Sheela Benedict

Acetylsalicylic acid-induced oxidative stress, cell cycle arrest, apoptosis and mitochondrial dysfunction in human hepatoma HepG2 cells

It is widely accepted that non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, reduce the risk of cancer. The anti-cancer and anti-inflammatory effects of NSAIDs are associated with the inhibition of prostaglandin synthesis and cyclooxygenase-2 activity. Several other mechanisms which contribute to the anti-cancer effect of these drugs in different cancer models both in vivo and in vitro are also presumed to be involved. The precise molecular mechanism, however, is still not clear. We investigated, therefore, the effects of acetylsalicylic acid (ASA, aspirin) on multiple cellular and functional targets, including mitochondrial bioenergetics, using human hepatoma HepG2 cancer cells in culture. Our results demonstrate that ASA induced G0/G1 cell cycle arrest and apoptosis in HepG2 cells. ASA increased the production of reactive oxygen species, reduced the cellular glutathione (GSH) pool and inhibited the activities of the mitochondrial respiratory enzyme complexes, NADH-ubiquinone oxidoreductase (complex I), cytochrome c oxidase (complex IV) and the mitochondrial matrix enzyme, aconitase. Apoptosis was triggered by alteration in mitochondrial permeability transition, inhibition of ATP synthesis, decreased expression of the anti-apoptotic protein Bcl-2, release of cytochrome c and activation of pro-apoptotic caspase-3 and the DNA repairing enzyme, poly (-ADP-ribose) polymerase (PARP). These findings strongly suggest that ASA-induced toxicity in human hepatoma HepG2 cells is mediated by increased metabolic and oxidative stress, accompanied by mitochondrial dysfunction which result in apoptosis.

Predictors and relationships of serum 25 hydroxyvitamin D concentration with bone turnover markers, bone mineral density, and vitamin D receptor genotype in Emirati women

OBJECTIVES To determine factors influencing serum 25 hydroxyvitamin D (25OHD) concentration and relationships between serum 25OHD concentration, bone turnover markers, bone mineral density (BMD), and vitamin D receptor (VDR) genotype in Emirati women. METHODS Serum 25OHD, parathyroid hormone (PTH), osteocalcin (OC), vitamin D binding protein (VDBP), and urinary deoxypyrdinoline (UDPD) concentrations and VDR genotype were determined in Emirati women volunteers who were participating in a study aiming at establishing a reference database for BMD. RESULTS Serum 25OHD concentration in the 259 women volunteers was 25.3 +/- 10.8 nmol/l (mean +/- SD), and all had vitamin D deficiency (25OHD <80 nmol/l). Mean serum 25OHD was highest in April (29.2 +/- 13.0 nmol/l), which marks the end of the short and cooler winter season, and lowest in August (18.2 +/- 5.9 nmol/l). No significant difference in 25OHD concentration was noted among Emirati women wearing different dress styles, but the mean serum 25OHD was significantly lower in comparison with non-Arab Caucasian women volunteers who dressed in a Western style (P < 0.001). Serum 25OHD correlated positively with age (r = 0.2), number of pregnancies (r = 0.16), dietary vitamin D intake (r = 0.15), serum calcium (r = 0.14), phosphorus (r = 0.14), VDBP (r = 0.15), and urinary calcium/creatinine (r = 0.2), and inversely with PTH (r = -0.22), OC (r = -0.13), and UDPD/creatinine (r = -0.15); P < 0.05 for all correlations. Multiple linear regression analysis showed that age, dietary vitamin D intake, multivitamin intake, and cooler season were independent positive predictors of serum 25OHD concentration (R(2) = 0.18). The frequencies of VDR genotypes were 36% GG, 44.1% AG, and 19.9% AA. Allele frequencies were 58% for G allele and 42% for A allele and were in Hardy-Weinberg equilibrium (x(2) = 1.44; P > 0.1). There was no statistically significant influence of VDR genotype on bone turnover or BMD. CONCLUSIONS Vitamin D deficiency is highly prevalent in Emirati women and appears largely attributable to insufficient sunlight exposure. It is associated with increased bone turnover. VDR genotype does not appear to influence bone turnover markers or BMD in Emirati women.

