GSTT1 polymorphisms with mild elevation of liver enzymes in Brazilian individuals under anti-tuberculosis drug therapy

Clinica Chimica Acta 415 (2013) 215–219

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Relationship of NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms with mild elevation of liver enzymes in Brazilian individuals under anti-tuberculosis drug therapy Francisco Jose Forestiero a, Leticia Cecon a, Mario Hirouki Hirata a, Fernando Fiuza de Melo b, Rosilene Fressatti Cardoso c, Alvaro Cerda a, Rosario Dominguez Crespo Hirata a,⁎ a b c

Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Clemente Ferreira Institute, Sao Paulo, SP, Brazil Department of Clinical Analysis and Biomedicine, State University of Maringa, Maringa, PR, Brazil

a r t i c l e

i n f o

Article history: Received 17 June 2012 Received in revised form 14 October 2012 Accepted 16 October 2012 Available online 22 October 2012 Keywords: NAT2 CYP2E1 GSTM1 GSTT1 Anti-tuberculosis drugs Liver enzymes

a b s t r a c t The relationship of NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms with mild elevation of liver biomarkers was investigated in individuals under anti-tuberculosis drug therapy. Tuberculosis outpatients (18–70 y) with (n = 59) and without (n = 40) mild increase of liver enzymes (MILE) at two-month treatment were selected. Blood samples were obtained for DNA extraction and evaluation of serum markers of liver function. NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms were detected by DNA sequencing, PCR-RFLP, and PCR multiplex. Frequency of NAT2*5/*5 genotype was higher in MILE than in non-MILE group (p = 0.04). Patients carrying NAT2*5/*5 genotype had increased susceptibility to MILE (OR: 9.00, 95CI: 1.46–55.48, p = 0.018). CYP2E1*5B allele (*1A/*5B plus *5B/*5B genotypes) carriers had a trend for reduced risk for MILE (OR: 0.34, 95CI: 0.11–1.03, p = 0.056) that was confirmed by lower levels of liver markers than CYP2E1*1A/*1A carriers after treatment (p b 0.05). Moreover, increased post-treatment ALT, AST and total bilirubin were associated with GSTM1*1/GSTT1*1 genotypes (p b 0.05). Patients taking CYP2E1 inhibitors had increased susceptibility to MILE (OR: 7.39, 95CI: 1.93–28.29, p = 0.003), which was independent of the studied polymorphisms. These results are suggestive that NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms and concomitant use of CYP2E1 inhibitors contribute to the susceptibility to mild alterations in liver enzymes in patients under anti-tuberculosis drug therapy. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Tuberculosis treatment is based on the use of isoniazid, rifampicin and pyrazinamide, which were shown to cause adverse drug reactions (ADRs), including hepatitis, gastrointestinal intolerance, kidney failure, and cutaneous and hematological reactions [1], which can lead to therapy discontinuation or more serious morbidity and mortality. The incidence of anti-tuberculosis drug-induced hepatotoxicity ranges from 1% to 36% [1–4]. Advanced age, gender, alcoholism, malnutrition, HIV co-infection, pre-existent liver disease and drug interactions are common risk factors for hepatotoxicity [4,5]. Genetic background, such as the isoniazid acetylator status, has also an important role on the likelihood of developing hepatotoxicity and other ADRs [2]. N-acetyltransferase 2 (NAT2) metabolizes isoniazid into acetyl isoniazid, which is hydrolyzed to acetyl hydrazine [6]. Acetyl hydrazine is ⁎ Corresponding author at: Universidade de Sao Paulo, Faculdade de Ciencias Farmaceuticas, Av Prof Lineu Prestes, 580 , 05508-000 Sao Paulo, SP, Brazil. Tel.: +55 11 3091 3660; fax: +55 11 3813 2197. E-mail address: [email protected] (R.D.C. Hirata). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2012.10.030

