**3.3. Glutathione S-transferases**

Glutathione S-transferases constitute a superfamily of multifunctional ubiquitous enzymes that play an important role in cellular detoxification by protecting macromolecules against reactive electrophilic attack. The GSTs are Phase II enzymes that catalyze the nucleophilic attack of glutathione (GSH) into components that contain an electrophilic carbon, nitrogen or sulfur atom. The combination of the GSH with these compounds often leads to formation of less reactive and more water soluble products, more easily excreted by the body [23, 78].

Glutathione transferases are of great interest to pharmacologists and toxicologists, since they are drug targets for the treatment of asthma and cancer, in addition to metabolize drugs, insecticides, herbicides, carcinogens and products of oxidative stress. Polymorphisms in *GST* genes are often correlated with susceptibility to various cancers, as well as alcoholic liver disease [23, 78-81].

In humans, eight gene families of soluble (or cytosolic) GSTs have been described: alpha (α) located on chromosome 6, mu (μ) on chromosome 1, theta (θ) on chromosome 22, pi (π) on chromosome 11; zeta (ζ) on chromosome 14, sigma (σ) on chromosome 4; kappa (κ) (chromo‐ somal location not given) and omega (Ω) on chromosome 10 [80]. This classification is based on amino acid sequences, substrate specificity, chemical affinity, protein structure and enzyme kinetics. These enzymes are highly expressed in the liver and constitute up to 4% of total soluble proteins but can be seen in several other tissues [82]. GSTs have an overlap of specific substrates and the deficiency in one isoform can be compensated by other isoforms. Gluta‐ thione S-transferase mu (GSTM), glutathione S-transferase theta (GSTT) and glutathione Stransferase Pi (GSTP) have been the most studied isoform [83-88].

Several studies have described the involvement of polymorphisms in *CYP2E1* in cancer development but results are controversial. The studies showed that the frequency of SNP -1053 C>T in the promoter region varies significantly in different ethnic groups. The mutant allele is present with a frequency of 2-8% in Euro-descendants but varies in Asia from 25 to 36% [75].

In 2003, Huang and coworkers showed an association of the wild-type genotype \*1A/\*1A with risk of developing liver damage induced by isoniazid in adult TB patients, regard‐ less of their profile of acetylation (OR 2.52; 95% CI 1.26 to 5.05) [76]. Later, Vuilleumier and colleagues showed association between this CYP and isoniazid-induced hepatotoxici‐ ty, without hepatitis, during chemoprophylaxis for TB (OR 3.4; 95% CI 1.1 to 12; *p* = 0.02). The risk of having high levels of liver enzymes was 3.4-fold higher when com‐ pared with all other *CYP2E1* genotypes [55]. Another study on Indian children with TB showed association between risk of hepatotoxicity and polymorphisms in *CYP2E1*, de‐ spite of low sample size [77]. However, a study with on a Korean population found no relationship between hepatic adverse effects with genotype \*1A/\*1A of *CYP2E1* during anti-TB treatment [51]. Lack of association between this CYP and antituberculosis druginduced liver injury was also observed in Brazil [58]. The discrepancy of these results may be due to differences in the frequencies of *CYP2E1\*1A* and *CYP2E1\*5* alleles among

Finally, CYP2E1 converts acetyl hydrazine into hepatotoxins like acetyldiazene, ketene and acetylonium ion. The reaction of acetyl hydrazine (at high levels) with CYP2E1 leads to covalent binding of these secondary metabolites with intracellular proteins (Figure 1). As a consequence, intracellular changes occur resulting in loss of ionic gradients and decrease of ATP levels and consequent disruption of actin followed by cell lysis. Further studies in different populations and with a larger sample size are needed to determine the true influence of CYP2E1 gene polymorphisms on the occurrence of liver injury during treatment for TB.

