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Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity in Korean patients with pulmonary tuberculosis
ARTICLE IN PRESS Tuberculosis (2007) 87, 551–556
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Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity in Korean patients with pulmonary tuberculosis Hyun-Jung Choa, Won-Jung Kohb, Yon-Ju Ryub, Chang-Seok Kia, Myung-Hyun Namc, Jong-Won Kima, Soo-Youn Leea, a
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, South Korea b Department of Pulmonary and Critical Care Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea c Department of Laboratory Medicine and Genetics, Korea University College of Medicine, Seoul, South Korea Received 12 October 2006; received in revised form 10 May 2007; accepted 21 May 2007
KEYWORDS Isoniazid; Hepatotoxicity; NAT2; CYP2E1; Korean
Summary Antituberculosis drug-induced hepatitis attributed to isoniazid (INH) is one of the most prevalent drug-induced liver injuries. INH is metabolized by hepatic N-acetyltransferase (NAT) and cytochrome P450 2E1 (CYP2E1) to form hepatotoxins. The aim of this study was to evaluate whether polymorphisms of the NAT2 and/or CYP2E1 genes were associated with antituberculosis drug-induced hepatotoxicity in Korean patients. A total of 132 patients with tuberculosis who received antituberculosis treatment were followed prospectively. Their NAT2 and CYP2E1 genotypes were determined using polymerase chain reaction (PCR) with or without sequencing. Eighteen (13.6%) patients developed antituberculosis drug-induced hepatotoxicity. Regarding NAT2, slow acetylators had a higher incidence of hepatotoxicity than rapid acetylators (36.8% vs. 9.7%, P ¼ 0.005) and there was a 3.8-fold risk of hepatotoxicity for the slow acetylators compared to the rapid acetylators. For the CYP2E1 gene, the RsaI polymorphism in the 50 untranslated region, and a polymorphic repetitive sequence at the CYP2E1 50 -flaking region were analyzed; there was no significant association between any CYP2E1 genotype and antituberculosis druginduced hepatotoxicity. In conclusion, slow acetylator status of NAT2 was a significant susceptibility risk factor for antituberculosis drug-induced hepatotoxicity; NAT2 genotyping may be a useful tool for predicting antituberculosis drug-induced hepatotoxicity. & 2007 Published by Elsevier Ltd.
Corresponding author. Tel.: +82 2 3410 1834; fax: +82 2 3410 2719.
E-mail address: [email protected] (S.-Y. Lee). 1472-9792/$ - see front matter & 2007 Published by Elsevier Ltd. doi:10.1016/j.tube.2007.05.012
ARTICLE IN PRESS 552
Introduction Despite the availability of effective chemotherapeutic agents to treat tuberculosis, the hepatotoxicity that results from use of these drugs remains a significant problem for clinical treatment.1–10 The incidence of antituberculosis drug-induced hepatotoxicity ranges from 1% to 36%, and mortality in such cases is not rare.1–10 Although there is a wide variability in reported incidence of antituberculosis drug-induced hepatotoxicity from different countries, there is a generally higher incidence of antituberculosis druginduced hepatotoxicity in Asian countries; this may be due to ethnic susceptibility and the inherent genetics of drug metabolism.4 Among first-line therapeutic drugs for tuberculosis, isoniazid (INH) is the major drug associated with drug-induced hepatotoxicity. INH is metabolized to acetylisoniazid via hepatic N-acetyltransferase2 (NAT2).6 In turn, acetylisoniazid is hydrolyzed to acetylhydrazine, which is oxidized by cytochrome P450 2E1 (CYP2E1) to form some hepatotoxic intermediates.11,12 Disposal of acetylhydrazine also depends on further acetylation by NAT2 to form a non-toxic metabolite, diacetylhydrazine.13,14 Previously reported studies have shown inconsistent results on whether slow acetylators or rapid acetylators cause antituberculosis drug-induced hepatotoxicity.2,6,8,13,15 Moreover the impact of CYP2E1, the major catalyzing enzyme in the formation of hepatotoxins, on the risk of developing antituberculosis drug-induced hepatotoxicity has not been fully explained. To determine the contribution of NAT2 and/or CYP2E1 gene polymorphisms in susceptibility to antituberculosis drug-induced hepatotoxicity in Koreans and to determine the genotype and phenotype for risk of antituberculosis drug-induced hepatotoxicity, we genotyped NAT2 and CYP2E1 in Korean patients with pulmonary tuberculosis.
Patients and methods Patients A total of 132 Korean patients with newly diagnosed tuberculosis at Samsung Medical Center from June 2004 to December 2005 were consecutively screened. Inclusion criteria consisted of: adult patients newly diagnosed with active tuberculosis, having evident lesion of tuberculosis by simple X-ray or computed tomography or positive results of sputum smear or culture for detection of mycobacteria. Patients with any of the following conditions were excluded from the study: (1) abnormal serum alanine transferase (ALT), aspartate transaminase (AST), or bilirubin levels or symptoms related to abnormal liver function such as jaundice before antituberculosis treatment; (2) alcoholic liver disease or habitual alcohol drinking; (3) any other hepatic or systemic diseases that may cause liver dysfunction. The Institutional Review Board at Samsung Medical Center Clinical Research Institute approved this study protocol, and signed informed consent was obtained from each subject prior to the commencement of the study. All patients received oral INH (300 mg), rifampicin (600 mg), pyrazinamide (20 mg/kg body weight), and ethambutol (800 mg) daily for the first 2 months. Pyrazinamide was
H.-J. Cho et al. then discontinued, while INH, rifampicin and ethambutol were continued for another 4 months. Except for 18 patients who had antituberculosis drug-induced hepatotoxicity, the duration of antituberculosis treatment was 6 months. Treatment compliance was assessed by comparing the number of administered treatment doses to the number of treatment doses scheduled each month. There were no consistently missing doses corresponding to more than 10 days of monthly scheduled doses of medication for all the patients. Before antituberculosis therapy, serum ALT, AST, alkaline phosphatase, direct and total bilirubin, hepatitis B virus tests including surface antigen, antibody to surface antigen, e antigen, antibody to e antigen and HBV DNA, immunoglobulin M antibody to hepatitis A virus, antibody to hepatitis C and antibody human immunodeficiency virus were measured. Serum AST, ALT, and total bilirubin levels were then monitored monthly until the end of treatment. Antituberculosis drug-induced hepatotoxicity was designated as an increase in serum ALT level more than two times the upper limit of the normal value (ULN) after antituberculosis treatment, according to the criteria of drug-induced liver injuries developed by the international consensus meeting.16 When antituberculosis drug-induced hepatotoxicity was identified, serum ALT, AST, and bilirubin were monitored every week thereafter.