further oxidized into hepatotoxic intermediates by cytochrome P450 2E1 (CYP2E1). Direct hydrolysis of isoniazid also generates hydrazine, a potent hepatotoxin. Glutathione S-transferases (GSTs) are detoxification enzymes that catalyze the conjugation of glutathione to several classes of molecules including toxic intermediates of the isoniazid metabolism. NAT2 polymorphisms have been shown to cause individual variation in isoniazid N-acetylation having the slow acetylators more susceptibility to develop hepatotoxicity than those with high NAT2 activity [6–11]. Common polymorphisms in the CYP2E1 have also shown to be associated with drug-induced hepatotoxicity in tuberculosis patients [6,12]. Homozygosis of GSTT1 or GSTM1 non-functional (null) genotypes, which cause lack of enzyme activity, was suggested to increase the risk for hepatotoxicity to anti-tuberculosis drugs [6,13,14]. Although a relationship between gene polymorphisms and antituberculosis drug-induced hepatotoxicity has been described in various studies, there is still limited information in our population, which has a heterogeneous genetic background. We have investigated the contribution of NAT2, CYP2E1, GSTM1 and GSTT1 variants to the mild increase in the liver enzymes in a sample of non-HIV outpatients under anti-tuberculosis drug therapy.

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2. Subjects and methods

2.4. DNA sequencing

2.1. Patients

NAT2 polymorphisms were also analyzed by DNA sequencing using Mega BACE 1000 (Amersham Pharmacia Biotech, Upsala, Sweden). Sequence data were assembled and edited using BioEdit Sequence Alignment Editor, version 5.0.9 (Tom Hall, Department of Microbiology, North Caroline State University, USA).

Tuberculosis outpatients, aged 18–70 years, were recruited at the Clemente Ferreira Institute (Sao Paulo, SP, Brazil) and at the Center for Health Sciences, State University of Maringa (Maringa, PR, Brazil) from 2006 to 2008. From this sample, we selected 59 individuals with mild increase of liver enzymes (MILE) at two-month anti-tuberculosis drug therapy and 40 without MILE (control group). MILE was considered an increase in the serum ALT 126–250 U/L (2.5–5 times the upper limit of normal) [4]. Active tuberculosis was diagnosed by either clinical (positive X-ray and symptoms) and laboratory data (positive sputum smear or culture). Individuals with HIV or viral hepatitis infection, liver dysfunction (increased baseline aminotransferases and bilirubin), alcoholic liver disease, history of chronic liver disease, concomitant use of hepatotoxic drugs, and pregnant women were not included in this study. The frequency of moderate (2%) and severe (1%) hepatotoxicity was low in the initial sample therefore these individuals were not considered in the analysis. Information on age, gender, self-reported ethnics, alcohol intake, and medication in use were recorded. Each individual declared his ethnical group during the interview and agreed to participate in the study by signing an informed consent. The study protocol was approved by the Ethics Committees of the School of Pharmaceutical Sciences (University of Sao Paulo, Sao Paulo, SP, Brazil), and the State University of Maringa.

2.2. Anti-tuberculosis therapy and clinical assessment All selected patients were treated with an anti-tuberculosis regimen (daily doses of isoniazid, up to 300 mg; rifampicin, up to 600 mg; pyrazinamide, up to 2000 mg) according to the Brazilian guidelines on tuberculosis [15], with discontinuation of pyrazinamide at twomonth treatment. Patients underwent routine follow-up of clinical assessments every two-weeks during the first month and monthly afterwards. Concomitant medications were also registered. Serum markers of liver function (aminotransferases, alkaline phosphatase, gamma-glutamyl transpeptidase and total bilirubin) were measured using routine laboratory methods, before and after 2, 4 and 6 months of therapy.

