* Laboratory of Metabolism, CCR, NCI, NIH, Bethesda, Maryland; Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut;
Veterinary and Tumor Pathology Section, Center for Cancer Research, NCI, Frederick, Maryland;
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas; and ¶ Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas
Received August 24, 2004; accepted August 25, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: PXR; acetaminophen; hepatotoxicity; nuclear receptor.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isoforms of CYP3A may play an important role in metabolizing acetaminophen to its toxic intermediate metabolite, NAPQI. The kinetics of NAPQI formation by reconstituted human liver CYP3A4 is compatible with the therapeutic doses of this drug (Thummel et al., 1993), indicating that CYP3A4 is involved in APAP metabolism. Inducers of CYP3A potentiate, while inhibitors of CYP3A prevent, APAP toxicity (DiPetrillo et al., 2002
; Madhu et al., 1992
; Sinclair et al., 1998
; van Bree et al., 1989
). However, the role of CYP3A in acetaminophen metabolism still remains controversial as revealed by other studies in which CYP3A is negligible in APAP metabolism in human, and CYP3A inducers protect hamsters from APAP induced hepatic injury (Madhu and Klaassen, 1991
; Manyike et al., 2000
). The CYP3A enzymes metabolize over half of the pharmaceuticals on the current market, and the expression levels of CYP3A enzymes are modulated by a variety of xenobiotics, including numerous chemicals and dietary compounds. Many of these chemicals induce CYP3A by acting as ligands for the pregnane X receptor (PXR) in rodents or for the steroid xenobiotic receptor (SXR), the PXR homologue in humans (Moore and Kliewer, 2000
). PXR is a ligand activated transcription factor and belongs to the orphan nuclear receptor superfamily. When activated, PXR induces a network of genes that encode phase I and phase II xenobiotic metabolizing enzymes and drug transporters by forming a heterodimer with the universal partner for class II nuclear receptors, retinoid X receptor (RXR), which subsequently binds to the promoter or enhancer regions of PXR target genes (Guo et al., 2002
; Kliewer, 2003
; Kliewer et al., 1998
; Lehmann et al., 1998
). The activation of PXR results in enhanced metabolism and/or excretion of many endogenous chemicals or xenobiotics. The role of orphan nuclear receptors in APAP metabolism was explored and recent studies show that peroxisome proliferators-activated receptor
(PPAR
) and the constitutive androstane receptor (CAR) play roles in APAP induced hepatic injury (Chen et al., 2000
; Zhang et al., 2002
).
In order to investigate the role of PXR in APAP-induced hepatic toxicity, wild-type or PXR-null mice were pretreated with pregnenalone 16-carbonitrile prior to the administration of a toxic dose of APAP. Pretreatment with PCN dramatically augmented APAP-induced hepatic injury in wild-type, but not in PXR-null mice. The severity of APAP toxicity correlated very well with the levels of CYP3A11 expression. In summary, the current study demonstrates an important role for PXR in APAP-induced hepatic toxicity via induction of CYP3A11, and therefore confirms that CYP3A11 is critical in APAP reactive intermediate metabolite formation in mice.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and treatments. Mice were housed in a pathogen-free animal facility under standard 12-h light/12-h dark cycle with access to chow and water ad libitum. All protocols and procedures were approved by the NCI Animal Care and Use Committee and are in accordance with the National Institute of Health and ALAC Guidelines. The 8- to 12-week-old male B6; 129-Pxrtm1GlaxoWellcome (PXR-null) mice or their corresponding wild-type controls were used throughout the study (n = 5 to 7 per group). The animals were pretreated with vehicle (corn oil, ip) or PCN (75 mg/kg, ip) for two days before one APAP administration. APAP was dissolved in alkaline solution, and delivered to animals at 350 mg/kg, ip.
Tissue collection (urine, serum, and liver). Pooled urine was collected from five animals placed in metabolic cages for 6 h following the administration of APAP. Blood was collected by orbital plexus bleeding at 6 and 24 h after APAP administration. Serum was separated from whole blood by centrifugation at 6000 x g using Microcontainer serum separating tubes (Becton Dickinson, Franklin Lakes, NJ). Following blood collection, the animals were euthanized. Livers and kidneys were collected, divided and either stored in 10% phosphate buffered formalin for histological analysis, or snap-frozen in liquid nitrogen and stored at 80°C for future analysis.