Genotyping of NAT2 and CYP2E1 Genomic DNA was extracted from peripheral blood leukocytes using the Wizard Genomic DNA Purification kit following the manufacturer’s instructions (Promega, Madison, WI, USA). Polymerase chain reaction (PCR) with direct sequencing was used to genotype NAT2 with the appropriate primer sets (these are available upon request). The presence of any two mutant alleles defined a slow acetylator, whereas rapid acetylators had one or two wildtype NTA2*4 alleles. A 371-bp fragment in the 50 untranslated region (50 UTR) including the RsaI polymorphism was amplified and direct sequencing analysis was performed with an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA) using primer sets that were designed by the authors (these are available upon request). For the RsaI polymorphism of CYP2E1, the wild-type allele was c1, and the mutant allele was c2, that lacks the RsaI restriction site. To detect a polymorphic repetitive sequence at the CYP2E1 50 -flanking region, PCR amplifications were performed with a thermal cycler (model 9700, Applied Biosystems, Foster City, CA, USA) with appropriate primer sets (these are available upon request) and the PCR products were isolated by agarose gel electrophoresis and the products: 346, 394, or 490 bp, depending on the CYP2E1 genotype, were designated as *1A (5 repeats), *1C (6 repeats), and *1D (8 repeats), respectively.
Statistical methods Odds ratios (OR) and confidence intervals (CI) were calculated using logistics regression. The Mann–Whitney U-test, w2 tests with Yates’ correction, or Fisher’s exact test were used for the univariate analysis as appropriate. Multivariate analysis was performed to evaluate the
ARTICLE IN PRESS Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity
(36.8% vs. 9.7%; OR, 5.41; 95% CI, 1.76–16.59; P ¼ 0.005; Table 2) and there was a 3.8-fold risk of hepatotoxicity for the slow acetylators compared to the rapid acetylators. Sensitivity, specificity and positive and negative predictive values of NAT2 slow acetylator genotype are as follows: 0.39 (95% CI: 0.20–0.61), 0.89 (95% CI: 0.83–0.94), 0.37 (95% CI: 0.19–0.59), and 0.90 (95% CI: 0.83–0.94). A total of 75 patients (56.8%) were genotyped as CYP2E1 c1/c1, 53 patients (40.2%) as CYP2E1 c1/c2 and 4 patients (3.0%) as CYP2E1 c2/c2. There was no significant difference in the frequencies of c1/c1, c1/c2, and c2/c2 genotypes of CYP2E1 between the patients with hepatotoxicity and those without hepatotoxicity (Table 3). The polymorphic repetitive sequence at the CYP2E1 50 -flaking region, in patients with hepatotoxicity and those without hepatotoxicity, showed similar genotypes and allele frequencies (Table 3.). CYP2E1*1C was found to be the predominant allele (77.2% allele frequency); the allele frequencies of CYP2E1*1A and CYP2E1*1D were 2.3% and 20.5%,
adjusted risk of antituberculosis-induced hepatitis using a stepwise logistic regression analysis (SPSS 8.0 for Windows; SPSS Inc., Chicago, IL). A two-tailed P-value less than 0.05 was considered statistically significant.
Results Eighteen patients (13.6%) were diagnosed with antituberculosis drug-induced hepatotoxicity. There was no statistical difference in the age, gender, or body mass index between the patients with hepatotoxicity and those without hepatotoxicity (Table 1). Eleven patients (8.3%) had more than three times the ULN of ALT; thus, INH was discontinued temporarily. A total of 113 patients (85.6%) were genotyped as rapid acetylators, and the other 19 patients (14.4%) were slow acetylators. The risk of having antituberculosis druginduced hepatotoxicity was significantly higher in the slow acetylators compared to the rapid acetylators
Table 1
553
Characteristics of patients with or without antituberculosis drug-induced hepatotoxicity. With hepatotoxicity (n ¼ 18)
Without hepatotoxicity (n ¼ 114)
P-value
Sex (F/M) Age (yr) Body mass index (kg/m2)
6/12 51.2717.5 20.272.5
51/63 46.7718.4 18.472.9
0.447 0.392 0.546
Initial laboratory findings,y ALT (U/L) AST (U/L) Total bilirubin (mg/dL)
28.4715.8 20.9710.3 0.670.3
19.4713.2 23.9713.5 0.670.9
0.224 0.546 0.207
30.9721.9 (8–151) 32.4714.2 (15–82) 0.870.9 (0.1–1.7)
o0.001 o0.001 0.069
Laboratory findings during antituberculosis treatment,z Peak ALT (U/L) 291.87456.7 (86–2067) Peak AST (U/L) 446.77955.9 (62–4173) Peak total bilirubin (mg/dL) 1.672.3 (0.3–9.1)
ALT: alanine transferase; AST: aspartate transaminase. Mean7SD. y Reference interval of ALT, 0–40 U/L; AST, 0–40 U/L; total bilirubin, 0.2–1.5 mg/dL. z Mean7SD (range).