2.3. Polymorphism analysis Genomic DNA was isolated from 1 mL EDTA-anticoagulated whole blood samples using a salting-out method previously described [16]. PCR-RFLP was used for detection of the NAT2 and CYP2E1 variants. Primer sequences and PCR thermal cycling conditions are displayed at the Supplementary table. NAT2*5, NAT2*6 and NAT2*7 amplicons were digested during 4 h with EcoRII (Promega, Madison, WI, USA) at 60 °C, TaqI (Jena Biosciences GmbH, Germany) at 65 °C, and XhoII (Fermentas International Inc., Ontario, Canada) at 37 °C. NAT2*4 allele has a differential RFLP profile for each endonuclease. CYP2E1 PCR products were digested with AluI and RsaI (New England BioLabs, USA), respectively, for 4 h at 37 °C. RFLP products were analyzed by 8.0% polyacrylamide gel electrophoresis stained with SYBR Gold® (Molecular Probes, Invitrogen Detection Technologies, OR, USA). RFLP strategies included a constitutive site to monitor endonuclease activity. A PCR multiplex assay was used to amplify GSTM1 and GSTT1 genes simultaneously (Supplementary table), including the 182-bp CYP2E1 amplicon, which was used as an internal control of PCR reactions. PCR products were analyzed by 0.8% agarose gel electrophoresis.

2.5. Statistical analysis Categorical variables were compared by chi-square or Fisher's exact test. Continuous variables were presented as mean ± standard error (SEM) and compared by t-test. Variables without normal distribution were log transformed for analysis. Univariate logistic regression analysis was used to evaluate the risk of independent variables for liver-related ADRs. Statistical tests were performed using SigmaStat v. 2.0 (San Rafael, CA, USA), Minitab v. 14 (Minitab Inc., USA), R 2.7.1 (Vienna, Austria), and SPSS v. 15 (Chicago, IL, USA). Statistical significance was considered at p b 0.05. 3. Results Demographic, clinical and laboratory data of the tuberculosis outpatients are shown in Table 1. Gender, age, ethnics and alcohol intake were not associated with the MILE in this sample (p > 0.05). On the other hand, hepatotoxicity and use of concomitant medications, such as CYP2E1 inhibitors, were more frequent in MILE than in non-MILE patients (p b 0.05). Liver markers in serum at two-month anti-tuberculosis treatment were higher in MILE than in non-MILE group (p b 0.05). Genotype distributions for NAT2, CYP2E1 and GSTM1 and GSTT1 variants were in Hardy–Weinberg equilibrium indicating the random selection of patients from the sample population. Frequencies of NAT2 Table 1 Demographic, clinical and laboratory data of tuberculosis patients. Variables

Total (99)

MILE (59)

Non-MILE (40)

p

Gender Male Female

53.5% (53) 46.5% (46)

49.2% (29) 50.8% (30)

60.0% (24) 40.0% (16)

0.288

Age b50 years ≥50 years

83.8% (83) 16.2% (16)

86.4% (51) 13.6% (8)

80.0% (32) 20.0% (8)

0.565

Ethnics White Black Alcohol intake (>1 g/day) Hepatotoxicitya

60.6% (60) 39.4% (39) 7.1% (7) 11.1% (11)

66.1% (39) 33.9% (20) 5.1% (3) 18.6% (11)

52.5% (21) 47.5% (19) 10.0% (4) 0% (0)

0.223

Concomitant medication CYP2E1 inducers Inhibitors Both No med/no effectb

16.2% 20.2% 10.1% 53.5%

18.6% 28.8% 13.6% 39.0%

12.5% (5) 7.5% (3) 5.0% (2) 75.0% (30)

Liver biomarkers ALT, U/L AST, U/L ALP, U/L GGT, U/L Total bilirubin, mg/dL

Basal 38 ± 5 39 ± 7 113 ± 5 85 ± 12 0.8 ± 0.7

(16) (20) (10) (53)

(11) (17) (8) (23)

Two-month treatment 141 ± 17 37 ± 4 118 ± 14 28 ± 4 224 ± 13 135 ± 10 172 ± 13 122 ± 10 3.3 ± 2.4 0.8 ± 0.5

0.349 0.003

0.004

0.011 0.013 0.018 0.022 0.007

Number of individuals in parenthesis. Continuous variables are expressed as mean ± SEM and compared by t-test. Categorical variables were compared by chi-square and Fisher's exact tests. MILE, mild increase in liver enzymes; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transpeptidase. a Hepatotoxicity: was considered as ALT or AST values >3 times the ULN, and/or total bilirubin >2 mg/dL, according to the Brazilian Consensus on Tuberculosis. b Without effect or use of concomitant medication.

