antituberculosis drug - induced hepatitis is one of the most prevalent drug - induced liver injuries ( 1 ) .
rifampin ( rif ) , isoniazid ( inh ) , pyrazinamide ( pza ) and ethambutol ( emb ) are first line chemotherapeutic agents in treatment of tuberculosis ( tb ) ( 2 - 5 ) . among these drugs ,
hepatotoxicity of inh can be presented as a mild transient elevation in aminotransferases in 10 to 20% of tuberculosis patients to rare cases of overt hepatitis ( 7 ) metabolism pathways and toxic metabolites of anti - tb agents play a central role in hepatotoxicity of these agents ( 8) .
acetyltransferase-2 ( nat2)-mediated acetylation , resulting in acetyl - inh , which is hydrolyzed to acetylhydrazine and isonicotinic acid ( 9 ) .  
another important factor in inh - induced hepatotoxicity is genetic variations ( 6 , 9 ) .
there are large variations in the inh acetylation capacity suggesting a pharmacogenetic polymorphism of nat2 ( 10 , 11 ) .
this variability in the acetylation capacity results from a wide range of n- acetylation activity and has been characterized by bimodal or trimodal distribution ( 12,13 ) .
the frequency of slow and rapid acetylators differs among ethnic populations ( 14 - 17 ) .  
although primary studies had suggested that the fast acetylators are more susceptible to developing inh induced hepatotoxicity ( 17 , 18 ) , recent studies showed that the slow acetylators are more vulnerable to inh induced hepatotoxicity ( 19 , 20 ) .
still the association between acetylation phenotype and drug - induced hepatotoxicity is controversial ( 6 , 10 ) . the goal of this study was to evaluate relationship of acetylator phenotype and the incidence of antituberculosis drugs - induced hepatotoxicity in iranian pulmonary tb patients .
this study was a prospective , cross - sectional one . a total of 50 unrelated patients ( age > 18 ) with newly diagnosed pulmonary tb from the infectious diseases ward from imam referral hospital affiliated to tehran university of medical sciences ( tehran , iran ) were entered into the study from september 2006 to september 2008 .
standard tuberculosis treatment regimen in our country is based on who recommendation and includes : inh ( 5 mg / kg ) , rif ( 10 mg / kg ) , pza ( 25 mg / kg ) , emb ( 15 mg / kg ) for the first 2 months followed by inh and rif daily for 4 additional months . who tb diagnosis criteria including ( i ) a positive culture for mycobacterium tb or ( ii ) negative culture patient with clinical and radiological features consistent with tb and response to anti - tb treatment or ( iii ) histological findings consistent with tb and response to anti - tb treatment were used for diagnosis of tb in our hospital ( 4 ) .
patients with human immunodeficiency virus ( hiv ) infection , hepatic insufficiency ( alt or ast > 2  upper limit normal or clinically symptoms of liver diseases such as jaundice and ascites ) and renal insufficiency ( creatinine clearance less than 50 ml / min based on cockcroft - gault equation ) , history of smoking ( only positive based on individual expression ) and chronic alcohol consumption ( persons that have history of regular alcohol drinking at least 30 g alcohol daily for 6 months before hospital admission ) excluded from the study .   for all the patients involved in the study , a complete history and physical examination were administered and patients demographic characteristics , history of smoking , alcohol drinking , drug abuse , concomitant diseases and drugs , status of viral infections and other treatment information were collected .  
the liver function tests ( lft ) including aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , alkaline phosphates ( alp ) , direct and total bilirubin and clinical symptoms of drug - induced hepatotoxicity such as anorexia , abdominal pain , nausea and vomiting and right upper quadrant pain were monitored prior to and during anti - tb therapy .  
the causality of a drug - induced hepatotoxicity was determined using the roussel uclaf causality assessment method ( rucam ) ( 21 ) . at the beginning of the anti - tb treatment course and after the overnight fasting , each patient received 300 mg inh ( one tablet ) orally and then 3 h later , 5 ml of venous blood sample was collected into an edta tube and immediately centrifuged .
the plasma samples were then separated and kept frozen at -70c , waiting for analysis .
plasma inh and acetyl - inh were measured by a gradient hplc method ( 22 ) .
acetylator phenotype was determined from the metabolic ratio ( mr ) of acetyl - inh to inh in the plasma samples ( 23 ) .
