Worldwide, tuberculosis (TB) is one biggest public health issues. In 2017, TB was estimated to have caused about 1.3 million deaths and among the top 10 causes of death in the planet, according to the World Health Organization (WHO). In Brazil, little less than 73,000 new cases were notified in 2018, representing an incidence coefficient of 34.8 cases/100,000 inhabitants. In the same year, the state of Rio de Janeiro reported an incidence coefficient of 66.3 cases/100,000 inhabitants, ranking second among states with the highest coefficient in the country. The state of Amazonas recorded the highest incidence, with 72.9 cases/100,000 inhabitants in 2018.1

In the current treatment of TB, adverse drug reactions (ADR) can vary in intensity, severity, and/or among populations.2

TB drug-induced liver injury (DILI), considered the most severe ADR, influences the current treatment with negative impact on patient treatment outcome.3

The current treatment of TB in Brazil,4 although effective, can induce adverse events, including DILI.3

Isoniazid (INH) is the main contributing drug for DILI development. It is metabolized by CYP2E1 and generates hepatotoxic metabolites. The GST enzyme reduces this toxicity because it can catalyze the conjugation reaction of the sulfhydryl terminal of these compounds (Fig. 1).5,6

Fig. 1.

Route of biotransformation of isoniazid (INH). R: Rifampicin. Adapted from Sotsuka et al.6 The use of this figure has been authorized by the copyright owner.

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CYP2E1 is expressed by CYP2E1, a polymorphic gene. In this study, four alleles of the gene were characterized. The CYP2E1*1A wild type allele presents a cleavage site for the Dra I and Rsa I restriction enzymes by polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP). The variant alleles are CYP2E1*5B, with polymorphism in the promoter region, with no site for Rsa I; CYP2E1*6, with polymorphism in intron 6, with no site for Dra I; and CYP2E1*5A, with polymorphism in the promoter and intronic regions, has no site for any of these two enzymes. It is not clear which allele has greater influence on the increased activity of CYP2E1.7

GST enzymes play a protective role on the mechanism of liver detoxification, catalyzing glutathione reaction, facilitating solubilization, and excretion of toxic substrates.8 The GSTM1 and GSTT1 genes present polymorphisms by partial or total deletion. Individuals with total deletion are more prone to toxic effects because the enzyme activity is absent; in partial deletion, it can display some activity.3

The objective of this study was to evaluate the genotype and the allele frequencies of the CYP2E1, GSTM1 and GSTT1 gene variants besides investigating the relation between these genetic polymorphisms and the development of DILI in patients treated for active pulmonary tuberculosis, admitted to the Hospital Estadual Santa Maria (HESM) [Santa Maria State Hospital], in the state of Rio de Janeiro, Brazil.

In this study, 13% of patients presented DILI, similar to studies that included viral hepatitis in their exclusion criteria (EC). Compared to other studies, the frequency of DILI can vary. In the Brazilian population, DILI frequency seems to be related to the rigor of the EC used by the authors. Apparently, these predisposed conditions can cause a selection bias in the study and be a contributing factor for higher rates of DILI. Studies that were less rigorous and included active smokers, patients with abusive alcohol consumption, and patients co-infected with HIV and viral hepatitis (B and/or C) reported higher DILI frequency. Therefore, including patients who have predictive factors for DILI during treatment of TB will likely have higher rates of DILI, as reported by Zaverucha et al.15 who found 40% and Monteiro et al.16 who found 33%. Other Brazilian studies (Vieira & Gomes;13 Possuelo et al.;17 Teixeira et al.;18 and Santos et al.19) characterized by more restrictive eligibility criteria (with no predictive factors for DILI), have found lower DILI frequencies (<20%). Liu et al.20 have argued that these factors influence the risk and severity of DILI.

The American Thoracic Society21 (ATS) criteria for defining DILI are more rigorous than Bénichou criteria.12 Among the parameters used, liver enzymes level should be five-fold higher than the ULN, while Bénichou12 parameters set up a level of two-fold. One would expected to find a relationship between the different criteria used to define DILI and its frequency in the analyzed studies. However, the choice of DILI definition had no impact on the frequency of DILI.

