Dual functionality of pyrimidine and flavone in targeting genomic variants of EGFR and ER receptors to influence the differential survival rates in breast cancer patients.

Breast cancer ranks as one of the most prevalent forms of cancer and stands as the primary global cause of mortality among women. Overexpression of EGFR and ER receptors or their genomic alterations leads to malignant transformation, disease aggression and is linked to poor patient survival outcomes. The clinical breast cancer patient's genomic expression, survival analysis, and computational drug-targeting approaches were used to identify best-hit phytochemicals for therapeutic purposes. Breast cancer patients have genomic alterations in EGFR (4%, n = 5699) and ER (9%, n = 8461), with the highest proportion being missense mutations. No statistically significant difference was observed in the patient survival rates between the altered and unaltered ER groups, unlike EGFR, with the lowest survival rates in the altered group. Computational screening of natural compound libraries (7711) against each EGFR (3POZ) and ER (3ERT) receptor shortlists the best-hit 3 compounds with minimum docking score (ΔG = -7.9 to -10.8), MMGBSA (-40.16 to -51.91 kcal/mol), strong intermolecular H-bonding, drug-like properties with least kd, and ki. MD simulation studies display stable RMSD, RMSF, and good residual correlation of best-hit common compounds (PubChem ID: 5281672 and 5280863) targeting both EGFR and ER receptors. In vitro, studies revealed that these common drugs exhibited a high anti-proliferative effect on MCF-7 and MDA-MB-231 breast cancer cells, with effective IC50 values (15-40 µM) and lower free energy, kd, and ki (5281672 > 5280863 > 5330286) much affecting HEK-293 non-cancerous cells, indicating the safety profile. The experimental and computational correlation studies suggest that the highly expressed EGFR and ER receptors in breast cancer patients having poor survival rates can be effectively targeted with best-hit common potent drugs with a multi-target therapeutic approach. Insight Box: The findings of this study provide valuable insights into the genomic/proteomic data, breast cancer patient's survival analysis, and EGFR and ER receptor variants structural analysis. The genetic alterations analysis of EGFR and ER/ESR1 in breast cancer patients reveals the high frequency of mutation types, which affect patient's survival rate and targeted therapies. The common best-hit compounds affect the cell survival patterns with effective IC50, drug-like properties having lower equilibrium and dissociation constants demonstrating the anti-proliferative effects. This work integrates altered receptor structural analysis, molecular interaction-based simulations, and ADMET properties to illuminate the identified best hits phytochemicals potential efficacy targeting both EGFR and ER receptors, demonstrating a multi-target therapeutic approach.

Structure and dynamic simulation-based interactions of benzenoids, pyrroles and organooxygen compounds for effective targeting of GPX4 in ischemic stroke.

The discovery of a novel drug for ischemic stroke is plagued by expensive and unsuccessful outcomes. FDA-approved drugs could be a viable repurposing strategy for stroke therapy. Emerging evidence suggests the regulating role of Glutathione peroxidase (GPX4) in stroke and attracts as a potential target. To overcome limited therapeutic interventions, a drug repurposing in silico investigation of FDA-approved drugs is proposed for the GPX4 receptor in distinctive species (Homo sapiens and Mus musculus). The GPX4 UniProt wild type ids, that is, P36969 (Homo sapiens), P36970 (Rattus norvegicus) and O70325 (Mus musculus) are Swiss modelled, and resultant templates are 2OBI and 6HN3 for Homo sapiens, and 5L71 for Mus musculus with a sequence identity of ∼88%. Enrichment analysis reveals high sensitivity and ranked actives with ROC and AUC values of 0.59 and 0.61, respectively. Virtual screening at extra precision resulted hit Acarbosum, is similar between 2OBI and 6HN3, demonstrating a multiple-target specificity and Iopromide, targeting 2OBI. MD simulation at 100 ns following trajectory analysis provides RMSD (∼1.2-1.8Å), RMSF (∼1.6-2.7Å), Rgyr (∼15-15.6Å) depicting stabilisation of receptor-ligand complexes. Furthermore, average B-factor value of 2OBI, 6HN3 and 5L71 is 25Å, 24Å and 60Å with a defined resolution of 1.55Å, 1.01Å and 1.80Å, respectively, depicting the thermodynamic stability of the protein structures. The dynamic cross-correlation and principal component analysis of residual fluctuations reveal more positive correlation, high atomic displacements and greater residual clustering of residues from atomic coordinates. Therefore, Acarbosum, an FDA-approved drug, could act as a potential repurposing drug with a multi-target approach translating from preclinical to clinical stages.Communicated by Ramaswamy H. Sarma.