F.J. Forestiero et al. / Clinica Chimica Acta 415 (2013) 215–219 Table 2 Frequencies of NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms in tuberculosis patients. Variants

MILE (59)

Non-MILE (40)

p-Value

NAT2*4/*4 NAT2*4/*5 NAT2*4/*6 NAT2*4/*7 NAT2*5/*5 NAT2*5/*6 NAT2*5/*7 NAT2*6/*6 NAT2*6/*7 CYP2E1*1A/*1A CYP2E*1A/*5B CYP2E*5B/*5B GSTM1/GSTT1 M1*1/T1*1 M1*1/T1 null M1 null/T1*1 M1 null/T1 null

10.2% (6) 28.8% (17) 11.9% (7) 1.7% (1) 20.3% (12) 16.9% (10) 1.7% (1) 6.8% (4) 1.7% (1) 89.8% (53) 8.5% (5) 1.7% (1)

22.5% 20.0% 17.5% 7.5% 5.0% 15.0% 7.5% 5.0% 0.0% 75.0% 22.5% 2.5%

(9) (8) (7) (3) (2) (6) (3) (2) (0) (30) (9) (1)

0.157

47.4% (28) 10.2% (6) 35.6% (21) 6.8% (4)

40.0% 7.5% 40.0% 12.5%

(16) (3) (16) (5)

0.690

0.199

217

p = 0.056) (Table 3). Furthermore, analysis of liver biomarkers showed that individuals carrying CYP2E1 *1A/*1A genotype had three times increase in serum ALT, AST and total bilirubin at two-month anti-tuberculosis treatment compared with CYP2E1*5B allele carriers (Fig. 1). The interaction between CYP2E1 and NAT2 variants on serum liver markers was analyzed but patients carrying the CYP2E1*1A/*1A genotype have similar frequencies of NAT2 genotypes than the CYP2E1*5B allele carriers (p > 0.05) (data not shown). Frequency of GSTM1/GSTT1 genotypes did not differ between the studied groups (Table 2) (p = 0.690). The presence of one or both null genotypes was not associated with MILE by logistic regression analysis (p > 0.05) (Table 3). Analysis of the liver marker profile showed that individuals carrying the GSTM1*1/GSTT1*1 genotypes had increased ALT and AST at two-month tuberculosis treatment compared with null genotype carriers (p b 0.05) (Fig. 2). These results were not influenced by NAT2 or CYP2E1 genotypes (data not shown). The use of CYP2E1 inhibitors was associated with seven times more chance to have MILE in this sample (OR: 7.39, 95CI: 1.93–28.29,

Categorical variables were compared by chi-square test. MILE, mild increase in liver enzymes.

*5B carriers

genotypes were similar between MILE and non-MILE tuberculosis patients (Table 2). However, the frequency of NAT2*5/*5 genotype was four times higher in MILE (20.3%) compared to non-MILE group (5.0%, p = 0.04). Logistic regression analysis showed that tuberculosis patients with slow acetylation profile display a trend of association with MILE (p = 0.061) (Table 3). Further analysis showed that patients carrying the NAT2*5/*5 genotype had increased susceptibility to MILE (OR: 9.00, 95CI: 1.46–55.48, p = 0.018) compared to the NAT2*4/*4 genotype carriers (fast acetylators) (data not shown). On the other hand, we could not observe a relationship between slow acetylation profile and altered serum liver markers at two-month anti-tuberculosis treatment (data not shown). A direct relationship between CYP2E1 polymorphism and MILE was not found in this sample (p = 0.199) (Table 2); the frequency of CYP2E1*1A/*5B genotype was slightly lower in MILE (8.5%) than in non-MILE patients (22.5%). Logistic regression analysis indicated that CYP2E1*5B allele (*1A/*5B plus *5B/*5B genotype) is slightly associated with a reduced risk for MILE (OR: 034, 95CI: 0.11–1.03,