In Brazilian studies (including the current study) the variation found in the DILI frequency as per GST genes was small. Monteiro et al.16 have suggested that the Null GSTM1 gene is probably associated with the intensity of liver toxicity. Teixeira et al.18 have found no association between GST polymorphisms and risk of DILI. Singla et al.5 have argued that patients with deletion in both GST genotypes have greater risk of DILI. In contrast, Tang et al.3 have not associated the deleted GST genotypes with the risk of DILI.

Some authors have reported that patients with DILI have higher frequency of the CYP2E1 variant alleles. In our study, patients with the presence of CYP2E1 variant alleles are at higher risk for DILI. In the literature, the findings of the Brazilian population show some discrepancies regarding the association of CYP2E1 genotype and risk for DILI. Zaverucha et al.15 found an increased risk for DILI in individuals with CYP2E1 variant alleles. However, Teixeira et al.18 did not find any relationship between DILI and the CYP2E1 genotype.

Deka et al.22 have found a significant association of the two variant alleles (c2 and C) with DILI. Bose et al.23 have found a significant association of the C variant allele with DILI in patients with viral hepatitis. A significant association of the c2 allele with DILI was reported by Singla et al.5 Thus, it has been suggested that CYP2E1 variant genotypes could be associated with increased risk of DILI during anti-TB treatment.

Roy P, Majumder & Roy B2 and Ramachandran & Swaminathan7 have proposed that INH can induce CYP2E1 activity in mice, an activity dependent on the plasma concentration of INH. On the other hand, INH can inhibit CYP2E1 activity, a fact more common in individuals of variant genotype. In addition, they have related the increase in enzyme activity to higher production of hepatotoxins, which increases the risk for DILI. Ramachandran & Swaminathan7 have pointed to the important existing discussion in the literature about which allele has greater influence on the increased activity of CYP2E1, variant or wild.

Most of the research subjects were male. According to data from the Sistema Único de Saúde (SUS) [Unified Health System]24 during the inclusion period, 230 patients were admitted to the HESM with active pulmonary TB, 72% males and 28% females. Data are consistent with the Brazilian TB cases according to the PNCT25 Epidemiological Bulletin that describes a two-fold higher incidence coefficient in males. The studied sample consisted of patients in productive age, mean of 44 years for males and 37 years for females.

Patients who had cure discharged remained in the hospital until completing treatment (about 180 days), while the other patients remained hospitalized for a shorter period. There was no follow-up of patients’ treatment after hospital discharge, a fact that could be considered a study limitation. The mean time of patients’ hospitalization was 61% shorter than the time needed to complete treatment. Mild anemia found in patients during hospitalization is compatible with anemia of chronic diseases, common in Tuberculosis.26,27

Among the 45 participants, five (11%) were co-infected with HIV, similar to that described by the PNCT28 Epidemiological Bulletin that reports a 10% rate of HIV co-infection among TB cases of Rio de Janeiro (RJ) state.

The ADR frequency found in this study is slightly higher (3 ADR/patient) than that reported by Vieira & Gomes13 who have pointed out that 49% of their patients had one or more ADR associated with TB treatment. The discrepancy can be explained by the characteristics of the studied population: no permanent residence, poor nutritional status and compromised immune system, besides having limited access to basic sanitation and hygienic conditions. The DILI group showed predominance of the female sex and an increased sensitivity to anti-TB drugs when compared to the No-DILI group. The studied sample is consistent with findings in the literature: higher DILI frequency among females, malnourished individuals with pre-existing liver disease and advanced age.29 The studied sample showed some of these characteristics except for prior liver disease, since it was one of the exclusion criteria. Piva et al.29 have observed that the rate of treatment default among malnourished patients was three-fold higher than what is acceptable because malnourished patients tend to have a compromised immune response more often than the normal weight patient.

This study had a small sample size (n). However, the other works cited had periods of inclusion greater than 13 months. Monteiro et al.16 have included 177 patients over five years, Zaverucha et al.15 have included 131 patients over seven years and Teixeira et al.18 have included 167 patients over 10 years. Even with reduced sample size, we have observed an important effect in the evaluation of the risk for DILI.

Patients who have the CYP2E1 variant genotype or Null GSTT1 have a four-fold greater risk of having DILI during anti-TB treatment. Those who have both the CYP2E1 variant genotype and Null GSTT1, have no increased risk of having liver injury compared to the individuals who have only one of these genotypes. More studies with larger sample sizes should be conducted for a better understanding of the role of these genetic factors in the association with the occurrence of DILI during treatment of TB.