Terpenoid analogues as putative therapeutic agents towards glutathione peroxidase (GPX4) in neurodegenerative disorders: a dynamic computational approach.

Carvacrol, a monoterpenoid phenolic phytochemical, a potent antioxidant, and neuroprotective agent is an emerging neuroprotective agent for neurodegenerative diseases (NDDs). Considering scarce information on carvacrol analogues, we hypothesized an in silico investigation emphasizing their preferential binding towards glutathione peroxidase (GPX4) as a target across different species for evaluating through preclinical to clinical studies (2OBI and 6HN3 for Homo sapiens; 5L71 for Mus musculus). Enrichment analysis suggests that ROC (0.59) and AUC (0.61) values have higher sensitivity and significant number of ranked actives. Extra Precision (XP) of 59 compounds was conducted, followed by molecular dynamics and trajectory analysis. Top three hits were chosen for each target i.e., 101203408, 101419546, 59294 (2OBI); 101419546, 100938426, and 28092 (6HN3); and 12059, 52434, 335 (5L71) implying high docking score. 101419546 is common among 2OBI and 6HN3 targets, indicating a multi-target approach. Trajectory analysis of hits provides a permissible range of RMSD, RMSF, Rgyr (∼1.3-2 Å, ∼0.84-1.09 Å, ∼15.05-15.29 Å). Overlapped dynamically simulated 3D-structures of Apo and complexes display significant conformational changes in RMSD of the complexes (∼1.40-2.0 Å) in contrast to Apo (∼1.3-1.8 Å), suggesting structural stability and compactness of the complexes within 45-90 ns. DCCM and PCA analysis shows positive correlation and residual clustering among residues of complexes. The establishment of firm H-bonding, favorable aromaticity and ADMET profile makes them promising drugs across various GPX4 targets among the species. Studies considering the targets across different species aids in anticipating and discovering a common compound for future NDDs therapeutics from bench to bedside.Communicated by Ramaswamy H. Sarma.

Secretory Phospholipase A2 (sPLA2) Isozymes as Potential Targets in Tobacco Condensate- induced Colon Damage.

AIMS: To find out the role of secretory phospholipase A2 (sPLA2) isozymes as potential targets in tobacco condensate-induced colon damage. BACKGROUND: The effects of cigarette smoke condensate (CSC) and the molecular mechanisms involved in the regulation of phospholipase A2 (PLA2) and its isozymes in colon cells, which are still unclear and emerging, are studied. OBJECTIVES: The study aimed to check the effect of CSC on cell viability and reactive oxygen species (ROS) and superoxide. Also, the effect of CSC on gene expression of different secretory phospholipase A2 (sPLA2) was evaluated. Moreover, the impact of inhibition of sPLA2 on various cell properties i.e. cell viability, cell proliferation, membrane damage and free radicals' generation is also studied. METHODS: CSC-induced changes were evaluated in cell viability by MTT assay, followed by the evaluation of membrane modulation by flow cytometry, free radical generation by fluorescent dyes, PLA2 isoforms gene expression patterns and their suppression by small interfering RNA (siRNA) studied in HCT-15 male and HT-29 female colon cells. RESULTS: Our results demonstrate that HCT-15 and HT-29 cells treated with CSC significantly reduced the cell viability by 50% within 48 h and significantly enhanced the total reactive oxygen species (ROS) by 2 to 10-fold, and mitochondrial ROS (mtROS) and superoxide radicals (SOR) by 2-fold each. Treatment with CSC significantly unregulated secretory phospholipase A2 (sPLA2) IID group and down-regulated IB and cytosolic phospholipase (cPLA2) IVA groups in HCT-15 cells without affecting them in HT-29 cells. Silencing the sPLA2 IID group results in an increase in cell viability and a decrease in ROS. Silencing the PLA2 IVA gene in the HCT-15 cells showed a reduced expression which had no impact on the CSC-induced cell proliferation, membrane damage and free radicals (ROS, mtROS, and SOR) generation. CONCLUSION: Therefore, identifying cell-specific sPLA2 isozymes seems to play a key role in controlling the ROSinduced damage by CSC and helps develop specific therapeutic strategies.

