GSTT 1 null and MPO-463 G > A Polymorphisms and Carboplatin Toxicity in an Indian Population

Platinum drugs are most commonly used in present day cancer chemotherapy. However, severe drugrelated toxicity which is often irreversible remains major limitation in their use. Carboplatin, an analogue of cisplatin has antitumour activity similar to cisplatin but different spectra of toxicity. It causes less nephrotoxicity, neurotoxicity, ototoxicity and gastrointestinal toxicity than cisplatin. However, myelosuppression, which is uncommon with cisplatin, is the doselimiting toxicity of carboplatin (O’dwyer et al., 1997). Among many potential causes of individual variability for drug response, genetic variation has been recognized as an important determining factor for more than five decades (Ma and Lu, 2011). Genetic alterations in drugmetabolizing enzymes may cause such variability in drugrelated adverse effects (Donnelly, 2004; Nakamura, 2008).


Introduction
Platinum drugs are most commonly used in present day cancer chemotherapy.However, severe drug-related toxicity which is often irreversible remains major limitation in their use.Carboplatin, an analogue of cisplatin has antitumour activity similar to cisplatin but different spectra of toxicity.It causes less nephrotoxicity, neurotoxicity, ototoxicity and gastrointestinal toxicity than cisplatin.However, myelosuppression, which is uncommon with cisplatin, is the dose-limiting toxicity of carboplatin (O'dwyer et al., 1997).
Among many potential causes of individual variability for drug response, genetic variation has been recognized as an important determining factor for more than five decades (Ma and Lu, 2011).Genetic alterations in drugmetabolizing enzymes may cause such variability in drugrelated adverse effects (Donnelly, 2004;Nakamura, 2008).

GSTT1 null and MPO -463G>A Polymorphisms and Carboplatin Toxicity in an Indian Population
Arundhati Bag 1 *, Nirdosh Kumar Pant 2 , Lalit Mohan Jeena 1 , Niladri Bag 3 , Narayan Singh Jyala 4 Glutathione S-transferases (GSTs) constitute a multigene family of metabolizing enzymes and are involved in phase II cellular detoxification of reactive metabolites by conjugating glutathione to electrophilic functional groups and rendering them more water soluble (Hayes and Pulford, 1995).GSTT1 gene is a member of GST theta class, the most ancient class among seven classes of GSTs (Di Pietro et al., 2010).A null polymorphism or total gene deletion (-/-) which abolishes gene activity is found for GSTT1.Since GSTT1 is constitutively expressed in the liver (Thorn et al., 2012) and is involved in detoxification process of platinum drugs (Moyer et al., 2010) the null genotype may have an important role in generating therapeutic outcome.
Further, carboplatin generates oxidative stress (Cheng et al., 2008;Lin et al., 2010) which can lead to toxic effects (Husain et al., 2001).Therefore, genetic polymorphisms of enzymes related to cellular oxidative stress may lead to adverse events in cancer patients.It has been found that platinum drugs like carboplatin are regulated by genes involved in DNA detoxification, e.g., myeloperoxidase (Marsh et al., 2007).Myeloperoxidase MPO, a lysosomal enzyme found abundantly in neutrophils, generates reactive oxygen species (ROS) that can be released outside the cell and have potential to damage extracellular target (Klebanoff, 1999).A single nucleotide polymorphism, -463G>A at its promoter site leads to loss of SP1 transcription factor binding site (Piedrafita et al., 1996) and subsequent reduced production of ROS.Thus A allele of MPO can be hypothesized to have a protective role from carboplatin-toxicity.
In the present study we investigated GSTT1 null (-/-) and MPO-463G>A polymorphisms in cancer patients treated with carboplatin and developing toxicity.

Selection of patients
Cancer patients taking treatment with carboplatin at Swami Ram Cancer Hospital and Research Center, Haldwani were recruited as study subjects.This is a referral hospital which caters people from hills and planes of Kumaun region of northern India and adjoining areas of neighboring states (Bag et al., 2012).Four hundred fifty milligram of carboplatin was given as standard dose to them either in single or combined regimen.Signed informed consent was collected from each study participant.Ethical approval for the study was obtained from Institutional Ethical Committee, Govt.Medical College, Haldwani.Toxicity was scored according to the NCI.CTC common toxicity criteria version 2.0.Information on toxicity was collected by researcher unaware of the genotypes of patients.

DNA extraction
Two to five millilitre of peripheral blood was collected in sterile EDTA tube.DNA was extracted from whole blood following standard phenol-chloroform method (Sambrook et al., 1989).

Detecion of GSTT1 (-/-)
Homozygous gene deletion polymorphism (-/-) for GSTT1 was detected by performing duplex polymerase chain reaction (PCR) as described earlier (Bag et al., 2013).Primer set for GSTT1 genotypes was within the GSTT1 gene and another set of primer was selected from human mitochondrial manganese superoxide dismutase (MnSOD) as positive control of PCR.Absence of a 131bp band indicated homozygous deletion (null; -/-) for GSTT1.Presence of this band indicated homozygous or heterozygous genotypes (+/+ or +/-).This protocol did not differentiate between genotypes either with one or both copies of the gene.A 175 bp band of positive control (MnSOD) indicated successful PCR.Duplex PCR were repeated for all samples showing null genotype.