Urinary analysis of APAP metabolites. Urine was analyzed for APAP and its metabolites using an HPLC method modified from that of Howie et al. (1977) as previously described (Manautou et al., 1996
). Retention times of APAP and its metabolites were determined by comparison with that of authentic standards. Since this HPLC method does not separate the cysteinylglycine and cysteine conjugates of APAP, they were quantitated together as APAP-CG/CYS. Preliminary chromatographic analysis of control urine samples shows no interfering peaks. Quantitation was based on integrated peak areas. The concentrations of APAP and its metabolites were calculated using an APAP standard curve since the molar extinction coefficients of APAP and its conjugated metabolites are approximately the same (Howie et al., 1977
).
Analysis of hepatic non-protein sulfhydryls (NPSH). Liver samples obtained from mice killed at 6 h after APAP challenge were homogenized (20% w/v) in 5% trichloroacetic acid/ethylenediamine tetraacetic acid (TCA/EDTA). Homogenates were centrifuged at 1500 x g for 15 min. NPSH concentration in supernatants was determined as an indicator of reduced glutathione (GSH) following the colorimetric procedure of Ellman (Boyne and Ellman, 1972; Ellman, 1959
). NPSH concentration was quantified by comparison with a GSH standard curve.
Northern blot analysis of gene expression. Total RNA was prepared using Trizol reagent (Amersham Biosciences; Piscataway, NJ) and analyzed by electrophoresis in 0.22 M formaldehyde-containing 1% agarose gel. The sequences for the cDNA probes (CYP3A11, CYP2E1, CYP1A2, GST and control 18 s) are available upon request. Probes were 32P-labeled by the random primer method using ready-to-Go DNA labeling beads (Amersham Biosciences, Piscataway, NJ). The details for the Northern blot analysis have been described in detail previously (Guo et al., 2003
).
Histological analysis. Tissues were fixed in 10% phosphate buffered formalin, and embedded in paraffin. Sections were prepared and stained with hematoxylin and eosin, and subjected to a blind review by a pathologist.
Statistical analysis. The data were expressed as mean ± SE. Statistical significance was determined by one-way analysis of variance, followed by Duncan's posthoc test. The significance level was set at p < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study clearly shows that PXR plays a pivotal role in modulating APAP metabolism. Several lines of evidence have suggested that hepatic nuclear receptors are involved in APAP-induced hepatotoxicity. For example, activation of peroxisomal proliferator-activated receptor (PPAR
) appears to reduce the severity of APAP-induced hepatic injury through induction of numerous genes involved in pathways such as stress response, DNA damage repair, and cell cycle regulation (Chen et al., 2000
; Shankar et al., 2003
). Similarly, APAP toxicity appears to be enhanced by phenobarbital (PB), which may be attributed to the activation of CAR, which induces expression of CYP3A and CYP1A2 that metabolize APAP to its reactive intermediate metabolite (Zhang et al., 2002
). Interestingly, treatment with androstanol, a specific CAR inhibitor but a modest PXR ligand, abrogated APAP induced-hepatotoxicity in wild-type mice, but produced an even greater toxicity in the CAR-null mice. This former study may be interpreted to suggest that while CAR activation critically affects APAP induced-hepatotoxicity, activation of PXR activity influences APAP hepatotoxicity consistent with the results seen here. Modulating PXR activity can result in a markedly different outcome for drug metabolism. PXR is activated by numerous structurally unrelated compounds (Kliewer et al., 1998
) and its activation affects the expression of a network of genes encoding critical enzymes or transporters involved in hepatic and enteric drug metabolism (Kliewer, 2003
). Thus it is reasonable to predict that drugdrug interactions are one of the consequences after modulating PXR activity by endogenous or xenobiotic factors.