Identification of homologous human miRNAs as antivirals towards COVID-19 genome.

The COVID-19 fatality rate is ~57% worldwide. The investigation of possible antiviral therapy using host microRNA (miRNA) to inhibit viral replication and transmission is the need of the hour. Computational techniques were used to predict the hairpin precursor miRNA (pre-miRNAs) of COVID-19 genome with high homology towards human (host) miRNA. Top 21 host miRNAs with >80% homology towards 18 viral pre miRNAs were identified. The Gibbs free energy (ΔG) between host miRNAs and viral pre-miRNAs hybridization resulted in the best 5 host miRNAs having the highest base-pair complementarity. miR-4476 had the strongest binding with viral pre-miRNA (ΔG = -21.8 kcal/mol) due to maximum base pairing in the seed sequence. Pre-miR-651 secondary structure was most stable due to the (1) least minimum free energy (ΔG = -24.4 kcal/mol), energy frequency, and noncanonical base pairing and (2) maximum number of stem base pairing and small loop size. Host miRNAs-viral mRNAs interaction can effectively inhibit viral transmission and replication. Furthermore, miRNAs gene network and gene-ontology studies indicate top 5 host miRNAs interaction with host genes involved in transmembrane-receptor signaling, cell migration, RNA splicing, nervous system formation, and tumor necrosis factor-mediated signaling in respiratory diseases. This study identifies host miRNA/virus pre-miRNAs strong interaction, structural stability, and their gene-network analysis provides strong evidence of host miRNAs as antiviral COVID-19 agents.

Cytosolic phospholipase A2 (cPLA2) IVA as a potential signature molecule in cigarette smoke condensate induced pathologies in alveolar epithelial lineages.

BACKGROUND: Smoking is one of the leading causes of millions of deaths worldwide. During cigarette smoking, most affected and highly exposed cells are the alveolar epithelium and generated oxidative stress in these cells leads to death and damage. Several studies suggested that oxidative stress causes membrane remodeling via Phospholipase A2s but in the case of cigarette smokers, mechanistically study is not yet fully defined. In view of present perspective, we evaluated the involvement of cytosolic phospholipase A2 (cPLA2) IVA as therapeutic target in cigarette smoke induced pathologies in transformed type I and type II alveolar epithelial cells. METHODS: Transformed type I (WI26) and type II (A549) alveolar epithelial cells were used for the present study. Cigarette smoke condensate (CSC) was prepared from most commonly used cigarette (Gold Flake with filter) by the Indian population. CSC-induced molecular changes were evaluated through cell viability using MTT assay, reactive oxygen species (ROS) measurement using 2,7 dichlorodihydrofluorescin diacetate (DCFH-DA), cell membrane integrity using fluorescein diacetate (FDA) and ethidium bromide (EtBr) staining, super oxide dismutase (SOD) levels, cPLA2 activity and molecular involvement of specific cPLA2s at selected 24 h time period. RESULTS: CSC-induced response on both type of epithelial cells shown significantly reduction in cell viability, declined membrane integrity, with differential escalation of ROS levels in the range of 1.5-15 folds and pointedly increased cPLA2 activity (p < 0.05). Likewise, we observed distinction antioxidant potential in these two types of lineages as type I cells had considerably higher SOD levels when compared to type II cells (p < 0.05). Further molecular expression of all cPLA2s increased significantly in a dose dependent manner, specifically cytosolic phospholipase A2 IVA with maximum manifestation of 3.8 folds. Interestingly, CSC-induced ROS levels and cPLA2s expression were relatively higher in A549 cells as compared to WI26 cells. CONCLUSIONS: The present study indicates that among all cPLA2s, specific cPLA2 IVA are the main enzymes involved in cigarette smoke induced anomalies in type I and type II lung epithelial cells and targeting them holds tremendous possibilities in cigarette smoke induced lung pathologies.