Association of TCF7L2 polymorphism with diabetes mellitus, metabolic syndrome, and markers of beta cell function and insulin resistance in a population-based sample of Emirati subjects

AIMS The prevalence of type 2 diabetes mellitus (DM) among Emirati subjects is one of the highest in the world. This has been attributed to rising prevalence of obesity acting on genetically susceptible individuals. We analyzed the associations between TCF7L2 polymorphism and DM, metabolic syndrome, and markers of beta cell function and insulin resistance in a population-based sample of Emirati subjects. METHODS We genotyped the two TCF7L2 single nucleotide polymorphisms (SNPs) rs12255372 and rs7903146 in 368 adult subjects. Homeostatic model assessment (HOMA) was used to assess beta cell function (HOMA2-%B) and insulin resistance (HOMA2-IR). The SNP genotypes were analyzed against disease stage [normal glucose=0 (n=188), pre-diabetes=1 (n=85), and DM=2 (n=95)] and against clinical and biochemical measures. Age and sex were included as covariates in all association analyses. Additional adjustments were made for body mass index (BMI) and waist circumference in several analyses. RESULTS Diabetes disease stage was marginally significantly associated with the frequency of the T variant at rs12255372 (p=0.057; adjusted p=0.017) but not at rs7903146 (p=0.5; adjusted p=0.2). Comparison between subjects with normal glucose and the combined DM/pre-diabetes showed a significant association with rs12255372 (OR 1.47, CI 1.04-2.08; p=0.03) but not with rs7903146 (OR 1.16, CI 0.81-1.64; p=0.4). We found no association with metabolic syndrome, or with insulin and glucose levels, HOMA2-%B or HOMA2-IR. The age-standardized prevalence rate for metabolic syndrome was 43.9% in men and 42.1% in women. CONCLUSION These data suggest that TCF7L2 variants are associated with increased risk for DM in Emirati subjects. We also demonstrate a high prevalence of the metabolic syndrome in this population.

Biocompatibility of calcined mesoporous silica particles with cellular bioenergetics in murine tissues.

A novel in vitro system was developed to investigate the effects of two forms of calcined mesoporous silica particles (MCM41-cal and SBA15-cal) on cellular respiration of mouse tissues. O(2) consumption by lung, liver, kidney, spleen, and pancreatic tissues was unaffected by exposure to 200 μg/mL MCM41-cal or SBA15-cal for several hours. Normal tissue histology was confirmed by light microscopy. Intracellular accumulation of the particles in the studied tissues was evident by electron microscopy. The results show reasonable in vitro biocompatibility of the mesoporous silicas with murine tissue bioenergetics.

Serum interleukin-5 levels are elevated in mild and moderate persistent asthma irrespective of regular inhaled glucocorticoid therapy

BackgroundInterleukin-5 (IL-5) is thought to play a pivotal role in the pathogenesis of asthma. High levels of circulating IL-5 have been documented in acute asthma. However, serum IL-5 levels in mild to moderate asthmatics and the influence of regular use of inhaled glucocorticoids, is not known.MethodsFifty-six asthmatics and 56 age and sex matched controls were recruited prospectively from an outpatient department. Information on asthma severity and treatment was gathered by a questionnaire. Serum IL-5, total IgE and specific IgE levels were measured in a blinded fashion.ResultsThere were 32 atopic and 24 non-atopic mild-to-moderate asthmatics. The median serum IL-5 levels in atopic asthmatics (9.5 pg/ml) and in non-atopic asthmatics (8.1 pg/ml) were significantly higher than in normal controls (4.4 pg/ml, both p < 0.003). However, median serum IL-5 levels in atopic and non-atopic asthmatics were not significantly different. The median serum IL-5 level was insignificantly higher in fourteen moderate persistent asthmatics (10.6 pg/ml) compared to forty-two mild persistent asthmatics (7.3 pg/ml) (p = 0.13). The median serum IL-5 levels in asthmatics using regular inhaled steroids (7.8 pg/ml) was not significantly different from those not using inhaled steroids (10.2 pg/ml). Furthermore, serum total IgE levels and eosinophil counts were not significantly different in those using versus those not using inhaled glucocorticoids.ConclusionSerum IL-5 levels are elevated in mild and moderate persistent atopic and non-atopic asthmatics. Regular use of inhaled glucocorticoids may not abrogate the systemic Th2 type of inflammatory response in mild-moderate persistent asthma.