The data presented here clearly suggests that CYP3A11 is involved in APAP-induced hepatic toxicity in mice. The expression levels of CYP3A11 correlated well with the severity of APAP-induced hepatic injury. For example, the basal levels of CYP3A11 expression were higher in PXR-null mice compared to wild-type mice, and correlated with a more severe APAP-induced hepatic toxicity as revealed by serum ALT levels and the appearance of hepatic centrilobular necrosis. Although the role of GST in NAPQI detoxification remains controversial, the reduced levels of GST
in the livers of PXR-null mice may also contribute to the more severe APAP-induced hepatic injury. The CYP2E1 and CYP1A2 enzymes are reported the two major forms of P450 s for the generation of NAPQI (Lee et al., 1996
; Tonge et al., 1998
; Zaher et al., 1998
), but there are studies suggesting that other mechanisms of bioactivation exist. For example, the CYP2E1-null mice are resistant to APAP toxicity even at high doses of APAP (Lee et al., 1996
). The CYP1A2-null mice, on the other hand, fail to demonstrate that they are less sensitive to APAP toxicity, even within a low dose range (Tonge et al., 1998
). Furthermore, despite relatively lower basal levels of CYP1A2 and similar levels of CYP2E1 in PXR-null mice livers compared to those in the wild-type mice, the APAP toxicity was more severe in the PXR-nulls, indicating that other P450 enzymes, probably CYP3A11, contributed significantly to NAPQI formation. The role of the CYP3A family in APAP metabolism has been long speculated upon, and is confirmed to a certain degree by studies using the so-called CYP3A-specific inhibitors; however, the lack of a CYP3A family gene knockout model prohibits the direct investigation of this issue. Nevertheless, by using an indirect approach in the study here, a PXR-null mouse model confirmed the critical role of PXR-regulated CYP3A11 in APAP metabolism.
PXR also plays a critical role in regulating phase II xenobiotic metabolizing enzymes and transporters that are important to detoxifying xenobiotics. Although the role of GST in NAPQI detoxification is not completely clear, it is reported to be the major phase II enzyme responsible for detoxifying NAPQI with APAP overdose. However, our study showed that GST
was induced by PCN in both wild-type and PXR-null mice; this is probably due to overlapping activation of other nuclear receptors, such as CAR that induces or stabilizes GST
mRNA. Therefore the effect of GST
by PCN in both wild-type and PXR-null mice on APAP detoxification is similar and can not count for the more severe APAP-induced hepatic injury in the PXR-null mice prior to PCN pretreatment. Possible activation of other nuclear receptors was also suggested by CYP1A2 expression, which was induced to a moderate degree by PCN and inhibited by APAP administration regardless of the genotypes or the combination with PCN, indicating that other nuclear receptors, such as CAR that cross-talks with PXR (Zhang et al., 2002
), might be involved in regulating CYP1A2 expression in these animals. The APAP metabolites are excreted out of the liver into the circulation or bile by hepatic transporters, such as Mrp3 and/or Mrp2 (Chen et al., 2003
). The expression of Mrp2 and Mrp3 is induced by PXR activation (Kast et al., 2002
; Teng et al., 2003
). Thus, activation of PXR enhances APAP-induced hepatic injury by induction of CYP3A isoforms, but on the other hand, also aids in the detoxification of APAP overdose by induction of phase II enzymes and transporters. However, data from this study showed that bioactivation of APAP to its reactive intermediate metabolite, NAPQI, by CYP3A11 appeared to overcome the detoxification process mediated by phase II enzymes and transporters, resulting in more severe APAP-induced hepatic injury.
In summary, this study demonstrated that activation of PXR dramatically enhanced APAP-induced hepatic injury mainly via induction of CYP3A. This finding implies that chemicals that modulate PXR activity will have a significant effect on the outcome following APAP administration, and it adds another layer of complexity in understanding APAP metabolism and treatment for APAP overdose patients.
![]() |
NOTES |
---|
2 To whom correspondence should be addressed at Bldg. 37, Rm 3106B, CCR, NCI, NIH, Bethesda, MD 20892. Fax: (301) 496-8419. E-mail: fjgonz{at}helix.nih.gov.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chen, C., Hennig, G. E., and Manautou, J. E. (2003). Hepatobiliary excretion of acetaminophen glutathione conjugate and its derivatives in transport-deficient (TR-) hyperbilirubinemic rats. Drug Metab. Dispos. 31, 798804.