Comparison of encapsulated versus nonencapsulated (14) C-urea breath test for the detection of Helicobacter pylori infection: a scintigraphy study.

BACKGROUND AND AIMS: (14) C-urea breath test ((14) C-UBT) is considered as "gold standard" for detection of active gastric H. pylori infection. However, till date no comparative study using encapsulated and non-encapsulated (14) C-UBT protocols has been conducted in same subjects in identical conditions. We monitored gastric fate of capsule containing (14) C-urea with real time display and compared sensitivities of these protocols at different time points of breath collection. METHODS: Non-encapsulated (14) C-UBT was performed using 74 kBq of (14) C-urea in 100 dyspeptic patients by collecting breath samples at 10, 15 and 20 minutes. Thereafter, within 2 days a gelatin capsule containing (14) C-urea along with 6.0 MBq of (99m) Tc-diethylene triamine penta-acetic acid was administered to each patient for real time display of capsule movement and its fate in gastrointestinal tract by gamma camera. Simultaneously, breath samples were collected for (14) CO2 measurement during image acquisition. RESULTS: Employing non-encapsulated (14) C-UBT, 74 out of 100 dyspeptic patients were found to be H. pylori positive. Discordant (14) C-UBT results were obtained in 4/74 (5.4%) cases using these two protocols. By employing encapsulated and nonencapsulated (14) C-UBT protocols, sensitivities of (14) C-UBT were found to be 90.5 versus 98.6% at 10 and 91.8 versus 97.2% at 15 minutes respectively; while these were 94.6 versus 100, 90.7 versus 98.6 and 83.7 versus 93.2% considering any one, two or all three positive values respectively. CONCLUSIONS: Incomplete/non-resolution of (14) C-urea capsule in stomach during the phase of breath collections appears to decrease sensitivity of encapsulated (14) C-UBT as compared to nonencapsulated protocol for detection of H. pylori infection.

Comparative evaluation of different doses of PPAR-γ agonist alone and in combination with sulfasalazine in experimentally induced inflammatory bowel disease in rats.

BACKGROUND: Inflammatory bowel disease (IBD) is an idiopathic, chronic inflammatory condition, which affects the gastrointestinal tract and has no curative treatment. The present study aimed to investigate the effect of different doses of pioglitazone alone and in combination with sulfasalazine in TNBS (trinitrobenzenesulfonic acid)-induced inflammatory bowel disease (IBD) in rats. METHODS: A total of 36 animals were included in the study. Animals were divided into five groups (n = 6): group I--vehicle (ethanol), group II--TNBS + ethanol, group IIIA--TNBS + pioglitazone (15 mg/kg), group IIIB--TNBS + pioglitazone (30 mg/kg), group IV--TNBS + sulfasalazine (360 mg/kg), group V--TNBS + sulfasalazine (360 mg/kg) + pioglitazone (least effective dose found in group III). Group III was divided into two subgroups, namely IIIA and IIIB, on the basis of different doses of pioglitazone used. After completion of two weeks of treatment, rats were sacrificed under ether anesthesia by cervical dislocation for assessment of intestinal inflammation, histological analysis, myeloperoxidase assay, malondialdehyde assay and TNF-α estimation. RESULTS: All the drug-treated groups showed both gross morphological and microscopic score either 1 or 2. None of them showed score of > 2 on both gross and microscopic morphological examination. Both MDA levels and MPO activity were significantly reduced in the drug-treated groups, with maximum reduction seen in the combination group. TNF-α was reduced in pioglitazone group. It was highly reduced in sulfasalazine group (group V) as compared to TNBS group thereby indicating that pioglitazone is protective in TNBS-induced inflammatory bowel disease. CONCLUSION: The present study showed reduction in lipid peroxidation, malondialdehyde levels and TNF-α levels in pioglitazone-treated group and hence, there was significant improvement in gross and microscopic features, too. However, combination of pioglitazone and sulfasalazine has shown greater efficacy.

