Hema Prasad Mundluru

Assessment of 8-oxo-7, 8-dihydro-2'-deoxyguanosine and malondialdehyde levels as oxidative stress markers and antioxidant status in non-small cell lung cancer.

Objective: The present investigation was taken up to evaluate the 8-oxo-7,8-dihydro-2′-deoxyguanosine and malondialdehyde as markers of oxidative stress, the levels of antioxidants and the correlations between these oxidative stress markers and antioxidants in lung cancer patients. Methods: The study included 222 patients (158 men and 64 women, age ranging from 32 to 85 years) and 207 control subjects (153 men and 54 women, aged 30–80 years) for the analysis of urinary excretion of 8-oxodG using an ELISA assay, plasma malondialdehyde using spectrophotometer and red cell Cu-Zn SOD and GPx activities by kit methods. Results: The levels of 8-oxodG and malondialdehyde were significantly higher (p < 0.001) and red cell superoxide dismutase and glutathione peroxidase activities (p < 0.001) were significantly lower in lung cancer patients than in controls. There was a significantly positive correlation between 8-oxodG and malondialdehyde (r = 0.912, p < 0.001) and a negative correlation between 8-oxodG and antioxidants. Conclusions: Our results demonstrate that an increased rate of oxidative stress might play a role in the pathogenesis of lung cancer as evidenced by a failure in the oxidant/antioxidant balance in favour of lipid peroxidation and DNA damage.

Mutational analysis of JAG1 gene in non-syndromic Tetralogy of Fallot children☆

BACKGROUND JAG1 is an evolutionarily conserved ligand for Notch receptor and functions in the cell fate decisions, cell-cell interactions throughout the development of heart especially right heart development. Tetralogy of Fallot (TOF) is essentially a right sided heart disease with characteristic features of ventricular septal defect, right ventricular outflow tract obstruction, aortic dextroposition and right ventricular hypertrophy. Hence, the present study was investigated to identify mutations of JAG1 gene in an Indian cohort of patients with TOF. METHODS The clinical data and blood samples from 84 unrelated subjects with TOF were collected and evaluated in comparison with 87 healthy individuals. PCR based single strand conformation polymorphism analysis and subsequent bidirectional DNA sequencing of conformers was carried in the exon 6 of JAG1 gene. RESULTS The DNA sequences aligned with NCBI-BLAST led to the identification of four novel variations including one nonsense 765 C>A, two missense 814 G>T, 834 G>T; and one silent alteration 816 G>T in TOF patients. The protein structure of JAG1 predicts that these variations effect first and second epidermal growth factor like repeat and might disturb ligand-receptor binding ability. The presence of similar variations was not observed in healthy controls. The software CLUSTAL-W showed the inter species conservation of altered amino acids in missense mutations. CONCLUSION Disease-associating novel JAG1 gene variations were found in TOF patients, and seem to play an important role in the causation of the disease.

Association of CYP1A1, GSTM1 and GSTT1 gene polymorphisms with risk of non-small cell lung cancer in Andhra Pradesh region of South India

BackgroundLung cancer is one of the most preventable causes of death globally both in developed and developing countries. Although it is well established that smokers develop lung cancer, there are some smokers who are free from the disease risk. The predisposition to lung cancer is attributed to genetic polymorphisms in xenobiotic metabolizing genes. Reports on assessment of xenobiotic metabolizing genes like Cytochrome P 450 1A1 (CYP1A1), Glutathione -S -transferase M1 (GSTM1) and T1 (GSTT1) polymorphisms from India are meagre, and reports from Andhra Pradesh are lacking.Methods and resultsAssessment of polymorphisms in CYP1A1, GSTM1 and GSTT1 in NSCLC patients and healthy individuals specific to population of Andhra Pradesh, a South Indian state was attempted by multiplex PCR and RFLP, and this is the first study which tried to correlate oxidative stress with the polymorphisms in xenobiotic metabolizing genes. Results showed that CYP1A1 m1 ‘CC’ genotype was significantly associated with lung cancer susceptibility with a 2.3-fold risk, CYP1A1 m2 ‘AG’ gene polymorphisms with 8.8-fold risk and GSTT1 (−/−) genotype demonstrated a twofold risk of disease susceptibility.ConclusionsA combined role of genetic polymorphisms and smoking status can be attributed for the cause of lung cancer. Further, the association between oxidative stress and genetic polymorphisms showed a correlation between GSTT1 and super oxide dismutase activity; CYP1A1 m1, m2 and GSTT1 with glutathione peroxidase activity; CYP1A1 m1 and GSTM1 with melondialdehyde levels; and CYP1A1 m1 and GSTT1 with 8-oxo-7,8-dihydro-2′-deoxyguanosine. A higher risk of lung cancer seems to be associated with combined gene polymorphisms of phase I and phase II enzymes than that ascribed to single gene polymorphism.