Table 2
NAT2 genotypes in tuberculosis patients with or without antituberculosis drug-induced hepatotoxicity. With hepatotoxicity (n ¼ 18)
Without hepatotixicity (n ¼ 114)
OR (95% CI)
P-value
Rapid or intermediate acetylator NAT2*4/*4 NAT2*4/*5A NAT2*4/*6A NAT2*4/*5B NAT2*4/*7B
11
102
0.18 (0.06–0.57)
0.005
7 1 2 0 1
45 2 32 4 19
Slow acetylator NAT2*5B/*7B NAT2*6A/*6A NAT2*6A/*7B NAT2*7B/*7B
7 0 3 4 0
12 1 2 7 2
5.41 (1.76–16.59)
0.005
OR: odds ratios; CI: confidence intervals.
ARTICLE IN PRESS 554
H.-J. Cho et al.
Table 3
Risk of antituberculosis drug-induced hepatotoxicity in relation to polymorphism in the CYP2E1 gene.
RsaI polymorphism c1/c1 c1/c2 or c2/c2
With hepatotoxicity (n ¼ 18)
Without hepatotixicity (n ¼ 114)
OR (95% CI)
P-value
10 8
65 49
0.94 (0.35–2.56) 1.06 (0.64–4.61)
1.000 1.000
0.83 (0.29–2.36)
0.322
1.38 (0.49–3.93)
0.322
Polymorphic repetitive sequence at the CYP2E1 50 -flaking region 6 43 *1D allele *1A/*1D 1 0 *1C/*1D 4 39 *1D/*1D 1 4 Non-*1D alleley *1A/*1A *1A/*1C *1C/*1C
12 0 0 12
71 1 3 67
OR: odds ratios; CI: confidence intervals. Group with any number of *1D alleles y Group with no *1D allele.
Table 4 status.
Combined risk for antituberculosis drug-induced hepatotoxicity associated with CYP2E1 genotype and acetylator
Acetylator status
CYP2E1 genotype
With hepatotoxicity (n ¼ 18)
Without hepatotoxicity (n ¼ 114)
OR (95% CI)
P-value
Slow Slow Slow Slow Rapid or intermediate Rapid or intermediate Rapid or intermediate Rapid or intermediate
c1/c2 or c2/c2 c1/c1 *1D allele Non-*1D alleley c1/c2 or c2/c2
3 4 3 4 5
4 7 5 6 44
5.5 (1.12–27) 3.96 (1.03–15.28) 4.36 (0.94–20.13) 5.14 (1.29–20.49) 0.61 (0.20–1.83)
0.053 0.044 0.077 0.031 0.441
c1/c1
6
59
0.47 (0.16–1.33)
0.205
*1D allele
2
39
0.24 (0.05–1.09)
0.057
Non-*1D allele
9
64
0.78 (0.29–2.11)
0.799
OR: odds ratios; CI: confidence intervals. Group with any number of *1D alleles. y Group with no *1D allele.
respectively. Combined analysis of the two polymorphisms in the CYP2E1 gene was performed but no synergistic effect was observed (data not shown). Considering the CYP2E1 genotype and the acetylator status together showed that the slow acetylator, regardless of CYP2E1 genotype, was associated with a higher incidence of hepatotoxicity compared to other combinations (Table 4).
Discussion Our study showed a significant association between the slow acetylator genotype of NAT2 and the risk of developing antituberculosis drug-induced hepatotoxicity in Korean tuberculosis patients (Table 2); these results are consistent
with those from previous reports.4,8,15,17,19 Among the many risk factors for antituberculosis drug-induced hepatotoxicity, acetylator status has been regarded to be the most important.2,6,8–10,13,15,17–19 However, there have been only a few reports to evaluate the association of acetylator status, determined by NAT2 genotyping, and the susceptibility of antituberculosis drug-induced hepatotoxicity.4,20–22 The NAT2 genotype has been proven to be one of the most important factors for inter-individual differences in plasma isoniazid (INH) concentration in Chinese tuberculosis patients.23 In our previous study, we showed that the concordance rate between NAT2 genotyping and phenotyping, by the plasma concentration ratio of acetylisoniazid (AcINH) to INH (pAcINH/INH), was 90.2%.24
ARTICLE IN PRESS Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity CYP2E1 is induced by ethanol and is critically important in the metabolic activation of many drugs and carcinogens.25,26 Among several CYP2E1 genetic polymorphisms, only the RsaI polymorphism has been evaluated in association with antituberculosis drug-induced hepatotoxicity27,28 and hepatocellular carcinoma.29 Recent studies have reported a significant association between the c1/c1 genotype and the risk of developing antituberculosis drug-induced hepatotoxicity, explained by a higher CYP2E1 activity with the c1/c1 genotype and the inhibitory effect of INH.27,28 We found no evidence for association of the c1/c1 genotype with antituberculosis drug-induced hepatotoxicity (Table 3). However, we evaluated the polymorphic repetitive sequence at the CYP2E1 50 -flaking region in addition to the RsaI polymorphism, which has not been previously reported. Of the CYP2E1 polymorphisms identified to date, only the CYP2E1*1D has been linked to altered function; it has been associated with increased metabolic capacity in obese individuals or those individuals consuming alcohol,30 consistent with increased induction of gene activity under these conditions. CYP2E1*1D allelic frequency, among our study population, was 20.5%, similar as those of previous reports in other Asian populations.31–34 Our results failed to show any significant association between the polymorphic repetitive sequence at the CYP2E1 50 -flaking region and susceptibility to hepatotoxicity, and there was no synergistic effect of the two polymorphisms of the CYP2E1 on the development of hepatotoxicity (Table 3). We then considered the combined risk, for antituberculosis drug-induced