T., Ishikawa, T., Meier, P. J., 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, G438–G446.
Ohmori, S., Kuriya, S., Uesugi, T., Horie, T., Sagami, F., Mikami, T., Kawaguchi, A., Rikihisa, T., and Kanakubo, Y. (1991). Decrease in the specific forms of cytochrome P-450 in liver microsomes of a mutant strain of rat with hyperbilirubinuria. Res. Commun. Chem. Pathol. Pharmacol. 72, 243–253.[Medline]
Oude Elferink, R. P., and Jansen, P. L. (1994). The role of the canalicular multispecific organic anion transporter in the disposal of endo- and xenobiotics. Pharmacol. Ther. 64, 77–97.[CrossRef][ISI][Medline]
Reichel, C., Gao, B., Van Montfoort, J., Cattori, V., Rahner, C., Hagenbuch, B., Stieger, B., Kamisako, T., and Meier, P. J. (1999). Localization and function of the organic anion-transporting polypeptide Oatp2 in rat liver. Gastroenterology 117, 688–695.[CrossRef][ISI][Medline]
Sasabe, H., Tsuji, A., and Sugiyama, Y. (1998). Carrier-mediated mechanism for the biliary excretion of the quinolone antibiotic grepafloxacin and its glucuronide in rats. J. Pharmacol. Exp. Ther. 284, 1033–1039.
Sato, H., Aono, S., Kashiwamata, S., and Koiwai, O. (1991). Genetic defect of bilirubin UDP-glucuronosyltransferase in the hyperbilirubinemic Gunn rat. Biochem. Biophys. Res. Commun. 177, 1161–1164.[CrossRef][Medline]
Sellman, R., and Parkki, M. G. (1983). Induction of microsomal epoxide hydrolase by tienilic acid in the rat. J. Appl. Toxicol. 3, 245–248.[Medline]
Soroka, C. J., Lee, J. M., Azzaroli, F., and Boyer, J. L. (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.[CrossRef][ISI][Medline]
Takagi, S., Takayama, S., and Onodera, T. (1991). Hepatotoxicity of DR-3438, tienilic acid, indacrinone and furosemide studied in vitro. Toxicol. Lett. 55, 287–293.[Medline]
Tenhunen, R., Marver, H. S., and Schmid, R. (1968). The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Natl. Acad. Sci. U.S.A. 61, 748–755.
Yamaguchi, T., Terakado, M., Horio, F., Aoki, K., Tanaka, M., and Nakajima, H. (1996). Role of bilirubin as an antioxidant in an ischemia-reperfusion of rat liver and induction of heme oxygenase. Biochem. Biophys. Res. Commun. 223, 129–135.[CrossRef][ISI][Medline]
Zimmerman, H. J., Abernathy, C. O., Lukacs, L., and Ezekiel, M. (1982). Effects of ticrynafen on hepatic excretory function in the isolated perfused rat liver. Hepatology 2, 255–257.[Medline]
Zimmerman, H. J., Lewis, J. H., Ishak, K. G., and Maddrey, W. C. (1984). Ticrynafen-associated hepatic injury: Analysis of 340 cases. Hepatology 4, 315–323.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||