Superiority of non-capsulated 14C-urea breath test over capsule based method for detection of Helicobacter pylori infection - a preliminary report.

BACKGROUND: 14C-urea breath test (14C-UBT) is employed as a 'gold standard' technique for the detection of active gastric Helicobacter pylori infection and is recommended as the best option for "test-and-treat" strategy in primary health care centers. AIM: To compare the performance of capsulated and non-capsulated 14C-UBT protocols for the detection of H. pylori infection in patients. METHODS: Fifty eight H. pylori infected patients underwent routine upper GI endoscopy and biopsies were processed for rapid urease test (RUT) and histopathology examination. Capsulated 14C-UBT was done in a novel way by using 74 kBq of 14C-urea along with 6.0 MBq of 99mTc-diethylene triamine penta-acetic acid (99mTc-DTPA) to simultaneously monitor the movement and the fate of ingested capsule after delineating the stomach contour by using 20.0 MBq of 99mTechnetium pertechnetate (99mTcO4-) under dual head gamma camera. Non-capsulated 14C-UBT was performed within 2 days of the previous test and the results of these protocols were compared. RESULTS: In 3 out of 58 H. pylori positive cases (5.17%), 14C-UBT results were found to be negative by using the capsulated method. Interestingly, on monitoring the real time images of the capsule in these cases it was found that misdiagnosis of H. pylori infection occurred mainly due to either rapid transit of the 14C-urea containing capsule from the upper gastric tract or its incomplete resolution in the stomach during the phase of breath collection. CONCLUSION: Use of non-capsulated '4C-UBT protocol appears to be a superior option than the conventional capsule based technique for the detection of H. pylori infection.

14C-urea breath test is safe for pediatric patients.

Helicobacter pylori (H. pylori) infection, quite prevalent in the developing countries, is considered to be one of the causative factors for various gastric pathologies and other nongastric diseases. It is believed that H. pylori infection is almost always acquired in early childhood and persists throughout life unless specific treatment is given. The (13/14)C-urea breath test (UBT) is now considered to be a 'gold standard' technique for the detection of H. pylori infection. However, because of the lack of facilities and high cost, the preferred nonradioactive ¹³C-UBT cannot be performed on pediatric patients in developing countries, whereas the radioactive ¹4C-UBT is not used on children because of the fear of radiation exposure. When using 37 kBq (1 µCi) of ¹4C-urea for the ¹4C-UBT, the patient is not exposed to more radiation than is acquired from the natural environment in one day, as almost all the ingested radioactivity is excreted from the body (urine and breath) within 72-120 h. This article reviews the importance of the ¹4C-UBT for the detection of H. pylori and justifies the radiation safety aspects of its use in children without any fear of 'radiation phobia' where the facility for ¹³C-UBT is lacking.

Involvement of various molecular events in cellular injury induced by smokeless tobacco.