mr was used to classify the subjects as slow ( mr < 1.0 ) or fast acetylators ( mr > 1.0 ) ( 23 ) .   hepatotoxicity was defined as ( i ) the increased levels of liver transaminases more than three times above the normal value ( < 40 ul for ast and alt ) with any other clinical signs and symptoms , ( ii ) and the elevation of transaminases more than five times above the normal limit if patients had no symptoms ( 24 ) . for assessing the effect of age on anti - tb induced hepatotoxicity ,
patients were divided into two groups : those whose age were < 35 ( group a ) and those whose age were  35 ( group b ) ( 25 ) .
data obtained from our study was expressed as mean - values  sd , numbers or percentages and was analyzed using the statistical package for social sciences version 16.0 .
group comparisons for categorical variables were carried out using the chi - square ( x ) test and fisher s exact test .
all statistical tests were based on a two - tailed probability , and a p - value  0.05 was considered significant .
the age of the patients ranged from 18 to 86 years with a mean of 47.8  21.9 years .
fourteen patients ( 28% ) , 8 of 28 males ( 28.6% ) and 6 of 22 females ( 27.3% ) developed hepatocellular type liver injury .
the mean age of the patients who developed hepatotoxicity , was 43.1  22.7 years ( the age range = 19 - 66 years ) . among the 50 patients ,
20 ( 40% ) were slow acetylators and 30 ( 60% ) were fast acetylators .   for evaluation of causality , we used rucam ( 21 ) .
hepatotoxicity induced by anti - tb drugs was classified as probable based on the causality assessment method .
hepatotoxicity was manifested in 9 of 20 slow acetylators ( 45% ) and only in 5 of 30 rapid acetylators ( 16.7% ) .
the mean duration of treatment before the start of hepatotoxicity was 14.6  6.4 days .
the application of x test showed a significant difference between the acetylation phenotype and hepatotoxicity ( x = 4.778 , and p = 0.03 ) .
frequencies of n- acetyl transferase 2 phenotype and anti - tuberculosis drug - induced hepatotoxicity in iranian pulmonary tuberculosis patients patients were divided into two groups , due to their ages . from 22 patients in group a ( age < 35 ) , seven developed hepatotoxicity , 4 of them were slow acetylators and 3 patients were fast acetylators .
twenty eight patients were  35 years old and consequently were categorized in group b. seven patients ( 5 slow and 2 fast acetylators ) in this group developed hepatotoxicity .
there was not statistically significant difference in the frequency of inh - induced hepatotoxicity between group a and b ( x = 0.284 , and p = 0.59 ) .
furthermore , there was no association between sex and hepatotoxicity in our study using fisher s exact test ( p = 1.00 ) .
summary of anti - tuberculosis drug - induced hepatotoxicity based on the age group and n- acetyl transferase-2 acetylation phenotype group a : age < 35 ; group b : age  35 frequency histogram of metabolic ratio ( acetyl inh / inh ) showed an apparent bimodal distribution with an apparent antimode of acetylation ratio of 1.0 , separating the slow acetylators from the fast ones ( figure 1 ) .
scatter plot of the metabolic ratio of acetyl - inh / inh as a function of inh concentration in plasma following 5 mg / kg dose of inh ( n = 50 ) .
moreover , as shown in figure 2 , there is a lack of correlation between the metabolic ratio ( acetyl - inh / inh ) and the age of the subjects ( r = 0.16 ) .
relationship between metabolic ratio ( acetyl - inh / inh ) and age of iranian .
various proportions of rapid and slow acetylators of different populations in relation to the ethnicity and geographic location were reported ( 26 ) .
we have determined the frequency of nat2 polymorphisms , nat2 acetylation profile and also its relationship with the occurrence of anti - tb drug induced hepatotoxicity .
our major finding is the association of the slow acetylation profile with anti - tb drug - induced hepatotoxicity in a population of tb patients from iran , which is in accordance with results described by other researchers ( 6 , 27 ) .
the present findings contrast sharply with the observations of mitchell , j.r . , et al .