Genetic instability in peripheral lymphocytes as biological marker for non-small cell lung cancer patients in the South Indian state of Andhra Pradesh.

Objectives This study aims, first, at evaluating the DNA and chromosomal damage in non-small cell lung cancer (NSCLC) patients from the South Indian state of Andhra Pradesh, and then at correlating these results with possible confounding factors that might potentially play a role in causing genetic damage. Methods The study included 246 NSCLC patients (177 men and 69 women) and 250 healthy controls (180 men and 70 women) for the analysis of DNA and chromosomal damage using the comet assay and micronucleus test. Results Both DNA and chromosomal damage were found to be increased in NSCLC patients compared to healthy controls, and the extent of the damage was higher in males than female patients. The smoking status had a profound effect on the extent of DNA and chromosomal damage in NSCLC patients. The degree of genetic damage correlated with the stage of the disease. However, the histological status had no effect on the extent of DNA and chromosomal damage among NSCLC patients. Conclusions We here report, for the first time, that the NSCLC patients selected form the Andhra Pradesh population had increased DNA damage and higher mean micronucleus frequencies in peripheral lymphocytes, indicating a strong background level of genetic instability.

Evaluation of oxidative stress and DNA damage in traffic policemen exposed to vehicle exhaust

Abstract Objective: We aimed to study the genotoxic effects in traffic police who are occupationally exposed due to higher free radical generation. Methods: Ambient and breathing zone air samples were analyzed blood samples were collected for analysis of antioxidant enzymes Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx) and free radicals – nitric oxide (NO) and malondialdehyde (MDA) levels using a spectrophotometer. DNA damage was measured with the comet assay. Results: Higher levels of benzene (BZ), toluene (TOL), carbon monoxide (CO), benzo([a])pyrene (BaP) and sulfur dioxide (SO2) was observed in traffic police. Elevated levels of NO, MDA and comet tail length and lower SOD and GPx levels observed in traffic police. Conclusion: The studied biomarkers, related to oxidative stress and DNA damage positively correlated in traffic police exposed to environmental air pollutants.

Identification of Small Molecule as a High Affinity β2 Agonist Promiscuously Targeting Wild and Mutated (Thr164Ile) β 2 Adrenergic Receptor in the Treatment of Bronchial Asthma.

BACKGROUND A subset of asthmatics shows refractoriness to Salbutamol owing to ADRB2 gene C.T polymorphism (rs 1800888) that substitutes Thr to Ile at the position 164 in the β2 adrenergic receptor leading to sub-optimal binding of Salbutamol. The present study aims to associate the Salbutamol (200 mcg) refractoriness with the polymorphism and select the best existing agonist with optimal binding affinity against wild and mutated receptor and further identify high affinity compound, irrespectively targeting wild and mutated receptor through virtual screening methods. METHODS Responders to Salbutamol were categorized, if percentage reversibility was greater than or equal to 12% in them, while those showing reversibility less than 12% were non-responders. The genotyping for polymorphism was performed by ARMS PCR method. Established agonists with consistent binding affinity against wild and mutated receptors formed query compound to identify high affinity molecule from Phase database through 7 point pharmacophore based screening. RESULTS Polymorphism was significantly associated with non-responders (p= < 0.05) demonstrating it as a major factor of Salbutamol refractoriness. Results from Glide Docking showed that Fenoterol had highest affinity for mutated receptor and stood as second best (after Salbutamol) high affinity agonist for wild receptor among the established β2 agonists. Therefore Fenoterol formed a query molecule (7 point pharmacophore) in identification of high affinity compound for virtual screening process. CONCLUSION Compound CACPD2011a-0001278239 identified through virtual screening against 4 million compounds in phase database was shown to irrespectively target both wild and mutated β2 adrenergic receptor with high and consistent affinity which was par greater than established β2agonists.