Smokeless tobacco (ST) consumption is implicated in the pathogenesis of oral diseases, including cancer. However, its pathological effect in other organs is not well understood. In the present study, the effect of aqueous extract of smokeless tobacco (AEST) prepared from "gutkha" (a form of ST) on the xenobiotic drug-metabolizing enzymes, histopathological changes, and damage to the genetic material in lung, liver, and kidney of rats was evaluated. Animals were orally administered AEST at a low dose (L-AEST, 96 mg/kg body wt/day) for 2 (L-AEST(2)) and 28 weeks (L-AEST(28)) and at a high dose (H-AEST, 960 mg/kg body wt/day) for 2 weeks (H-AEST(2)). Real-time PCR and immunohistological studies showed that administration of L-AEST(2) did not induce the expression of phase I cytochrome P450s (CYP1A1, 1A2, and 2E1) and phase II mu-glutathione-s-transferase (GST-mu) drug-metabolizing enzymes in lung, liver, and kidney. Although H-AEST(2) administration significantly induced both gene and protein expression of CYP1A1, 1A2, and 2E1 in all of the above organs, it mildly expressed the phase II detoxifying enzyme, GST-mu, in type I and type II epithelial cells of lung and in proximal tubular cells of kidney. L-AEST(28) enhanced the gene and protein expression of CYP1A1, 1A2, and 2E1 in lung, liver, and kidney in a differential manner and induced the expression of GST-mu in lung and kidney. L-AEST(28) induced the micronuclei formation in the peripheral blood mononuclear cells, TNF-alpha in plasma, and myeloperoxidase activity in the organs. L-AEST(28) significantly enhanced Bax, p53, and NF-kappaB and decreased Bcl-2 gene expressions differentially in an organ-specific manner. The differential changes in these organs due to AEST might be due to their different physiological functions and variable sensitivities toward the metabolites of AEST, which create a microenvironment favorable for AEST-induced pathogenesis. This study broadens the insight into the different molecular mechanisms in various organs, which appear to be deregulated due to AEST. Understanding these processes may help in clinical treatment planning strategies for tobacco-related diseases.

Allopregnanolone, the active metabolite of progesterone protects against neuronal damage in picrotoxin-induced seizure model in mice.

Progesterone exerts anti-seizure effect against several chemoconvulsants. However, there is no published report on the interaction between progesterone and picrotoxin (PTX). The present study evaluated the effects of progesterone and its active metabolite, allopregnanolone against PTX-induced seizures, brain lipid peroxidation and DNA fragmentation in male mice. Finasteride, a 5alpha-reductase inhibitor and indomethacin, an inhibitor of 3infinity-hydroxysteroid dehydrogenase were assessed against progesterone's effects on PTX-induced seizures, brain lipid peroxidation and DNA fragmentation. PTX produced clonic-tonic seizures in mice with CD50 and CD97 of 2.4 and 4.0mg/kg, i.p. respectively. Progesterone significantly countered PTX-induced seizures, with ED50 of 78.30mg/kg and ED97 of 200mg/kg. Progesterone antagonized PTX-induced DNA fragmentation. Finasteride (200mg/kg) and indomethacin (1mg/kg) reversed the anti-seizure and anti-DNA fragmentation effects of progesterone. Allopregnanolone, also protected against PTX-induced seizures and DNA fragmentation. There was no significant change in the brain lipid peroxidation parameters in any of the treatment groups. It may be concluded that progesterone protects against PTX-induced seizures and DNA fragmentation through its active metabolites allopregnanolone and 5alpha-pregnan-3,20-dione. However, it appears from the present study that, the neuroprotection with progesterone is primarily on account of allopregnalone. The therapeutic potential of allopregnanolone deserves to be evaluated clinically.

Intestinal inflammation and seizure susceptibility: understanding the role of tumour necrosis factor-alpha in a rat model.

OBJECTIVES: The aim of the study was to evaluate the correlation between colitis and susceptibility to seizures. METHODS: Colitis was induced in Wistar rats by a single intracolonic administration of trinitrobenzene sulfonic acid (TNBS; 20 mg in 35% ethanol). The control group were given intracolonic vehicle. One group of rats with colitis were treated with thalidomide (150 mg/kg p.o.) daily for 14 days. The other colitis group received vehicle only. On day 15, seizure susceptibility was tested by administration of pentylenetetrazole (40 mg/kg i.p.). Colonic tissue was collected for estimation of morphological score, and malondialdehyde, superoxide dismutase, catalase and glutathione peroxidase. Tumour necrosis factor (TNF)-alpha levels were measured in serum and brain samples. KEY FINDINGS: The colitis group showed a significant increase in seizure score and reduction in onset time compared with the control group. Thalidomide was protective against seizures, resulting in decreased seizure score and significantly delaying the onset of seizures. Thalidomide also provided significant protection against TNBS-induced colonic damage in terms of morphological and histological score and levels of lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase in colonic tissue. The level of TNF-alpha in serum was also reduced significantly whereas brain TNF-alpha level was reduced but not significantly. CONCLUSIONS: TNBS-induced colitis increased seizure susceptibility to a subconvulsive dose of pentylenetetrazole; the immunomodulator thalidomide was protective.