0

Odds ratio

95CI

p

NAT2 acetylation profile Fast (*4/*4, n = 15) [ref] Intermediate (*4/*5, *4/*6; *4/*7, n = 43) Slow (*5/*5, *5/*6, *5/*7, *6/*6 , *6*7, n = 41)

1.00 2.08 3.23

– [0.63–6.90] [0.96–10.99]

– 0.230 0.061

CYP2E1 genotypes *1A/*1A (n = 83) [ref] *1A/*5B and *5B/*5B (n = 16)

1.00 0.34

– [0.11–1.03]

– 0.056

GSTM1/GSTT1 genotypes M1*1/T1*1 (n = 44) [ref.] M1*1/T1 null (n = 9) M1 null/T1*1 (n = 37) M1 null/T1 null (n = 9)

1.00 1.14 0.75 0.46

– [0.25–5.20] [0.31–1.83] [0.11–1.95]

– 0.863 0.529 0.290

Concomitant medication No med/no effect (n = 53)a [ref] CYP2E1 inducers (n = 16) Inhibitors (n = 20) Both (n = 10)

1.00 2.82 7.39 5.22

– [0.87–9.42] [1.93–28.29] [1.01–26.95]

– 0.082 0.003 0.049

100

150

200

*5B carriers

*

*1A/*1A

0

50

100

150

200

AST (U/L)

Basal 2 months

*5B carriers

*

*1A/*1A

0

95CI, 95% Confidence Interval. a Without effect or use of concomitant medication.

50

ALT (U/L)

Table 3 Logistic regression analysis of variables associated with MILE in tuberculosis patients. Variables

*

*1A/*1A

2

4

6

8

10

Total bilirubin (mg/dL) Fig. 1. Serum aminotransferases and total bilirubin according to CYP2E1 genotypes in patients on anti-tuberculosis therapy. Results are expressed as mean± SEM and compared by t-test (*p b 0.05). ALT: alanine aminotransferase; AST: aspartate transaminase. *5B carriers: *1A/*5B plus *5B/*5B genotypes.

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Null

*

M1*1/T1*1

0

50

100

150

200

250

ALT (U/L)

Null

*

M1*1/T1*1

0

50

100

150

200

250

AST (U/L)

Basal 2 months Null

M1*1/T1*1

0

2

4

6

8

10

Total bilirubin (mg/dL) Fig. 2. Serum aminotransferases and total bilirubin according to GSTM1/GSTT1 genotypes in patients on anti-tuberculosis therapy. Results are expressed as mean ± SEM and compared by t-test (*p b 0.05). ALT: alanine aminotransferase; AST: aspartate transaminase. Null: GSTM1 and/or GSTT1 null genotypes.

p = 0.003) compared with individuals without concomitant medication or taking medication without effect on CYP2E1 activity. Moreover individuals taking CYP2E1 inhibitors plus inducers have also increased susceptibility to MILE (OR: 5.22, 95CI: 1.01–26.95, p = 0.049). Further analysis showed that these relationships were not influenced by NAT2, CYP2E1 or GSTM1/GSTT1 polymorphisms (p> 0.05, data not shown). 4. Discussion Exposition to anti-tuberculosis drugs over a long period of time can result in severe hepatotoxicity in susceptible patients. Gender, age, ethnics background and alcohol intake have been proposed to play important roles in the pathogenesis of anti-tuberculosis drug induced hepatotoxicity (ADIH) [5,17], however we could not find a significant relationship of this variables with MILE. The use of CYP2E1 inhibitors was an important variable associated with MILE in this sample, suggesting their potential interaction with anti-tuberculosis drugs to develop liver injury. Inhibition of CYP2E1 could increase plasma concentration of other hepatotoxic drugs metabolized by CYP2E1, and therefore increase the susceptibility to MILE.

