Genetic Susceptibility to Diclofenac-Induced Hepatotoxicity: Contribution of UGT2B7, CYP2C8, and ABCC2 Genotypes

doi:10.1053/j.gastro.2006.11.023

Gastroenterology

Volume 132, Issue 1, January 2007, Pages 272-281

https://doi.org/10.1053/j.gastro.2006.11.023 Get rights and content

Background & Aims: Diclofenac is a widely used nonsteroidal anti-inflammatory drug and is among the most common drugs causing idiosyncratic hepatotoxicity in several recent series with up to 20% mortality in jaundiced subjects. We hypothesized that susceptibility to hepatotoxicity would be associated with genetic polymorphisms in the genes encoding the enzymes UGT2B7 and CYP2C8, which determine the formation of reactive diclofenac metabolites and in ABCC2 encoding the transporter MRP2 contributing to the biliary excretion of the reactive metabolite. Methods: Twenty-four patients (19 female) aged 24–70 (mean, 50.8) years who had suffered diclofenac hepatotoxicity, 48 subjects (35 female) aged 22–77 (mean, 52) years who were taking diclofenac for 0.3–20 (mean, 4) years without developing hepatotoxicity (hospital controls), and 112 healthy controls were investigated. Genotyping for several polymorphisms in the genes encoding UGT2B7, CYP2C8, and ABCC2 was performed and haplotypes assigned. Results: The UGT2B7*2 allele was more common in diclofenac hepatotoxicity patients compared with hospital controls (odds ratio [OR], 8.5, P = .03) and healthy controls (OR, 7.7, P = .03). The ABCC2 C-24T variant was more common in hepatotoxicity patients compared with hospital (OR, 5.0, P = .005) and healthy controls OR: 6.3, P = .0002). Haplotype distributions for CYP2C8 were different in patients compared with hospital controls (P = .04). Conclusions: Allelic variants of UGT2B7, CYP2C8, and ABCC2, which may predispose to the formation and accumulation of reactive diclofenac metabolites are associated with diclofenac hepatotoxicity. Increased level of reactive metabolites may lead to higher levels of protein-diclofenac adducts and subsequently hepatotoxicity.

Section snippets

Materials and Methods

Ethical approval for the study was obtained from Newcastle upon Tyne and North Tyneside local ethics committee, and all the subjects who took part in the study gave informed consent.

Group 1

Forty-eight white subjects (35 female, 75%), aged 22–77 (mean, 52) years who were taking diclofenac for 0.3–20 (mean, 4) years duration without developing hepatotoxicity on the basis of normal liver function tests at the time of recruitment were recruited from the rheumatology outpatient clinic of the Newcastle Hospitals Trust. Underlying diagnoses were rheumatoid arthritis in 34, psoriatic arthritis in 7, osteoarthritis in 4, ankylosing spondylitis in 2, and juvenile arthritis in 1.

Group 2

A local

Polymerase Chain Reactions

Polymerase chain reactions (PCRs) were performed in a final volume of 25 μL with 0.25 μmol/L each primer, 0.1 mmol/L each dNTP, 0.2–0.5 μg genomic DNA, 0.625 U Taq polymerase (Molzyme, Bremen, Germany) in 50 mmol/L potassium chloride, 10 mmol/L Tris-HCl pH 9.0, 0.1% (vol/vol) Triton X-100, and 1.5 mmol/L MgCl₂. Amplification normally involved 35 cycles of 94°C for 1 minute, a primer set-specific annealing temperature for 1 minute (Table 1), and 72°C for 1 minute followed by a final extension of

Restriction Fragment Length Polymorphism-PCR Assays

Restriction fragment length polymorphism (RFLP)-PCR assays were developed for a range of polymorphisms as summarized in Table 1. When possible, natural restriction sites were used to differentiate between genotypes, but when this was not feasible, sites were engineered using mismatched primers. For the majority of the assays, restriction fragments were separated by electrophoresis on 10% polyacrylamide gels in 1×TBE buffer followed by staining with ethidium bromide except for the assays for