Glutathione S-transferases constitute a superfamily of multifunctional ubiquitous enzymes that play an important role in cellular detoxification by protecting macromolecules against reactive electrophilic attack. The GSTs are Phase II enzymes that catalyze the nucleophilic attack of glutathione (GSH) into components that contain an electrophilic carbon, nitrogen or sulfur atom. The combination of the GSH with these compounds often leads to formation of less reactive and more water soluble products, more easily excreted by the body [23, 78].

Glutathione transferases are of great interest to pharmacologists and toxicologists, since they are drug targets for the treatment of asthma and cancer, in addition to metabolize drugs, insecticides, herbicides, carcinogens and products of oxidative stress. Polymorphisms in *GST* genes are often correlated with susceptibility to various cancers, as well as alcoholic liver

In humans, eight gene families of soluble (or cytosolic) GSTs have been described: alpha (α) located on chromosome 6, mu (μ) on chromosome 1, theta (θ) on chromosome 22, pi (π) on chromosome 11; zeta (ζ) on chromosome 14, sigma (σ) on chromosome 4; kappa (κ) (chromo‐

the populations and the different criteria to define hepatotoxicity used.

**3.3. Glutathione S-transferases**

114 Tuberculosis - Current Issues in Diagnosis and Management

disease [23, 78-81].

The subfamily GST mu is encoded by five genes arranged in tandem (5\_-*GSTM4-GSTM2- GSTM1-GSTM5-GSTM3*-\_3), forming a 100 kb gene cluster on chromosome 1p13.3 (Figure 4). Polymorphisms have been identified and clinical consequences of genotypes resulting from combinations of alleles *GSTM1\*0*, *GSTM1\*A*, and *GSTM1\*B* have been widely investigated [78, 81, 89, 90]. Individuals who possess the homozygous null for *GSTM1* (GSTM1\*0/GSTM1\*0) do not express this protein. Thus, the absence of this gene can cause an increased accumulation of reactive metabolites in the body, increasing the interaction with cellular macromolecules and tumor initiation process. *GSTM1*\**A* and *GSTM1\*B* differ in only one base in exon 7 and encode monomers that form active dimers. The catalytic activity of these enzymes are very similar [91].

The *GSTM1* gene is flanked by two almost identical 4.2-kb regions. *GSTM1\*0* originates from homologous recombination between the two repeat regions which results in a 16 Kb deletion containing the entire gene *GSTM1* (Figure 4). *GSTM1* is precisely excised leaving the adjacent *GSTM2* and *GSTM5* genes intact [78]. In a study of liver specimens of 168 autopsied Japanese subjects, observed was that the *GSTM1\*0* null allele was more frequent in livers with hepatitis and hepatocellular carcinoma compared to control livers [92].

**Figure 4.** Structural localization of 100 kb gene cluster encoding the GST mu subfamily (chromosome 1p13.3). The figure indicates the homologous recombination event that can happen causing the null allele (*GSTM1\*0* - no *GSTM1*). Figure adapted from [78].

The subfamily GST theta consists of two genes, *GSTT1* and *GSTT2*, located on chromosome 22q11.2 and separated by approximately 50 Kb (Figure 5). Analysis of the 119 Kb portion containing these genes revealed two regions flanking *GSTT1*, HA3 and HA5, with more than 90% homology. HA3 and HA5 contain two identical 403-bp repeats and the occurence of *GSTT1\*0* allele is probably caused by homologous recombination between the two regions [78]. In humans, *GSTT1* is also expressed in erythrocytes and probably plays a global role in early detoxification of xenobiotics and carcinogens.

These controversal results may be due to the small sample size in many studies and the different frequencies of the null genotypes. New populations should be evaluated with large sample size to see which of these polymorphisms can be used as genetic markers for the risk

Tuberculosis Pharmacogenetics: State of The Art

http://dx.doi.org/10.5772/54984

117

The concept of personalized medicine is not really new, but it has been receiving increasing attention in recent years for improval of drug regulation and medical guidelines. There is considerable interindividual variability in metabolism, partly due to human differences on a genetic level. Genetic polymorphisms in drug-metabolizing enzymes can affect enzyme activity and may cause differences in treatment response or drug toxicity, for example, due to an increased formation of reactive metabolites. Such polymorphisms may explain differences