Analysis of polymorphisms showed that frequencies of NAT2 functional and non-functional (slow acetylators) genotypes were similar to the overall frequencies previously described in Brazil [10,18] and in other populations [19–24]. Distribution of CYP2E1, GSTM1 and GSTT1 genotypes did not also differ from that reported in other studies [19,22,25–30]. Gene candidate association analysis showed a significant relationship between NAT2*5/*5 genotype and MILE. Reduced hepato-protective effect in slow acetylators, including NAT2*5/*5 carriers, has been shown in patients under anti-tuberculosis therapy from other populations [21–23,31,32]. Recently slow acetylation status was also found to be an increased risk factor for anti-tuberculosis drug-induced hepatotoxicity in another sample of Brazilian population (OR: 3.59, 95%CI: 2.53–4.68, p=0.02) [30]. NAT2*5/*5 and other non-functional genotypes lack the normal acetylation rate of isoniazid and its metabolites causing accumulation of hydrazine, a potent hepatotoxic compound [19,29,33–35]. The results from this study are suggestive that the susceptibility of NAT2 slow acetylators to anti-tuberculosis drug-induced liver injury shown in a number of studies [21–23,30–32,36] may depend on other risk factors. However, we could not detect any interaction of NAT2 variants with CYP2E1 and GSTM1/GSTT1 polymorphisms, concomitant medications and other variables, which could be related to MILE, suggesting a major role of the NAT2*5*5 genotype. In this sample, increased serum ALT, AST and bilirubin was found in CYP2E1*1A/*1A (wild type) genotype carriers suggesting that these individuals are more susceptible to liver injury under anti-tuberculosis therapy. Likewise, increased ALT activity was also found in pulmonary tuberculosis patients carrying CYP2E1 c1/c1 genotype [37]. Results from a meta-analysis study have shown that CYP2E1*1A/*1A genotype increases the risk for ADIH [38]. CYP2E1 c1/c1 RsaI genotype (wild type) was also suggested to be a potential risk factor for ADIH in the Chinese population [28]. Moreover, Lee and colleagues [22] have reported that CYP2E1 c1/c1 genotype carriers are prone to developing more severe hepatotoxicity (increased AST levels) than other c1/c2 and c2/c2 genotypes. Even though CYP2E1 promoter variant has not been associated with basal enzymatic levels, CYP2E1 activity is less inhibited by isoniazid in CYP2E1*1A/*1A genotype carriers than those individuals carrying CYP2E1*1A/*5 or CYP2E1*5/*5 genotypes [39]. This effect may explain the relationship between CYP2E1*1A/*1A genotype and increased serum biomarkers of liver injury under anti-tuberculosis drug therapy. Therefore, CYP2E1*1A/*1A genotype carriers are prone to produce more hepatotoxins and increased risk for hepatotoxicity under anti-tuberculosis therapy [6]. A recent study has shown that the combination of the CYP2E1 c1/c1 genotype with a slow acetylator NAT2 genotype increased the risk of ADIH in Chinese tuberculosis patients [40]. In our sample, NAT2 slow acetylator profile was not associated with CYP2E1 variants suggesting their independent contribution to MILE susceptibility in patients under anti-tuberculosis treatment. However, further studies are necessary to evaluate NAT2 and CYP2E1 interactions in larger sample populations. It is likely that co-administration with CYP2E1 substrates or inducers, such as acetaminophen and ethanol, may exert additional liver injury effect on individuals carrying CYP2E1*1A/*1A genotype treated with anti-tuberculosis drugs. However we could not demonstrate a relationship between alcohol consumption and/or concomitant use of CYP2E1 inducers or inhibitors and MILE in this sample. GSTM1 and GSTT1 have an important role in the detoxification of hydrazine and other toxic reactive compounds. Even though the GSTM1 and GSTT1 null genotypes have been associated with increased ADIH [6,13,14], a modest effect of GSTM1 null genotype on genetic susceptibility to ADIH was reported by a meta-analysis, but no significant evidence for GSTT1 null [40]. Moreover, the relationship of these null genotypes with ADIH was not confirmed in recent case–control association studies [27–30].