Kinetics of 14carbon dioxide excretion from 14C-urea by oral commensal flora.

BACKGROUND AND AIM: Previous studies have shown that while performing the (14)C-urea breath test ((14)C-UBT) for the detection of Helicobacter pylori (H. pylori), there is possibility of false-positive results due to the other urease producing bacteria present in oropharynx, if breath samples are obtained within 30 min after administration of non-capsulated (14)C-urea. Therefore, we have exclusively evaluated the kinetics of (14)carbon dioxide ((14)CO(2)) excretion by oral commensal flora to theoretically propose optimum breath collection timings for (14)C-UBT. METHODS: Multiple breath samples up to 15 min were collected in 0.25 mmol benzethonium hydroxide from 25 healthy volunteers after they withheld 37 kBq (1 muCi) of (14)C-urea in their mouths for 15 s and then expectorated the tracer. The test was repeated on the same subjects without and with mouth cleansing protocols. Breath (14)CO(2) content was measured by the Liquid Scintillation Counter (1409; Wallac, Turku, Finland) and results were expressed as (14)CO(2) excretion per mmol breath CO(2) (% administered dose). RESULTS: Peak breath radioactivity at 1 min in the former protocol was 3.53 times higher than the latter which declined subsequently with a half time of 1 min and 2.5 min, and reached baseline levels by 15 and 10 min, respectively. The peak radioactivity (100%) at 1 min declined by 94% and 97.8% in the former and later protocols, respectively, at 15 min. Although magnitude of the peak varied in different subjects, the shape of curve remained almost similar in all cases. CONCLUSIONS: Without mouth cleansing, oral micro flora excreted more (14)CO(2) up to 15 min after administration of non-capsulated (14)C-urea. Therefore, it is proposed that two breath samples may be obtained either at 15 and 20 min without or at 10 and 15 min with mouth cleansing protocols for reliable analysis of (14)C-UBT data for H. pylori detection.

Changes in gastric environment with test meals affect the performance of 14C-urea breath test.

BACKGROUND: (14)C-urea breath test (UBT) is considered to be an accurate diagnostic test for the detection of active Helicobacter pylori infection. Various test meals are used in (14)C-UBT to slow down gastric emptying, and to enhance the gastric distribution, in order to increase the time and area of contact between microorganisms and the tracer substrate. The aim of the present paper was to evaluate the effect of gastric environment on the performance of (14)C-UBT using an alkaline and an acidic liquid test meal having gastric emptying retardant effect. METHODS: The comparison of (14)C-UBT was done with liquid test meals (200 mL water) comprising (i) plain drinking water (PDW); (ii) 1.3 g or 3.0 g citric acid (CA); and (iii) 3.0 g trisodium citrate (TSC). Eighteen patients (37 +/- 12 years, range 18-57 years) with complaints of dyspepsia participated in the study. The status of H. pylori was confirmed by histology and rapid urease test. A total of 93 kBq of (14)C-urea (0.5 mL) in a gelatin capsule was orally administered along with liquid test meals to the overnight fasting subjects. Breath samples were collected and radioactivity measured. Results were expressed as (14)CO(2)/mmol exhaled CO(2) as percentage of administered radioactive urea. RESULTS: Higher acidic gastric environment (pH approx. 2.0) with CA was found to increase the exhaled (14)CO(2) level in a dose-dependent manner as compared to PDW and TSC meal (P < 0.05) at all time points. With TSC test meal, the expired (14)CO(2) level decreased in the lower acidic gastric environment (pH approx. 5.3). The peaks of exhaled (14)CO(2) with TSC test meal were observed at the same time points as that with PDW and CA test meals. The (14)C-UBT with TSC was found to be positive in 77% of patients (10/13). CONCLUSION: Better interaction between the microbial urease and (14)C-urea, caused by a test meal that retards gastric emptying and that changes gastric pH, plays an important role in hydrolysis of the administered (14)C-urea by H. pylori urease.