hepatotoxicity, associated with the CYP2E1 genotype and the acetylator status (Table 4); considering that genetic effect of CYP2E1 on hepatotoxicity would be inconspicuous compared to that of the NAT2. The statistically nonsignificant results regarding the CYP2E1 polymorphism and antituberculosis drug-induced hepatotoxicity may be due to the small sample size and low frequency of patients with antituberculosis drug-induced hepatotoxicity; re-evaluation and confirmation are needed in a large-scale population study. Potential limitations of the present study should be considered. The criteria in this study may be less strict than other criteria35,36 to assess antituberculosis drug-induced hepatotoxicity. Although these criteria may be low for defining clinically evident hepatotoxicity, we believe that they are clinically useful for finding adverse hepatic reactions earlier and for preventing the development of overt hepatotoxicity. Patients in this study were various concomitant drugs with INH. Co-administration of drugs may result in quantitative and qualitative alteration of the drug metabolism and it is very difficult to exclude the presence of a confounding factor in accessing the drug-induced hepatotoxicity. As rifampicin is known to reduce NAT2 activity,37,38 as INH is known to have a biphasic effect on CYP2E1 activity (initial inhibition followed by an induction)9 and because a combination of these drugs is known to increase the incidence of antituberculosis drug-induced hepatotoxicity up to 35%,3,9,37,38 drug–drug pharmacokinetic and pharmacodynamic interactions certainly did occur. There have been several reports about the relationship between NAT2 and antituberculosis drug-induced hepatotoxicity in Asian tuberculosis patients.4,20–22 Their reports and our study confirm that slow acetylators are significantly
555
associated with antituberculosis drug-induced hepatotoxicity in Asian population. In contrast, results of the study performed in Caucasians28 failed to show any significant association between NAT2 susceptibility to antituberculosis drug-induced hepatotoxicity. These discrepancies might well result from a different genotype distribution among Asian and Caucasian patients. Therefore, the determination of NAT2 genotypes for antituberculosis treatment may be clinically useful for the prediction and prevention of antituberculosis drug-induced hepatotoxicity in Asian tuberculosis patients. Funding: This study was supported by a grant of the Korean Health 21 R&D Project. Ministry of Health & Welfare, R.O.K. (03-PJ10-PG13-GD01-0002). Competing interests: None declared Ethical approval: Not required
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ARTICLE IN PRESS 556 14. Lauterburg BH, Smith CV, Todd EL, Mitchell JR. Pharmacokinetics of the toxic hydrazino metabolites formed from isoniazid in humans. J Pharmacol Exp Ther 1985;235:566–70. 15. Sarma GR, Immanuel C, Kailasam S, Narayana AS, Venkatesan P. Rifampin-induced release of hydrazine from isoniazid. A possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifampin. Am Rev Respir Dis 1986;133:1072–5. 16. Benichou C. Criteria of drug-induced liver disorders. Report of an international consensus meeting. J Hepatol 1990;11: 272–6. 17. Gronhagen-Riska C, Hellstrom PE, Froseth B. Predisposing factors in hepatitis induced by isoniazid-rifampin treatment of tuberculosis. Am Rev Respir Dis 1978;118:461–6. 18. Gurumurthy P, Krishnamurthy MS, Nazareth O, et al. Lack of relationship between hepatic toxicity and acetylator phenotype in three thousand South Indian patients during treatment with isoniazid for tuberculosis. Am Rev Respir Dis 1984;129:58–61. 19. Parthasarathy R, Sarma GR, Janardhanam B, et al. Hepatic toxicity in South Indian patients during treatment of tuberculosis with short-course regimens containing isoniazid, rifampicin and pyrazinamide. Tubercle 1986;67:99–108. 20. Hiratsuka M, Kishikawa Y, Takekuma Y, et al. Genotyping of the N-acetyltransferase2 polymorphism in the prediction of adverse drug reactions to isoniazid in Japanese patients. Drug Metab Pharmacokinet 2002;17:357–62. 21. Ohno M, Yamaguchi I, Yamamoto I, et al. Slow N-acetyltransferase 2 genotype affects the incidence of isoniazid and rifampicin-induced hepatotoxicity. Int J Tuberc Lung Dis 2000;4: 256–61. 22. Schimizu Y, Dobashi K, Mita Y, et al. DNA microarray genotyping of N-acetyltransferase 2 polymorphism using carbodiimide as the linker for assessment of isoniazid hepatotoxicity. Tuberculosis 2006;86:374–81. 23. Chen B, Li JH, Xu YM, Wang J, Cao XM. The influence of NAT2 genotypes on the plasma concentration of isoniazid and acetylisoniazid in Chinese pulmonary tuberculosis patients. Clin Chim Acta 2006;365:104–8. 24. Lee SY, Lee KA, Ki CS, et al. Complete sequencing of a genetic polymorphism in NAT2 in the Korean population. Clin Chem 2002;48:775–7. 25. Lucas D, Menez C, Girre C, et al. Cytochrome P450 2E1 genotype and chlorzoxazone metabolism in healthy and alcoholic Caucasian subjects. Pharmacogenetics 1995;5:298–304.