Genotyping cannot completely predict the phenotype on an individual level because of to the additional contribution of epigenetic, endogenous and environmental factors. However, pharmacogenetics is able to add important information in many cases where therapeutic drug scheme is inappropriate or not sufficient. Nowadays, we can cite three examples of personal‐ ized medicine application in clinical practice, (i) AIDS treatment (abavir / skin hypersensitiv‐ ity / *HLA-B\*5701*), (ii) anticoagulation (warfarin / bleeding / *CYP2C9*) and (iii) treatment of

Although limited information exists regarding isoniazid concentrations that cause toxic reactions, it has been proposed to adjust isoniazid dosage depending on individuals acetylator status: a lower dosage for slow acetylators to reduce the risk of liver injury and a higher isoniazid dosage for fast acetylators to increase the early bactericidal activity and thereby lower the probability of treatment failure [50]. However, more robust clinical prospective studies are needed to evaluate the real contribution of these different polyporphirms in the occurence of liver side effects during anti-TB treatment. Future studies should include larger sample size, different ethnic population, simultaneous analysis of different genetic markers, different

Laboratory of Molecular Biology Applied to Mycobacteria – Oswaldo Cruz Institute – Fiocruz,

, Márcia Quinhones Pires Lopes, Philip Noel Suffys and

in incidence of anti-TB drugs induced hepatotoxicity between different populations.

acute lymphoblastic leukemia (azathioprine / treatment resistence / *TPMT*) [98].

degrees of liver injury and consideration of possible confounding factors.

of side effects during anti-TB treatment.

**4. Conclusion**

**Author details**

Raquel Lima de Figueiredo Teixeira\*

\*Address all correspondence to: raquelft@ioc.fiocruz.br

Adalberto Rezende Santos

Rio de Janeiro, Brazil

**Figure 5.** Structural localization of gene cluster encoding the GST subfamily theta (chromosome 22q11.2). The *GSTT1* null allele (*GSTT1\*0*) arises by homologous recombination of the left and right 403-bp repeats, which results in a 54 kb deletion containing the entire *GSTT1* gene. Figure adapted from [78].

Deficiencies in the GST activity due to the null genotypes of *GSTM1* and *GSTT1* may modulate susceptibility to the development of hepatotoxicity induced by drugs and xenobiotics. Furthermore, it was observed that the frequencies of *GSTT1\*0* and *GSTM1\*0* alleles vary within different ethnic groups [78, 82]. Liver injury induced by INH has been associated with the depletion of glutathione content and reduction of GST activity in an animal model for hepatotoxicity by anti-TB drugs [22].

In 2001, Roy and colleagues demonstrated that individuals, homozygous for the null *GSTM1,* had a relative risk of 2.12 for developing hepatotoxicity induced by anti-TB drugs. However, these authors found no association of the *GSTT1* null genotype with this side effects [54]. Similarly, another study in the Thai population found that only the *GSTM1* null genotype increases the risk of liver injury (OR 2.23, 95% CI 1.07 to 4.67) [93]. The opposite was observed by Leiro and colleagues: individuals with the *GSTT1* null genotype had an increased risk of developing hepatotoxicity induced by anti-TB drugs and no significant association was observed between GSTM1\*0/\*0 genotype and liver injury [94]. These studies suggest a protective effect of glutathione S-transferases to the hepatotoxic effects of isoniazid.

On the other hand, recent studies in different population showed no relationship between GSTM1\*0/\*0 or GSTT1\*0/\*0 genotypes and liver injury during anti-TB treatment [58, 95, 96]. In a population-based prospective antituberculosis treatment coort in China, a more robust case-control study was conducted and there was no statistically significant association between null genotypes and hepatotoxicity induced by anti-TB drugs [97].

These controversal results may be due to the small sample size in many studies and the different frequencies of the null genotypes. New populations should be evaluated with large sample size to see which of these polymorphisms can be used as genetic markers for the risk of side effects during anti-TB treatment.