Analysis of plasma myeloperoxidase levels and functional gene –463G>A and –129G>A polymorphisms with early onset of coronary artery disease in South Indian population

Introduction. The present investigation is pursued to study the possible association of –463G>A and –129G>A polymorphism in MPO gene and assessment of plasma MPO levels with the risk of developing coronary artery disease. Material and methods. A total of 200 angiographically documented CAD patients and 200 age, gender ethnicity matched healthy controls were recruited for the study. Plasma MPO levels were assessed using enzyme-linked immunosorbent assay (ELISA) kit and genotypes were determined by PCR-RFLP technique. Results. The MPO levels were found to be significantly increased in CAD patients when compared with controls (p A gene polymorphism on MPO levels. A significant association of –463G>A polymorphism was observed with coronary artery disease. The frequency of recessive genotype “AA” at –463 promoter site was considerably lesser in patients (4%) relative to controls (11%) (odds ratio [OR] = 0.3371, 95% confidence interval [CI] 0.1463–0.7766, p = 0.012). However we did not find significant association of –129G>A polymorphism with CAD. Additionally, haplotype analysis revealed that single nucleotide polymorphisms (SNP) 1 of AA genotype and SNP 2 of GG genotype showed significant protective effect with disease (OR = 0.64; 95% CI [0.42–0.96], p = 0.032). Conclusion. The results revealed that –463G>A polymorphism in the MPO gene lowers the CAD related condition in patients by down regulating serum MPO concentration, which is known to aggravate the atherosclerotic events observed in CAD.

Association of Beta 2 adrenergic receptor (Thr164Ile) polymorphism with Salbutamol refractoriness in severe asthmatics from Indian population

BACKGROUND Thr164Ile polymorphism in the ADRB2 gene encoding β2 adrenergic receptor (β2AR) has its functional consequence in declining ligand-receptor interactions and depressed coupling of β2AR to adenylcyclase. In the present study, we sought to evaluate the possible association of Thr164Ile polymorphism with asthma susceptibility, pharmacogenetic response to Salbutamol and varying degrees of severity. METHODS Three hundred and ninety eight clinically diagnosed patients and four hundred and fifty six healthy controls were enrolled in the study. Patients were classified into severity classes according to the Global Initiative for Asthma (GINA) guidelines. To assess bronchodilator response, spirometry was performed before and 15min after Salbutamol (200μg) delivery. Responders to Salbutamol were categorized if percentage reversibility was greater than or equal to 12% in them, while those showing reversibility <12% were classified as non-responders. Further, responding phenotypes were stratified into severity groups. Genotyping was carried out by ARMS-PCR technique. Statistical methods were applied to test the significance of the results. RESULTS In the present study, polymorphism was not associated with disease susceptibility however; there was significant association with non-responding asthmatics. In case of severity subsets, the polymorphism was not associated with milder subtypes; although, notable association was observed with moderate and severe asthma subtypes. In addition, the polymorphism was significantly associated with non-responding patients with severe asthma. CONCLUSIONS In south Indian population, the ADRB2 Thr164Ile polymorphism may not form susceptible variant to develop asthma, however, it can form a predictive maker for bronchodilator (Salbutamol) response in severe asthmatics.

Association of Transforming Growth Factor-Beta 1 Promoter Variant -509 C/T with Bronchial Asthma in South Indian Population

Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine that plays a pivotal role in airway remodeling observed in the asthmatic airways. C to T base substitution at -509 promoter position in the TGF-β1 gene leads to its increased expression which contributes to airway remodeling in bronchial asthma. We sought to evaluate the association of TGF-β1 -509 C/T promoter variant with clinical asthma and varying degrees of disease severity. Three hundred and eighty-two clinically diagnosed asthma patients and 410 healthy controls were enrolled for the study. Patients were classified into severity classes according to the Global Initiative for Asthma (GINA) guidelines. TGF-β1 -509 C/T genotyping was carried out by amplification refractory mutation system polymerase chain reaction (ARMS-PCR) technique. In the present study, we found significantly higher frequency of TT genotype in asthma patients compared to controls (for TT vs. CC, p = 0.020). In addition, a significant difference was observed in the frequency of C and T allele in patients and controls (for T vs. C, p = 0.029). The heterozygous “CT” genotype was higher in moderate and severe asthmatics compared to mild subset of patients (for mild vs. moderate, p = 0.037). However, there was no significant distribution and association of variant allele with the severity subsets.

Association of eNOS and ACE gene polymorphisms and plasma nitric oxide with risk of non-small cell lung cancer in South India.

The role of ACE and eNOS gene polymorphisms and their association with various cancers were reported. However, their role in the lung cancer is unclear.