H.-J. Cho et al. 26. Powell H, Kitteringham NR, Pirmohamed M, Smith DA, Park BK. Expression of cytochrome P4502E1 in human liver: assessment by mRNA, genotype and phenotype. Pharmacogenetics 1998;8:411–21. 27. Huang YS, Chern HD, Su WJ, et al. Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis druginduced hepatitis. Hepatology 2003;37:924–30. 28. Vuilleumier N, Rossier MF, Chiappe A, et al. CYP2E1 genotype and isoniazid-induced hepatotoxicity in patients treated for latent tuberculosis. Eur J Clin Pharmacol 2006;62:423–9. 29. Yu MW, Gladek-Yarborough A, Chiamprasert S, Santella RM, Liaw YF, Chen CJ. Cytochrome P450 2E1 and glutathione S-transferase M1 polymorphisms and susceptibility to hepatocellular carcinoma. Gastroenterology 1995;109:1266–73. 30. McCarver DG, Byun R, Hines RN, Hichme M, Wegenek W. A genetic polymorphism in the regulatory sequences of human CYP2E1: association with increased chlorzoxazone hydroxylation in the presence of obesity and ethanol intake. Toxicol Appl Pharmacol 1998;152:276–81. 31. Hu Y, Hakkola J, Oscarson M, Ingelman-Sundberg M. Structural and functional characterization of the 50 -flanking region of the rat and human cytochrome P450 2E1 genes: identification of a polymorphic repeat in the human gene. Biochem Biophys Res Commun 1999;263:286–93. 32. Fritsche E, Pittman GS, Bell DA. Localization, sequence analysis, and ethnic distribution of a 96-bp insertion in the promoter of the human CYP2E1 gene. Mutat Res 2000;432:1–5. 33. Itoga S, Harada S, Nomura F. Polymorphism of the 50 -flanking region of the CYP2E1 gene: an association study with alcoholism. Alcohol Clin Exp Res 2001;25:11S–5S. 34. Howard LA, Ahluwalia JS, Lin SK, Sellers EM, Tyndale RF. CYP2E1*1D regulatory polymorphism: association with alcohol and nicotine dependence. Pharmacogenetics 2003;13:321–8. 35. Maria VAJ, Victorino RMM. Development and validation of a clinical scale for the diagnosis of drug-induced hepatitis. Hepatology 1997;26:664–9. 36. Aithal GP, Rawlins MD, Day CP. Clinical diagnostic scale: a useful tool in the evaluation of suspected hepatotoxic adverse drug reactions. J Hepatol 2003;33:942–52. 37. Branch RA, Adedoyin A, Frye RF, Wilson JW, Romkes M. In vivo modulation of CYP enzymes by quinidine and rifampicin. Clin Pharmacol Ther 2000;68:401–11. 38. Zand R, Nelson SD, Slattery JT, et al. Inhibition and induction of cytochrome P4502E1-catalysed oxidation by isoniazid in humans. Clin Pharmacol Ther 1993;54:142–9.
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Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity in Korean patients with pulmonary tuberculosis Hyun-Jung Choa, Won-Jung Kohb, Yon-Ju Ryub, Chang-Seok Kia, Myung-Hyun Namc, Jong-Won Kima, Soo-Youn Leea, a
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, South Korea b Department of Pulmonary and Critical Care Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea c Department of Laboratory Medicine and Genetics, Korea University College of Medicine, Seoul, South Korea Received 12 October 2006; received in revised form 10 May 2007; accepted 21 May 2007
KEYWORDS Isoniazid; Hepatotoxicity; NAT2; CYP2E1; Korean
Summary Antituberculosis drug-induced hepatitis attributed to isoniazid (INH) is one of the most prevalent drug-induced liver injuries. INH is metabolized by hepatic N-acetyltransferase (NAT) and cytochrome P450 2E1 (CYP2E1) to form hepatotoxins. The aim of this study was to evaluate whether polymorphisms of the NAT2 and/or CYP2E1 genes were associated with antituberculosis drug-induced hepatotoxicity in Korean patients. A total of 132 patients with tuberculosis who received antituberculosis treatment were followed prospectively. Their NAT2 and CYP2E1 genotypes were determined using polymerase chain reaction (PCR) with or without sequencing. Eighteen (13.6%) patients developed antituberculosis drug-induced hepatotoxicity. Regarding NAT2, slow acetylators had a higher incidence of hepatotoxicity than rapid acetylators (36.8% vs. 9.7%, P ¼ 0.005) and there was a 3.8-fold risk of hepatotoxicity for the slow acetylators compared to the rapid acetylators. For the CYP2E1 gene, the RsaI polymorphism in the 50 untranslated region, and a polymorphic repetitive sequence at the CYP2E1 50 -flaking region were analyzed; there was no significant association between any CYP2E1 genotype and antituberculosis druginduced hepatotoxicity. In conclusion, slow acetylator status of NAT2 was a significant susceptibility risk factor for antituberculosis drug-induced hepatotoxicity; NAT2 genotyping may be a useful tool for predicting antituberculosis drug-induced hepatotoxicity. & 2007 Published by Elsevier Ltd.
Corresponding author. Tel.: +82 2 3410 1834; fax: +82 2 3410 2719.