( 28 ) , who reported increased susceptibility of anti - tb drug hepatotoxicity in fast acetylators .
in contrast , a number of other studies did not find any relationship between acetylator status and drug - induced hepatotoxicity ( 9 , 29 ) . this clear discrepancy among the results of previous studies on acetylation status and anti - tb hepatotoxicity may be due to the different designs of the studies , especially in terms of the methodology for nat2 typing , the anti - tb drugs used , and the criteria for defining anti - tb drug hepatotoxicity .
the incidence of antituberculosis drug - induced hepatic dysfunction , ranged from 1% to 36% ( 6 , 9 , 28 ) .
the rate of anti - tb induced hepatitis in this study was 28% , comparable with 27.7% that reported in iranian patients by sharifzadeh m. , et al .
the wide variation of incidence may depend on the definition of hepatic dysfunction , study design , race , sex , and different concomitant use of antituberculosis drugs .
there are possible causes for the higher incidence of antituberculosis drug - induced hepatitis in our study in comparison with some other studies ( 1 ) . unlike our prospective regular monitoring of liver enzyme test results , kopanoff de et al .
( 30 ) checked the liver function test results only on clinical indication , not for routine monitoring .
some studies have evaluated and reported only inh hepatotoxicity ( 9 , 31 ) but it was documented that concomitant use of other hepatotoxic drugs such as rif and pza can increase the incidence and severity of inh - related hepatic dysfunction ( 32 ) .
this is supported by the following observations . the highest incidence ( 36% ) of anti - tb
the prevalence of nat 2 phenotype in a sample of healthy iranian individuals including 88 samples was 32.9% slow , 48.9% intermediate , and 18.2% rapid using pcr - rflp method ( 33 ) . in another study using pcr - rflp method , the frequency of slow , intermediate and rapid acetylator phenotype was 49.4% , 41.5% , and 9.1% respectively in 229 unrelated healthy subjects from the general tehran population ( 34 ) .
frequency of slow acetylator phenotype in these studies is approximately near to our finding ( 32.9% , and 49.4% vs. 40% ) .
the discrepancy probably originated from the difference in the methodology used , variations in study subjects ( healthy individuals versus patients ) , the effects of unidentified genetic or environmental factors , and incomplete correlation between reported acetylator phenotypes and corresponding nat2 genotypes . in another study , polymorphism of nat 2 was studied in 74 unrelated healthy iranian volunteers , using sulfamethazine as metabolic probe .
the frequency of slow acetylators , determined by using free and total plasma sulfamethazine concentrations , was 78.4% ( 35 ) .
the major finding is that phenotype assignment was dependent on the probe substrate ( 36 ) .
in addition to inh and sulfamethazine , other acetyl - transferase substrates such as caffeine ( 37 ) and dapsone ( 38 ) have been used as phenotypication probe .
on the other hand , in previous studies over iranian population , subjects were healthy volunteers , whereas the tb ones were studied in here .
our findings are in agreement with the previous study ( 9 ) , but another study reported old age as a risk factor for development of hepatotoxicity during the treatment of tb ( 39 ) . for women ,
it was reported increased risk of hepatotoxicity but this did not achieve statistical significance ( 40 ) .
 the aim of this study was to determine the association of n - acetyltransferase-2 polymorphisms and anti - tuberculosis drug - induced hepatotoxicity in iranian pulmonary tuberculosis patients . 
 acetylating phenotypes was studied in 50 iranian pulmonary tuberculosis patients using metabolic ratio of plasma acetyl - isoniazid to isoniazid . 
 the association between hepatotoxicity and the n - acetyltransferase-2 phenotype was evaluated by using the chi - square ( x2 ) test.the metabolic ratio had a bimodal distribution with an antimode value of 1.0 . 
 based on the metabolic ratio of the mentioned patients , 20 ( 40% ) were slow acetylators and 30 ( 60% ) were fast ones . 
 hepatotoxicity was manifested in 9 of 20 slow acetylators ( 45% ) and only in 5 of 30 rapid acetylators ( 16.7% ) . 
 there was a significant difference in the frequency of hepatotoxicity between the slow and fast acetylators ( x2 = 4.778 , and p = 0.03 ) . 
 sex and age were not found to be risk factors for hepatotoxicity . 
 our findings show that slow acetylation profile is significantly associated with a higher risk of developing hepatotoxicity due to the anti - tb drugs in iranian pulmonary tuberculosis patients . 
Introduction
Experimental
Results and Discussion