Detection of -463G>A of MPO
PCR was carried out using 10 pmol of each forward and reverse primer, 5?-GGTATAGGCACACAATGGTGAG-3?
, respectively (Eurofins, Bangalore).PCR master mix (Promega) was used.PCR condition followed an initial denaturation at 94 0 C for 2 mins followed by 30 cycles of 30 sec denaturation at 94 0 C, 30 sec annealing at 58 0 C, 30 sec extension at 72 0 C, and final extension at 72 0 C for 5 mins.Each set of reaction had negative control.
SNP of MPO was detected by digesting 6 µl of PCR product with 0.5U of AciI restriction enzyme (New England Biolab) at 37℃ for 1½ hr.Digestion product was visualized in polyacrylamide gel.Three bands at 169,120 and 61 were produced for G/G homozygotes, two bands at 289 and 61 bp were produced for A/A homozygotes and heterozygotes (G/A) produced four bands at 289, 169, 120 and 61 bp.Randomly chosen 5 samples were sequenced (SciGenome, India) for confirmation of the restriction digestion results and the reproducibility was 100%.

Results
During this study period from Dec, 2011 to Nov., 2012 total 33 patients were identified who were treated with carboplatin.We were able to note toxicity status for 10 patients which is presented in Table 1.Patient 1 to 5 had hematological toxicity ranging from grade 2 to grade 4. Patients 6 to 10 did not have hematological toxicity although had other types of toxicities.Out of 5 patients with hematological toxicity, 4 were homozygous for G allele at -463 position of MPO.While in the patient group without hematological toxicity, majority (80%) had AG alleles.This indicates that A allele of MPO -463G>A polymorphism may be related to reduced hematological toxicity.GSTT1 genotype did not show any relation with a particular type of toxicity.However, in patient 1 GSTT1 null genotype in combination with risky genotype of MPO may have contributed in generating severe hematologic toxicity, and also renal toxicity which is less frequently found for carboplatin.

Discussion
The second-generation platinum drug carboplatin is widely used to treat lung, ovarian, head and neck cancer (Lin et al., 2010).However, myelosuppression often limits its use.In this study we noted grade 4 and 3 hematologic toxicity in two patients and grade 2 toxicity in 3 of them It is known that genetic factors potentially influence the pharmacokinetics of anticancer drugs (Undevia et al., 2005).Thus polymorphisms in drug metabolizing enzyme like GSTT1 may be a putative influencing factor in therapeutic outcome.GSTT1 has been found to be a probable factor for therapy-related adverse events in medulloblastoma (Barahmani et al., 2009).However, in a study on malignant mesothelioma patients treated with either cisplatin or carboplatin combined regimen, GSTT1 null was found to have no influence on treatment-related leucopenia or thrombocytopenia (Erculj et al., 2012).In this study no role for GSTT1 in producing hematologic toxicity or other carboplatin-related toxicity was observed.
Myeloperoxidase MPO catalyzes the formation of strong oxidant hypochlorous acid (HOCl) from hydrogen peroxide (H 2 O 2 ).In normal condition HOCl is required for defense against microbial infection.However, its overproduction may have potential to cause unwanted damage to normal cells.In vitro experiment revealed that carboplatin generates H 2 O 2 (Palma and Aggarwal, 1994) that helps in its tumoricidal activity.But at the same time it increases cellular H 2 O 2 pool that may lead to enhanced production of HOCl.In addition, both H 2 O 2 and HOCl function as inflammatory mediators (Ochoa et al., 1997).Therefore, they may lead to carboplatin -related toxicity.Although MPO-463G>A promoter polymorphism has been studied for risk of different types of cancers (He et al., 2009), it has rarely been studied for predisposition to platinum drug related toxicity.In the present study AG genotype was found in the patients without hematologic toxicity indicating a plausible relation with reduced hematological toxicity.
To the best of our knowledge, association between these two genetic polymorphisms and carboplatintoxicity has been studied for the first time in an Indian population.Although this was a preliminary study with small number of individuals studied.Larger studies are required to demonstrate actual role of GSTT1 null and MPO -463G>A polymorphisms with carboplatin-toxicity.It has been found that treatment outcome can vary among ethnicities (Millward et al., 2003;Soo et al., 2012), and is an important factor to be considered during assessment of drug efficacy.Therefore, more experiments in this population of northern India can be carried out which may reveal potential differences in carboplatin-treatment outcome between this and other populations.Further, not only MPO but also other enzymes that balance cellular free radical levels by neutralizing them, can be studied for carboplatin toxicity.
In conclusion, the present study indicates a probable link between variant morph of MPO-463 G>A and reduced carboplatin-related hematological toxicity in a north Indian population, which requires larger study including other polymorphisms in oxidative stress pathway for a better comprehensive understanding.

Table 1 . Carboplatin Toxicity and Genetic Polymorphisms in Cancer Patients
Polymorphisms and Carboplatin Toxicity in Indianswhile other 5 patients did not have this toxicity.