E-mail address: [email protected] (S.-Y. Lee). 1472-9792/$ - see front matter & 2007 Published by Elsevier Ltd. doi:10.1016/j.tube.2007.05.012
ARTICLE IN PRESS 552
Introduction Despite the availability of effective chemotherapeutic agents to treat tuberculosis, the hepatotoxicity that results from use of these drugs remains a significant problem for clinical treatment.1–10 The incidence of antituberculosis drug-induced hepatotoxicity ranges from 1% to 36%, and mortality in such cases is not rare.1–10 Although there is a wide variability in reported incidence of antituberculosis drug-induced hepatotoxicity from different countries, there is a generally higher incidence of antituberculosis druginduced hepatotoxicity in Asian countries; this may be due to ethnic susceptibility and the inherent genetics of drug metabolism.4 Among first-line therapeutic drugs for tuberculosis, isoniazid (INH) is the major drug associated with drug-induced hepatotoxicity. INH is metabolized to acetylisoniazid via hepatic N-acetyltransferase2 (NAT2).6 In turn, acetylisoniazid is hydrolyzed to acetylhydrazine, which is oxidized by cytochrome P450 2E1 (CYP2E1) to form some hepatotoxic intermediates.11,12 Disposal of acetylhydrazine also depends on further acetylation by NAT2 to form a non-toxic metabolite, diacetylhydrazine.13,14 Previously reported studies have shown inconsistent results on whether slow acetylators or rapid acetylators cause antituberculosis drug-induced hepatotoxicity.2,6,8,13,15 Moreover the impact of CYP2E1, the major catalyzing enzyme in the formation of hepatotoxins, on the risk of developing antituberculosis drug-induced hepatotoxicity has not been fully explained. To determine the contribution of NAT2 and/or CYP2E1 gene polymorphisms in susceptibility to antituberculosis drug-induced hepatotoxicity in Koreans and to determine the genotype and phenotype for risk of antituberculosis drug-induced hepatotoxicity, we genotyped NAT2 and CYP2E1 in Korean patients with pulmonary tuberculosis.
Patients and methods Patients A total of 132 Korean patients with newly diagnosed tuberculosis at Samsung Medical Center from June 2004 to December 2005 were consecutively screened. Inclusion criteria consisted of: adult patients newly diagnosed with active tuberculosis, having evident lesion of tuberculosis by simple X-ray or computed tomography or positive results of sputum smear or culture for detection of mycobacteria. Patients with any of the following conditions were excluded from the study: (1) abnormal serum alanine transferase (ALT), aspartate transaminase (AST), or bilirubin levels or symptoms related to abnormal liver function such as jaundice before antituberculosis treatment; (2) alcoholic liver disease or habitual alcohol drinking; (3) any other hepatic or systemic diseases that may cause liver dysfunction. The Institutional Review Board at Samsung Medical Center Clinical Research Institute approved this study protocol, and signed informed consent was obtained from each subject prior to the commencement of the study. All patients received oral INH (300 mg), rifampicin (600 mg), pyrazinamide (20 mg/kg body weight), and ethambutol (800 mg) daily for the first 2 months. Pyrazinamide was
H.-J. Cho et al. then discontinued, while INH, rifampicin and ethambutol were continued for another 4 months. Except for 18 patients who had antituberculosis drug-induced hepatotoxicity, the duration of antituberculosis treatment was 6 months. Treatment compliance was assessed by comparing the number of administered treatment doses to the number of treatment doses scheduled each month. There were no consistently missing doses corresponding to more than 10 days of monthly scheduled doses of medication for all the patients. Before antituberculosis therapy, serum ALT, AST, alkaline phosphatase, direct and total bilirubin, hepatitis B virus tests including surface antigen, antibody to surface antigen, e antigen, antibody to e antigen and HBV DNA, immunoglobulin M antibody to hepatitis A virus, antibody to hepatitis C and antibody human immunodeficiency virus were measured. Serum AST, ALT, and total bilirubin levels were then monitored monthly until the end of treatment. Antituberculosis drug-induced hepatotoxicity was designated as an increase in serum ALT level more than two times the upper limit of the normal value (ULN) after antituberculosis treatment, according to the criteria of drug-induced liver injuries developed by the international consensus meeting.16 When antituberculosis drug-induced hepatotoxicity was identified, serum ALT, AST, and bilirubin were monitored every week thereafter.
Genotyping of NAT2 and CYP2E1 Genomic DNA was extracted from peripheral blood leukocytes using the Wizard Genomic DNA Purification kit following the manufacturer’s instructions (Promega, Madison, WI, USA). Polymerase chain reaction (PCR) with direct sequencing was used to genotype NAT2 with the appropriate primer sets (these are available upon request). The presence of any two mutant alleles defined a slow acetylator, whereas rapid acetylators had one or two wildtype NTA2*4 alleles. A 371-bp fragment in the 50 untranslated region (50 UTR) including the RsaI polymorphism was amplified and direct sequencing analysis was performed with an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA) using primer sets that were designed by the authors (these are available upon request). For the RsaI polymorphism of CYP2E1, the wild-type allele was c1, and the mutant allele was c2, that lacks the RsaI restriction site. To detect a polymorphic repetitive sequence at the CYP2E1 50 -flanking region, PCR amplifications were performed with a thermal cycler (model 9700, Applied Biosystems, Foster City, CA, USA) with appropriate primer sets (these are available upon request) and the PCR products were isolated by agarose gel electrophoresis and the products: 346, 394, or 490 bp, depending on the CYP2E1 genotype, were designated as *1A (5 repeats), *1C (6 repeats), and *1D (8 repeats), respectively.