PCR Single-Strand Conformation Polymorphism Assay

For the CYP2C8 A1196G polymorphism, a minor modification of the single-strand conformation polymorphism (SSCP)-based method described previously²⁹ was used. Following PCR as summarized in Table 1, SSCP analysis was performed on 1X Mutation Detection Enhancement (MDE; BioWhittaker Molecular Applications, Rockland, ME) in TME buffer (0.3 mol/L Tris, 0.35 mol/L MES, 10 mmol/L EDTA, pH 6.8) containing 2.5 mol/L urea as previously described.²⁹ Eight-μL PCR product was denatured at 95°C for 3 minutes

Relationship Between C801T and C-161T UGT2B7 Polymorphisms

To assess the relationship between the 2 previously reported UGT2B7 polymorphisms, PCR-RFLP assays using the restriction enzymes FokI and BbsI were developed for each. Each of 112 healthy control DNA samples was analyzed for both polymorphisms. As shown in Table 2 for all but 4 alleles in the group, the polymorphisms at -161 and 801 were in complete linkage disequilibrium, with T at -161 associated with T at position 801 and vice versa.

Relationship Between UGT2B7 Genotype and Susceptibility to Diclofenac Hepatotoxicity

All 24 DNA samples from diclofenac hepatotoxicity patients

Discussion

Although diclofenac is one of the most common causes of drug-induced liver injury,⁴³ this disease is very rare, making recruitment of large patient cohorts extremely difficult. We have succeeded in collecting 24 DNA samples from cases in which the phenotype is well defined, and because of the difficulty in accruing larger numbers of cases, we have aimed to recruit higher numbers of controls, including a group of patients who have not suffered the toxicity when exposed to the drug. It has been

References (50)

M.I. Lucena et al.
Comparison of two clinical scales for causality assessment in hepatotoxicity
Hepatology
(2001)
L. Ibanez et al.
Prospective surveillance of acute serious liver disease unrelated to infectious, obstructive, or metabolic diseases: epidemiological and clinical features, and exposure to drugs
J Hepatol
(2002)
R.J. Andrade et al.
Drug-induced liver injury: an analysis of 461 incidences submitted to the Spanish registry over a 10-year period
Gastroenterology
(2005)
U.A. Boelsterli
Diclofenac-induced liver injury: a paradigm of idiosyncratic drug toxicity
Toxicol Appl Pharmacol
(2003)
R. Bort et al.
Hepatic metabolism of diclofenac: Role of human CYP in the minor oxidative pathways
Biochem Pharmacol
(1999)
W.E. Gall et al.
Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7
J Steroid Biochem Mol Biol
(1999)
N. Bahadur et al.
CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6a-hydroxylase activity in human liver microsomes
Biochem Pharmacol
(2002)
G.P. Aithal et al.
Clinical diagnostic scale: a useful tool in the evaluation of suspected hepatotoxic adverse drug reactions
J Hepatol
(2000)
M. Stephens et al.
A new statistical method for haplotype reconstruction from population data
Am J Hum Genet
(2001)
A.T. Banks et al.
Diclofenac-associated hepatotoxicity: analysis of 180 cases reported to the Food and Drug Administration as adverse reactions
Hepatology
(1995)

A. Kretz-Rommel et al.

Cytotoxic activity of T cells and non-T cells from diclofenac-immunized mice against cultured syngeneic hepatocytes exposed to diclofenac

Hepatology

(1995)

V. Keitel et al.

Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome

Hepatology

(2000)

U. Yasar et al.

Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms

Biochem Biophys Res Commun

(2002)

F.J. de Abajo et al.

Acute and clinically relevant drug-induced liver injury: a population based case-control study

Br J Clin Pharmacol

(2004)

E. Bjornsson et al.

Outcome and prognostic markers in severe drug-induced liver disease

Hepatology

(2005)

E. Bjornsson et al.

Fulminant drug-induced hepatic failure leading to death or liver transplantation in Sweden

Scand J Gastroenterology

(2005)

U.A. Boelsterli et al.

Idiosyncratic liver toxicity of nonsteroidal antiinflammatory drugs: molecular mechanisms and pathology

Crit Rev Toxicol

(1995)

G.P. Aithal et al.