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The relationship between GSTM1*1/GSTT1*1 genotype and increased serum ALT and AST during anti-tuberculosis therapy found in this study was not influenced by interaction with NAT2 and CYP2E1 polymorphisms. Effects of GSTM1 and GSTT1 null genotypes on hydrazine serum levels were investigated by Fukino and colleagues [19]. Hydrazine was prone to be accumulated in NAT2 intermediate acetylators carrying the GSTM1 null genotype. Conversely, GSTT1 null genotype carriers also have increased hydrazine in intermediate acetylator group. It is likely that the GSTM1 and GSTT1 null variants have a minor contribution to the liver injury caused by exposure to anti-tuberculosis drugs in comparison with NAT2 non-functional alleles or even CYP2E1 common genotypes. In conclusion, besides the NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms, the use of CYP2E1 inhibitors contributes to the susceptibility to mild alterations in liver enzymes in patients under anti-tuberculosis drug therapy. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.cca.2012.10.030. Conflict of interest/financial disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. Acknowledgments This study was supported by grants from FAPESP (# 2004/08406-7), Sao Paulo, Brazil. A. Cerda is recipient of a fellowship from CONICYT— Chile. M.H. Hirata and R.D.C. Hirata are recipients of fellowships from CNPq, Brazil. References [1] Forget EJ, Menzies D. Adverse reactions to first-line antituberculosis drugs. Expert Opin Drug Saf 2006;5:231-49. [2] Saukkonen JJ, Cohn DL, Jasmer RM, et al. An official ATS statement: hepatotoxicity of antituberculosis therapy. Am J Respir Crit Care Med 2006;174:935-52. [3] Marra F, Marra CA, Bruchet N, et al. Adverse drug reactions associated with first-line anti-tuberculosis drug regimens. Int J Tuberc Lung Dis 2007;11:868-75. [4] Tostmann A, Boeree MJ, Aarnoutse RE, de Lange WC, van der Ven AJ, Dekhuijzen R. Antituberculosis drug-induced hepatotoxicity: concise up-to-date review. J Gastroenterol Hepatol 2008;23:192-202. [5] Arbex MA, Varella MC, de Siqueira HR, de Mello FA. Antituberculosis drugs: drug interactions, adverse effects, and use in special situations. Part 1: first-line drugs. J Bras Pneumol 2010;36:626-40. [6] Huang YS. Genetic polymorphisms of drug-metabolizing enzymes and the susceptibility to antituberculosis drug-induced liver injury. Expert Opin Drug Metab Toxicol 2007;3:1-8. [7] Hiratsuka M, Kishikawa Y, Takekuma Y, et al. Genotyping of the N-acetyltransferase 2 polymorphism in the prediction of adverse drug reactions to isoniazid in Japanese patients. Drug Metab Pharmacokinet 2002;17:357-62. [8] Cho HJ, Koh WJ, Ryu YJ, et al. Genetic polymorphisms of NAT2 and CYP2E1 associated with antituberculosis drug-induced hepatotoxicity in Korean patients with pulmonary tuberculosis. Tuberculosis 2007;87:551-6. [9] Higuchi N, Tahara N, Yanagihara K, et al. NAT2*6A, a haplotype of the N-acetyltransferase 2 gene, is an important biomarker for risk of anti-tuberculosis drug-induced hepatotoxicity in Japanese patients with tuberculosis. World J Gastroenterol 2007;13:6003-8. [10] Possuelo LG, Castelan JA, de Brito TC, et al. Association of slow N-acetyltransferase 2 profile and anti-TB drug-induced hepatotoxicity in patients from Southern Brazil. Eur J Clin Pharmacol 2008;64:673-81. [11] Sim E, Walters K, Boukouvala S. Arylamine N-acetyltransferases: from structure to function. Drug Metab Rev 2008;40:479-510. [12] 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. [13] Bolt HM, Thier R. Relevance of the deletion polymorphisms of glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology. Curr Drug Metab 2006;7:613-28. [14] Leiro V, Fernández-Villar A, Valverde D, et al. Influence of glutathione S-transferase M1 and T1 homozygous null mutations on the risk of antituberculosis drug-induced hepatotoxicity in a Caucasian population. Liver Int 2008;28:835-9.

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