Statistical methods Odds ratios (OR) and confidence intervals (CI) were calculated using logistics regression. The Mann–Whitney U-test, w2 tests with Yates’ correction, or Fisher’s exact test were used for the univariate analysis as appropriate. Multivariate analysis was performed to evaluate the
ARTICLE IN PRESS Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity
(36.8% vs. 9.7%; OR, 5.41; 95% CI, 1.76–16.59; P ¼ 0.005; Table 2) and there was a 3.8-fold risk of hepatotoxicity for the slow acetylators compared to the rapid acetylators. Sensitivity, specificity and positive and negative predictive values of NAT2 slow acetylator genotype are as follows: 0.39 (95% CI: 0.20–0.61), 0.89 (95% CI: 0.83–0.94), 0.37 (95% CI: 0.19–0.59), and 0.90 (95% CI: 0.83–0.94). A total of 75 patients (56.8%) were genotyped as CYP2E1 c1/c1, 53 patients (40.2%) as CYP2E1 c1/c2 and 4 patients (3.0%) as CYP2E1 c2/c2. There was no significant difference in the frequencies of c1/c1, c1/c2, and c2/c2 genotypes of CYP2E1 between the patients with hepatotoxicity and those without hepatotoxicity (Table 3). The polymorphic repetitive sequence at the CYP2E1 50 -flaking region, in patients with hepatotoxicity and those without hepatotoxicity, showed similar genotypes and allele frequencies (Table 3.). CYP2E1*1C was found to be the predominant allele (77.2% allele frequency); the allele frequencies of CYP2E1*1A and CYP2E1*1D were 2.3% and 20.5%,
adjusted risk of antituberculosis-induced hepatitis using a stepwise logistic regression analysis (SPSS 8.0 for Windows; SPSS Inc., Chicago, IL). A two-tailed P-value less than 0.05 was considered statistically significant.
Results Eighteen patients (13.6%) were diagnosed with antituberculosis drug-induced hepatotoxicity. There was no statistical difference in the age, gender, or body mass index between the patients with hepatotoxicity and those without hepatotoxicity (Table 1). Eleven patients (8.3%) had more than three times the ULN of ALT; thus, INH was discontinued temporarily. A total of 113 patients (85.6%) were genotyped as rapid acetylators, and the other 19 patients (14.4%) were slow acetylators. The risk of having antituberculosis druginduced hepatotoxicity was significantly higher in the slow acetylators compared to the rapid acetylators
Table 1
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Characteristics of patients with or without antituberculosis drug-induced hepatotoxicity. With hepatotoxicity (n ¼ 18)
Without hepatotoxicity (n ¼ 114)
P-value
Sex (F/M) Age (yr) Body mass index (kg/m2)
6/12 51.2717.5 20.272.5
51/63 46.7718.4 18.472.9
0.447 0.392 0.546
Initial laboratory findings,y ALT (U/L) AST (U/L) Total bilirubin (mg/dL)
28.4715.8 20.9710.3 0.670.3
19.4713.2 23.9713.5 0.670.9
0.224 0.546 0.207
30.9721.9 (8–151) 32.4714.2 (15–82) 0.870.9 (0.1–1.7)
o0.001 o0.001 0.069
Laboratory findings during antituberculosis treatment,z Peak ALT (U/L) 291.87456.7 (86–2067) Peak AST (U/L) 446.77955.9 (62–4173) Peak total bilirubin (mg/dL) 1.672.3 (0.3–9.1)
ALT: alanine transferase; AST: aspartate transaminase. Mean7SD. y Reference interval of ALT, 0–40 U/L; AST, 0–40 U/L; total bilirubin, 0.2–1.5 mg/dL. z Mean7SD (range).
Table 2
NAT2 genotypes in tuberculosis patients with or without antituberculosis drug-induced hepatotoxicity. With hepatotoxicity (n ¼ 18)
Without hepatotixicity (n ¼ 114)
OR (95% CI)
P-value
Rapid or intermediate acetylator NAT2*4/*4 NAT2*4/*5A NAT2*4/*6A NAT2*4/*5B NAT2*4/*7B
11
102
0.18 (0.06–0.57)
0.005
7 1 2 0 1
45 2 32 4 19
Slow acetylator NAT2*5B/*7B NAT2*6A/*6A NAT2*6A/*7B NAT2*7B/*7B
7 0 3 4 0
12 1 2 7 2
5.41 (1.76–16.59)
0.005
OR: odds ratios; CI: confidence intervals.
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H.-J. Cho et al.
Table 3
Risk of antituberculosis drug-induced hepatotoxicity in relation to polymorphism in the CYP2E1 gene.
RsaI polymorphism c1/c1 c1/c2 or c2/c2
With hepatotoxicity (n ¼ 18)
Without hepatotixicity (n ¼ 114)
OR (95% CI)
P-value
10 8
65 49
0.94 (0.35–2.56) 1.06 (0.64–4.61)
1.000 1.000
0.83 (0.29–2.36)
0.322
1.38 (0.49–3.93)
0.322
Polymorphic repetitive sequence at the CYP2E1 50 -flaking region 6 43 *1D allele *1A/*1D 1 0 *1C/*1D 4 39 *1D/*1D 1 4 Non-*1D alleley *1A/*1A *1A/*1C *1C/*1C
12 0 0 12
71 1 3 67
OR: odds ratios; CI: confidence intervals. Group with any number of *1D alleles y Group with no *1D allele.
Table 4 status.