Measurement of oxygen consumption by murine tissues in vitro

INTRODUCTION A novel in vitro system was developed to measure O₂ consumption by murine tissues over several hours. METHODS Tissue specimens (7-35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer, saturated with 95% O₂:5% CO₂. The specimens were incubated at 37 °C in the buffer, continuously gassed with O₂:CO₂ (95:5). [O₂] was determined as a function of time from the phosphorescence decay rates (1/τ) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The values of 1/τ were linear with [O₂]: 1/τ=1/τo + kq [O₂]; 1/τo=the decay rate for zero O₂, kq=the rate constant in s⁻¹ μM⁻¹. RESULTS NaCN inhibited O₂ consumption, confirming oxidation occurred in the mitochondrial respiratory chain. The rate of respiration in lung specimens incubated in vitro for 3.9≤t≤12.4 h was 0.24±0.03 μM O₂ min⁻¹ mg⁻¹ (mean±SD, n=28). The corresponding rate for the liver was 0.27±0.13 (n=11, t≤4.7 h), spleen 0.28± 0.07 (n=10, t≤5h), kidney 0.34±0.12 (n=7, t≤5h) and pancreas 0.35±0.09 (n=10, t≤4h). Normal tissue histology at hour 5 was confirmed by light and electron microscopy. There was negligible number of apoptotic cells by caspase 3 staining. DISCUSSION This approach allows accurate assessment of tissue bioenergetics in vitro.

Elevation of plasma transforming growth factor β1 levels in stable nonatopic asthma

BACKGROUND Increased transforming growth factor beta1 (TGF-beta1) levels have been reported in bronchoalveolar lavage fluid and bronchial biopsy specimens from asthmatic patients. However, systemic TGF-beta1 levels have not been reported in asthma. OBJECTIVE To evaluate the levels of plasma TGF-beta1 in asthmatic patients and matched, healthy controls to determine the associations with atopic status, disease severity, and duration. METHODS Asthmatic patients and healthy controls were recruited prospectively from a university hospital outpatient department between January 2001 and May 2002. Plasma TGF-beta1 and serum IgE levels were estimated using established methods. Patients were classified as atopic or nonatopic based on the presence or absence of serum specific IgE directed to common allergens. RESULTS Of the 56 patients recruited for the study, 32 were atopic and 24 nonatopic. The median value of plasma TGF-beta1 was significantly higher in nonatopic asthmatic patients (2.5 ng/mL) compared with controls (1.5 ng/mL, P = .002) and atopic asthmatic patients (1.4 ng/mL, P = .008). The median absolute neutrophil count in the nonatopic asthmatic patients (4.0 x 10(9)/L) was significantly higher compared with atopic asthmatic patients (3.0 x 10(9)/L) and healthy controls (3.5 x 10(9)/L) (P = .01 and P = .04). There was no significant correlation between the duration or severity of asthma and plasma TGF-beta1 levels. The distribution of moderate-persistent asthma cases was similar in atopic and nonatopic groups. CONCLUSION Compared with atopic asthmatic patients and healthy controls, patients with nonatopic asthma have elevated plasma TGF-beta1 levels and leukocytosis. These data suggest that nonatopic asthmatic patients exhibit an altered inflammatory response, perhaps to a respiratory infection.