Relationship of polymorphism in CYP2C9 to genetic susceptibility to diclofenac-induced hepatitis

Pharmacogenetics

(2000)

W. Tang

The metabolism of diclofenac—enzymology and toxicology perspectives

Curr Drug Metab

(2003)

G.P. Aithal et al.

Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity

Hepatology

(2004)

S. Kumar et al.

Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide

J Pharmacol Exp Ther

(2002)

S. Seitz et al.

Selective protein adduct formation of diclofenac glucuronide is critically dependent on the rat canalicular conjugate export pump (Mrp2)

Chem Res Toxicol

(1998)

H. Stierlin et al.

Biotransformation of diclofenac sodium (Voltaren) in animals and in man. II. Quantitative determination of the unchanged drug and principal phenolic metabolites, in urine and bile

Xenobiotica

(1979)

C. Jin et al.

Complementary deoxyribonucleic acid cloning and expression of a human liver uridine diphosphate-glucuronosyltransferase glucuronidating carboxylic acid-containing drugs

J Pharmacol Exp Ther

(1993)

M. Holthe et al.

Sequence variations in the UDP-glucuronosyltransferase 2B7 (UGT2B7) gene: identification of 10 novel single nucleotide polymorphisms (SNPs) and analysis of their relevance to morphine glucuronidation in cancer patients

Pharmacogenomics J

(2003)

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: Supported by Newcastle University Hospitals Special Trustees.

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Basic–liver, pancreas, and biliary tractGenetic Susceptibility to Diclofenac-Induced Hepatotoxicity: Contribution of UGT2B7, CYP2C8, and ABCC2 Genotypes

Section snippets

Materials and Methods

Group 1

Group 2

Polymerase Chain Reactions

Restriction Fragment Length Polymorphism-PCR Assays

PCR Single-Strand Conformation Polymorphism Assay

Relationship Between C801T and C-161T UGT2B7 Polymorphisms

Relationship Between UGT2B7 Genotype and Susceptibility to Diclofenac Hepatotoxicity

Discussion

Hepatology

J Hepatol

Gastroenterology

Toxicol Appl Pharmacol

Biochem Pharmacol

J Steroid Biochem Mol Biol

Biochem Pharmacol

J Hepatol

Am J Hum Genet

Hepatology

Hepatology

Hepatology

Biochem Biophys Res Commun

Acute and clinically relevant drug-induced liver injury: a population based case-control study

Br J Clin Pharmacol

Outcome and prognostic markers in severe drug-induced liver disease

Hepatology

Fulminant drug-induced hepatic failure leading to death or liver transplantation in Sweden

Scand J Gastroenterology

Idiosyncratic liver toxicity of nonsteroidal antiinflammatory drugs: molecular mechanisms and pathology

Crit Rev Toxicol

Relationship of polymorphism in CYP2C9 to genetic susceptibility to diclofenac-induced hepatitis

Pharmacogenetics

The metabolism of diclofenac—enzymology and toxicology perspectives

Curr Drug Metab

Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity

Hepatology

Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide

J Pharmacol Exp Ther

Selective protein adduct formation of diclofenac glucuronide is critically dependent on the rat canalicular conjugate export pump (Mrp2)

Chem Res Toxicol

Biotransformation of diclofenac sodium (Voltaren) in animals and in man. II. Quantitative determination of the unchanged drug and principal phenolic metabolites, in urine and bile

Xenobiotica

Complementary deoxyribonucleic acid cloning and expression of a human liver uridine diphosphate-glucuronosyltransferase glucuronidating carboxylic acid-containing drugs

J Pharmacol Exp Ther

Sequence variations in the UDP-glucuronosyltransferase 2B7 (UGT2B7) gene: identification of 10 novel single nucleotide polymorphisms (SNPs) and analysis of their relevance to morphine glucuronidation in cancer patients

Pharmacogenomics J

Basic–liver, pancreas, and biliary tract
Genetic Susceptibility to Diclofenac-Induced Hepatotoxicity: Contribution of UGT2B7, CYP2C8, and ABCC2 Genotypes