Chen, C., Hennig, G. E., Whiteley, H. E., Corton, J. C., and Manautou, J. E. (2000). Peroxisome proliferator-activated receptor alpha-null mice lack resistance to acetaminophen hepatotoxicity following clofibrate exposure. Toxicol. Sci. 57, 338344.
Dahlin, D. C., Miwa, G. T., Lu, A. Y., and Nelson, S. D. (1984). N-acetyl-p-benzoquinone imine: A cytochrome P-450-mediated oxidation product of acetaminophen. Proc. Natl. Acad. Sci. U.S.A. 81, 13271331.[Abstract]
DiPetrillo, K., Wood, S., Kostrubsky, V., Chatfield, K., Bement, J., Wrighton, S., Jeffery, E., Sinclair, P., and Sinclair, J. (2002). Effect of caffeine on acetaminophen hepatotoxicity in cultured hepatocytes treated with ethanol and isopentanol. Toxicol. Appl. Pharmacol. 185, 9197.[CrossRef][ISI][Medline]
Ellman, G. L. (1959). Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 7077.[ISI][Medline]
Gonzalez, F. J., and Kimura, S. (2003). Study of P450 function using gene knockout and transgenic mice. Arch. Biochem. Biophys. 409, 153158.[CrossRef][ISI][Medline]
Gregus, Z., Madhu, C., and Klaassen, C. D. (1988). Species variation in toxication and detoxication of acetaminophen in vivo: A comparative study of biliary and urinary excretion of acetaminophen metabolites. J. Pharmacol. Exp. Ther. 244, 9199.[Abstract]
Guo, G. L., Lambert, G., Negishi, M., Ward, J. M., Brewer, H. B., Jr., Kliewer, S. A., Gonzalez, F. J., and Sinal, C. J. (2003). Complementary roles of farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor in protection against bile acid toxicity. J. Biol. Chem. 278, 4506245071.
Guo, G. L., Staudinger, J., Ogura, K., and Klaassen, C. D. (2002). Induction of rat organic anion transporting polypeptide 2 by pregnenolone-16alpha-carbonitrile is via interaction with pregnanexreceptor. Mol. Pharmacol. 61, 832839.
Henderson, C. J., Wolf, C. R., Kitteringham, N., Powell, H., Otto, D., and Park, B. K. (2000). Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi. Proc. Natl. Acad. Sci. U.S.A. 97, 1274112745.
Howie, D., Adriaenssens, P. I., and Prescott, L. F. (1977). Paracetamol metabolism following overdosage: Application of high performance liquid chromatography. J. Pharm. Pharmacol. 29, 235237.[ISI][Medline]
Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z., Hashimoto, M., and Mitchell, J. R. (1974). Acetaminophen-induced hepatic necrosis. VI. Metabolic disposition of toxic and non-toxic dose of acetaminophen. Pharmacology 12, 251271.[CrossRef][ISI][Medline]
Kast, H. R., Goodwin, B., Tarr, P. T., Jones, S. A., Anisfeld, A. M., Stoltz, C. M., Tontonoz, P., Kliewer, S., Willson, T. M., and Edwards, P. A. (2002). Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane x receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J. Biol. Chem. 277, 29082915.
Kliewer, S. A. (2003). The nuclear pregnane X receptor regulates xenobiotic detoxification. J. Nutr. 133, 2444S2447S.
Kliewer, S. A., Moore, J. T., Wade, L., Staudinger, J. L., Watson, M. A., Jones, S. A., McKee, D. D., Oliver, B. B., Willson, T. M., Zetterstrom, R. H., Perlmann, T., and Lehmann, J. M. (1998). An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92, 7382.[ISI][Medline]
Lee, S. S. T., Buters, J. T., Pineau, T., Fernandez-Salguero, P., and Gonzalez, F. J. (1996). Role of CYP2E1 in the hepatotoxicity of acetaminophen. J. Biol. Chem. 271, 1206312067.
Lehmann, J. M., McKee, D. D., Watson, M. A., Willson, T. M., Moore, J. T., and Kliewer, S. A. (1998). The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 102, 10161023.