Resveratrol inhibits N-nitrosodiethylamine-induced ornithine decarboxylase and cyclooxygenase in mice.

Increased levels or overexpression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis pathway, is characteristic of tumor cells. Similarly, prostaglandins (PGs) appear to be important in the pathogenesis of cancer because these affect mitogenesis, cellular adhesion, immune surveillance and apoptosis. Cancers form much more PGs than the original tissue from which they have arisen. This study has revealed that pretreatment of mice with resveratrol at a dose of 2.5 mg/kg body weight for two weeks blocked the N-nitrosodiethylamine(NDEA)-induced cytosolic ODC levels in the liver and lungs. The blockage was pronounced in hepatic tissue compared to pulmonary tissue. Resveratrol feeding caused a significant reduction in microsomal cyclooxygenase (COX) activities in the liver and lungs, while the dosage of NDEA (200 mg/kg body weight) induced COX activity 24 h after its administration. In any case, resveratrol pretreatment turned out to effectively block the induction of COX activity in the lungs by NDEA.

Gallic acid, an antioxidant, exhibits antiapoptotic potential in normal human lymphocytes: A Bcl-2 independent mechanism.

Oxidative stress, produced as a consequence of normal metabolism or induced by extraneous stimuli, has been proved to be a mediator of cell death. The inherent antioxidant defense system and exogenous antioxidants can help the body to combat this oxidative stress-induced cell death. In this study, we explored the antiapoptotic potential of gallic acid, a dietary phenolic having antioxidative and anticarcinogenic properties, in normal human peripheral blood lymphocytes (PBLs). Incubation of PBLs with 100 microM H2O2 for 1.5-2.0 h induced phosphatidyl serine externalisation, lipid peroxidation and high molecular weight DNA fragmentation. Pretreatment of lymphocytes with gallic acid for 18 h could effectively inhibit lipid peroxidation and apoptosis induced by oxidative stress. Treatment of PBLs with gallic acid failed to induce any change in the expression of Bcl-2, an antiapoptotic protein. It seems that the protection provided by gallic acid was due to its direct action in the scavenging of free radicals as it was found to be a stronger antiradical than trolox, a water- soluble analogue of vitamin E.

Nimesulide affects antioxidant status during acute lung inflammation in rats.

Antiradical activity of nimesulide, a commonly used COX-2 specific inhibitor, was estimated in vitro by 1, 1 diphenyl-2-picrylhydrazyl (DPPH) assay, nitroblue tetrazolium reduction assay and lipid peroxidation assay, respectively. The biochemistry of antioxidant functions of nimesulide was also investigated under control and inflammatory conditions, caused by intratracheal instillation of lipopolysaccharide (LPS). Pro-inflammatory conditions generally end up in oxidative insults, which have been suggested to be the cause of multiple organ failure in inflammation. A primary defense, constituted of antioxidant enzymes, against this oxidative damage has evolved in the body. In this study, male Wistar rats were orally administered with nimesulide (9 mg/kg/twice daily for 1 week), followed by intratracheal instillation with 2 microg of LPS and after 18 hr, antioxidant defense system and lipid peroxidation were measured in liver, lungs and kidneys. Nimesulide pretreatment was found to protect the tissue from enhanced levels of lipid peroxidation, and also stimulated the levels of glutathione-S-transferase (GST) in liver and glutathione reductase in kidneys. Surprisingly, nimesulide oral feeding also significantly suppressed superoxide dismutase (SOD) activity in all the three organs. Although, in our study, nimesulide proved to be an inducer of GST (a marker for chemoprevention) and a scavenger of superoxide anions at higher concentrations (> 250 microM), but the relevance of suppression of SOD enzyme activity, which may contribute to the drug's toxic effects cannot be ignored. The work suggests that further long term studies are needed to confirm nimesulide as a safe drug.