Responses to Dehydration in the One-Humped Camel and Effects of Blocking the Renin-Angiotensin System

Our objectives were to compare the levels of circulating electrolytes, hormones, and renal function during 20 days of dehydration in camels versus the level in non-dehydrated camels and to record the effect of blocking angiotensin II AT1 receptors with losartan during dehydration. Dehydration induced significant increments in serum sodium, creatinine, urea, a substantial fall in body weight, and a doubling in plasma arginine vasopressin (AVP) levels. Plasma aldosterone, however, was unaltered compared with time-matched controls. Losartan significantly enhanced the effect of dehydration to reduce body weight and increase serum levels of creatinine and urea, whilst also impairing the rise in plasma AVP and reducing aldosterone levels. We conclude that dehydration in the camel induces substantial increments in serum sodium, creatinine, urea and AVP levels; that aldosterone levels are altered little by dehydration; that blockade of angiotensin II type 1 receptors enhances the dehydration-induced fall in body weight and increase in serum creatinine and urea levels whilst reducing aldosterone and attenuating the rise in plasma AVP.

Alterations in atrial natriuretic peptide and its receptors in streptozotocin-induced diabetic rat kidneys

In this study the effect of diabetes mellitus on atrial natriuretic peptide (ANP) receptors in streptozotocin- (STZ-) induced diabetic rat kidneys was studied. Moreover, plasma ANP concentration was evaluated in diabetic and control rats by using radioimmunoassay. In addition, the expression of ANP in the kidneys of control and diabetic rats was evaluated by immunohistochemistry. Body-weight loss and increased glucose levels were used as indices of diabetes mellitus in the STZ-induced rats. There was a significant loss in the body weight of the diabetic rats compared to controls. The efficacy of STZ administration was confirmed by rising blood glucose levels, which were significantly higher in diabetic rats compared to controls. Plasma ANP concentration was significantly greater in the diabetic rats in comparison with controls. Moreover, our immunohistochemical results show that the expression of ANP in diabetic rats was higher than that in age-matched controls. ANP was observed in the cells lining the proximal convoluted tubules in the cortex. The distribution and levels of ANP receptors in the kidneys of diabetic rats and age-matched controls were investigated using quantitative receptor autoradiography. Our results demonstrate significant decrease in ANP receptors in the kidneys of the diabetic rats compared to controls. The significant decrease was found in the juxtaglomerular medulla, inner medulla, and the papillae. The decrease in ANP receptors observed in the diabetic kidneys could have pathological consequences resulting in renal resistance to ANP in diabetes. (Mol Cell Biochem 261: 3–8, 2004)

ANP and BNP Responses to Dehydration in the One-Humped Camel and Effects of Blocking the Renin-Angiotensin System

The objectives of this study were to investigate and compare the responses of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in the circulation of hydrated, dehydrated, and dehydrated losartan - treated camels; and to document the cardiac storage form of B-type natriuretic peptide in the camel heart. Eighteen male camels were used in the study: control or hydrated camels (n = 6), dehydrated camels (n = 6) and dehydrated losartan-treated camels (n = 6) which were dehydrated and received the angiotensin II (Ang II) AT-1 receptor blocker, losartan, at a dose of 5 mg/kg body weight intravenously for 20 days. Control animals were supplied with feed and water ad-libitum while both dehydrated and dehydrated-losartan treated groups were supplied with feed ad-libitum but no water for 20 days. Compared with time-matched controls, dehydrated camels exhibited a significant decrease in plasma levels of both ANP and BNP. Losartan-treated camels also exhibited a significant decline in ANP and BNP levels across 20 days of dehydration but the changes were not different from those seen with dehydration alone. Size exclusion high performance liquid chromatography of extracts of camel heart indicated that proB-type natriuretic peptide is the storage form of the peptide. We conclude first, that dehydration in the camel induces vigorous decrements in circulating levels of ANP and BNP; second, blockade of the renin-angiotensin system has little or no modulatory effect on the ANP and BNP responses to dehydration; third, proB-type natriuretic peptide is the storage form of this hormone in the heart of the one-humped camel.