Madhu, C., and Klaassen, C. D. (1991). Protective effect of pregnenolone-16 alpha-carbonitrile on acetaminophen-induced hepatotoxicity in hamsters. Toxicol. Appl. Pharmacol. 109, 305313.[ISI][Medline]
Madhu, C., Maziasz, T., and Klaassen, C. D. (1992). Effect of pregnenolone-16 alpha-carbonitrile and dexamethasone on acetaminophen-induced hepatotoxicity in mice. Toxicol. Appl. Pharmacol. 115, 191198.[ISI][Medline]
Manautou, J. E., Tveit, A., Hoivik, D. J., Khairallah, E. A., and Cohen, S. D. (1996). Protection by clofibrate against acetaminophen hepatotoxicity in male CD-1 mice is associated with an early increase in biliary concentration of acetaminophen-glutathione adducts. Toxicol. Appl. Pharmacol. 140, 3038.[CrossRef][ISI][Medline]
Manyike, P. T., Kharasch, E. D., Kalhorn, T. F., and Slattery, J. T. (2000). Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation. Clin. Pharmacol. Ther. 67, 275282.[ISI][Medline]
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis.IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211217.[ISI][Medline]
Moore, J. T., and Kliewer, S. A. (2000). Use of the nuclear receptor PXR to predict drug interactions. Toxicology 153, 110.[CrossRef][ISI][Medline]
Ostapowicz, G., Fontana, R. J., Schiodt, F. V., Larson, A., Davern, T. J., Han, S. H., McCashland, T. M., Shakil, A. O., Hay, J. E., Hynan, L., Crippin, J. S., Blei, A. T., Samuel, G., Reisch, J., and Lee, W. M. (2002). Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann. Intern. Med. 137, 947954.
Shankar, K., Vaidya, V. S., Corton, J. C., Bucci, T. J., Liu, J., Waalkes, M. P., and Mehendale, H. M. (2003). Activation of PPAR-alpha in streptozotocin-induced diabetes is essential for resistance against acetaminophen toxicity. Faseb J. 17, 17481750.
Sinclair, J., Jeffery, E., Wrighton, S., Kostrubsky, V., Szakacs, J., Wood, S., and Sinclair, P. (1998). Alcohol-mediated increases in acetaminophen hepatotoxicity: role of CYP2E and CYP3A. Biochem. Pharmacol. 55, 15571565.[CrossRef][ISI][Medline]
Teng, S., Jekerle, V., and Piquette-Miller, M. (2003). Induction of ABCC3 (MRP3) by pregnane X receptor activators. Drug Metab. Dispos. 31, 12961299.
Thummel, K. E., Lee, C. A., Kunze, K. L., Nelson, S. D., and Slattery, J. T. (1993). Oxidation of acetaminophen to N-acetyl-p-aminobenzoquinone imine by human CYP3A4. Biochem. Pharmacol. 45, 15631569.[CrossRef][ISI][Medline]
Tonge, R. P., Kelly, E. J., Bruschi, S. A., Kalhorn, T., Eaton, D. L., Nebert, D. W., and Nelson, S. D. (1998). Role of CYP1A2 in the hepatotoxicity of acetaminophen: investigations using Cyp1a2 null mice. Toxicol. Appl. Pharmacol. 153, 102108.[CrossRef][ISI][Medline]
van Bree, L., Groot, E. J., and De Vries, J. (1989). Reduction by acetylsalicylic acid of paracetamol-induced hepatic glutathione depletion in rats treated with 4,4'-dichlorobiphenyl, phenobarbitone and pregnenolone-16-alpha-carbonitrile. J. Pharm. Pharmacol. 41, 343345.[ISI][Medline]
Zaher, H., Buters, J. T., Ward, J. M., Bruno, M. K., Lucas, A. M., Stern, S. T., Cohen, S. D., and Gonzalez, F. J. (1998). Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice. Toxicol. Appl. Pharmacol. 152, 193199.[CrossRef][ISI][Medline]
Zhang, J., Huang, W., Chua, S. S., Wei, P., and Moore, D. D. (2002). Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science 298, 422424.