Combined risk for antituberculosis drug-induced hepatotoxicity associated with CYP2E1 genotype and acetylator
Acetylator status
CYP2E1 genotype
With hepatotoxicity (n ¼ 18)
Without hepatotoxicity (n ¼ 114)
OR (95% CI)
P-value
Slow Slow Slow Slow Rapid or intermediate Rapid or intermediate Rapid or intermediate Rapid or intermediate
c1/c2 or c2/c2 c1/c1 *1D allele Non-*1D alleley c1/c2 or c2/c2
3 4 3 4 5
4 7 5 6 44
5.5 (1.12–27) 3.96 (1.03–15.28) 4.36 (0.94–20.13) 5.14 (1.29–20.49) 0.61 (0.20–1.83)
0.053 0.044 0.077 0.031 0.441
c1/c1
6
59
0.47 (0.16–1.33)
0.205
*1D allele
2
39
0.24 (0.05–1.09)
0.057
Non-*1D allele
9
64
0.78 (0.29–2.11)
0.799
OR: odds ratios; CI: confidence intervals. Group with any number of *1D alleles. y Group with no *1D allele.
respectively. Combined analysis of the two polymorphisms in the CYP2E1 gene was performed but no synergistic effect was observed (data not shown). Considering the CYP2E1 genotype and the acetylator status together showed that the slow acetylator, regardless of CYP2E1 genotype, was associated with a higher incidence of hepatotoxicity compared to other combinations (Table 4).
Discussion Our study showed a significant association between the slow acetylator genotype of NAT2 and the risk of developing antituberculosis drug-induced hepatotoxicity in Korean tuberculosis patients (Table 2); these results are consistent
with those from previous reports.4,8,15,17,19 Among the many risk factors for antituberculosis drug-induced hepatotoxicity, acetylator status has been regarded to be the most important.2,6,8–10,13,15,17–19 However, there have been only a few reports to evaluate the association of acetylator status, determined by NAT2 genotyping, and the susceptibility of antituberculosis drug-induced hepatotoxicity.4,20–22 The NAT2 genotype has been proven to be one of the most important factors for inter-individual differences in plasma isoniazid (INH) concentration in Chinese tuberculosis patients.23 In our previous study, we showed that the concordance rate between NAT2 genotyping and phenotyping, by the plasma concentration ratio of acetylisoniazid (AcINH) to INH (pAcINH/INH), was 90.2%.24
ARTICLE IN PRESS Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity CYP2E1 is induced by ethanol and is critically important in the metabolic activation of many drugs and carcinogens.25,26 Among several CYP2E1 genetic polymorphisms, only the RsaI polymorphism has been evaluated in association with antituberculosis drug-induced hepatotoxicity27,28 and hepatocellular carcinoma.29 Recent studies have reported a significant association between the c1/c1 genotype and the risk of developing antituberculosis drug-induced hepatotoxicity, explained by a higher CYP2E1 activity with the c1/c1 genotype and the inhibitory effect of INH.27,28 We found no evidence for association of the c1/c1 genotype with antituberculosis drug-induced hepatotoxicity (Table 3). However, we evaluated the polymorphic repetitive sequence at the CYP2E1 50 -flaking region in addition to the RsaI polymorphism, which has not been previously reported. Of the CYP2E1 polymorphisms identified to date, only the CYP2E1*1D has been linked to altered function; it has been associated with increased metabolic capacity in obese individuals or those individuals consuming alcohol,30 consistent with increased induction of gene activity under these conditions. CYP2E1*1D allelic frequency, among our study population, was 20.5%, similar as those of previous reports in other Asian populations.31–34 Our results failed to show any significant association between the polymorphic repetitive sequence at the CYP2E1 50 -flaking region and susceptibility to hepatotoxicity, and there was no synergistic effect of the two polymorphisms of the CYP2E1 on the development of hepatotoxicity (Table 3). We then considered the combined risk, for antituberculosis drug-induced hepatotoxicity, associated with the CYP2E1 genotype and the acetylator status (Table 4); considering that genetic effect of CYP2E1 on hepatotoxicity would be inconspicuous compared to that of the NAT2. The statistically nonsignificant results regarding the CYP2E1 polymorphism and antituberculosis drug-induced hepatotoxicity may be due to the small sample size and low frequency of patients with antituberculosis drug-induced hepatotoxicity; re-evaluation and confirmation are needed in a large-scale population study. Potential limitations of the present study should be considered. The criteria in this study may be less strict than other criteria35,36 to assess antituberculosis drug-induced hepatotoxicity. Although these criteria may be low for defining clinically evident hepatotoxicity, we believe that they are clinically useful for finding adverse hepatic reactions earlier and for preventing the development of overt hepatotoxicity. Patients in this study were various concomitant drugs with INH. Co-administration of drugs may result in quantitative and qualitative alteration of the drug metabolism and it is very difficult to exclude the presence of a confounding factor in accessing the drug-induced hepatotoxicity. As rifampicin is known to reduce NAT2 activity,37,38 as INH is known to have a biphasic effect on CYP2E1 activity (initial inhibition followed by an induction)9 and because a combination of these drugs is known to increase the incidence of antituberculosis drug-induced hepatotoxicity up to 35%,3,9,37,38 drug–drug pharmacokinetic and pharmacodynamic interactions certainly did occur. There have been several reports about the relationship between NAT2 and antituberculosis drug-induced hepatotoxicity in Asian tuberculosis patients.4,20–22 Their reports and our study confirm that slow acetylators are significantly
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associated with antituberculosis drug-induced hepatotoxicity in Asian population. In contrast, results of the study performed in Caucasians28 failed to show any significant association between NAT2 susceptibility to antituberculosis drug-induced hepatotoxicity. These discrepancies might well result from a different genotype distribution among Asian and Caucasian patients. Therefore, the determination of NAT2 genotypes for antituberculosis treatment may be clinically useful for the prediction and prevention of antituberculosis drug-induced hepatotoxicity in Asian tuberculosis patients. Funding: This study was supported by a grant of the Korean Health 21 R&D Project. Ministry of Health & Welfare, R.O.K. (03-PJ10-PG13-GD01-0002). Competing interests: None declared Ethical approval: Not required
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