A metabolomic study of low estimated GFR in non-proteinuric type 2 diabetes mellitus.

AIMS/HYPOTHESIS: We carried out a urinary metabolomic study to gain insight into low estimated GFR (eGFR) in patients with non-proteinuric type 2 diabetes. METHODS: Patients were identified as being non-proteinuric using multiple urinalyses. Cases (n = 44) with low eGFR and controls (n = 46) had eGFR values <60 and ≥60 ml min(-1) 1.73 m(-2), respectively, as calculated using the Modification of Diet in Renal Disease formula. Urine samples were analysed by liquid chromatography/mass spectrometry (LC/MS) and GC/MS. False discovery rates were used to adjust for multiple hypotheses testing, and selection of metabolites that best predicted low eGFR status was achieved using least absolute shrinkage and selection operator logistic regression. RESULTS: Eleven GC/MS metabolites were strongly associated with low eGFR after correction for multiple hypotheses testing (smallest adjusted p value = 2.62 × 10(-14), largest adjusted p value = 3.84 × 10(-2)). In regression analysis, octanol, oxalic acid, phosphoric acid, benzamide, creatinine, 3,5-dimethoxymandelic amide and N-acetylglutamine were selected as the best subset for prediction and allowed excellent classification of low eGFR (AUC = 0.996). In LC/MS, 19 metabolites remained significant after multiple hypotheses testing had been taken into account (smallest adjusted p value = 2.04 × 10(-4), largest adjusted p value = 4.48 × 10(-2)), and several metabolites showed stronger evidence of association relative to the uraemic toxin, indoxyl sulphate (adjusted p value = 3.03 × 10(-2)). The potential effect of confounding on the association between metabolites was excluded. CONCLUSIONS/INTERPRETATION: Our study has yielded substantial new insight into low eGFR and provided a collection of potential urinary biomarkers for its detection.

Plasma carotenoids and risk of acute myocardial infarction in the Singapore Chinese Health Study.

BACKGROUND AND AIM: Modification of low-density lipoprotein due to oxidative stress is essential in the development of coronary atherosclerosis. Data of specific carotenoids except ß-carotene on cardioprotective effects in humans are limited. METHODS AND RESULTS: This study examined the associations between plasma concentrations of specific carotenoids and incidence of acute myocardial infarction. The study included 280 incident cases of acute myocardial infarction and 560 matched controls nested within the Singapore Chinese Health Study, a prospective cohort of 63,257 Chinese men and women aged 45-74 years old enrolled in 1993-1998 in Singapore. Retinol and carotenoids in prediagnostic plasma were quantified using high-performance liquid chromatography. High levels of plasma ß-cryptoxanthin and lutein were associated with decreased risk of acute myocardial infarction after adjustment for multiple risk factors for coronary heart disease. For ß-cryptoxanthin, the odds ratio (95% confidence interval) for the highest (Q5) versus the lowest (Q1) quintile was 0.67 (0.37-1.21) (P for trend=0.03). For lutein, the odds ratios (95% confidence intervals) for the combined Q2-Q3 and the combined Q4-Q5 versus Q1 were 0.71 (0.45-1.12) and 0.58 (0.35-0.94) respectively (P for trend=0.03). There was no statistically significant association between other carotenoids or retinol and risk of acute myocardial infarction. CONCLUSIONS: High plasma levels of ß-cryptoxanthin and lutein were associated with decreased risk of acute myocardial infarction. The findings of this study support a cardioprotective role of these two carotenoids in humans.

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death.

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.

Deformability study of breast cancer cells using microfluidics.

Cell deformability is an important biomarker which can be used to distinguish between healthy and diseased cells. In this study, microfluidics is used to probe the biorheological behaviour of breast cancer cells in an attempt to develop a method to distinguish between non-malignant and malignant cells. A microfabricated fluidic channel design consisting of a straight channel and two reservoirs was used to study the biorheological behaviour of benign breast epithelial cells (MCF-10A) and non-metastatic tumor breast cells (MCF-7). Quantitative parameters such as entry time (time taken for the cell to squeeze into the microchannel) and transit velocity (speed of the cell flowing through the microchannel) were defined and measured from these studies. Our results demonstrated that a simple microfluidic device can be used to distinguish the difference in stiffness between benign and cancerous breast cells. This work lays the foundation for the development of potential microfluidic devices which can subsequently be used in the detection of cancer cells.

AFM indentation study of breast cancer cells.

Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Young's modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 degrees C), and their apparent Young's modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis.

Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1.

Accumulating evidence demonstrates that oxidative damage is one of the underlying mechanisms to the cytotoxicity and carcinogenicity of AFB1. The main objective of this study is to show that AFB1 increases reactive oxygen species (ROS) formation in hepatocytes. The ROS level was detected using a fluorescence probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS. It was found that AFB1 exposure significantly enhanced DCF fluorescence formation in cultured rat hepatocytes. A dose-response of AFB1 was also observed within the range of 10 nM to 1000 nM. Catalase (CAT) was able to completely prevent the increase of DCF fluorescence in AFB1-treated cells in a dose-dependent manner (from 500 to 2000 U/ml). Moreover, the significant inhibitory effects of desferrioxamine (DFO) and dimethyl sulfoxide (DMSO) on DCF fluorescence formation were also observed in both control and AFB1-treated hepatocytes. Therefore, results from the present study provide in vitro evidence indicating the generation of ROS in cultured rat hepatocytes caused by AFB1 exposure. It is postulated that the metabolic process of AFB1 by cytochrome P450 might be the possible source of the elevated ROS level in AFB1-treated hepatocytes. The enhanced level of ROS may be responsible for the oxidative damage caused by AFB1, which may ultimately contribute to the cytotoxic and carcinogenic effects of AFB1.

Caloric restriction prevents oxidative damage induced by the carcinogen clofibrate in mouse liver.

Long-term caloric restriction in rodents is known to decrease levels of oxidative damage, which may contribute to an 'anti-ageing' effect. We show here that a shorter period (10 months) of caloric restriction had only small effects on levels of oxidative DNA and protein damage in the livers of mice, but completely attenuated increased oxidative damage caused by the carcinogen clofibrate. Since clofibrate is thought to exert its actions by increasing oxidative damage, our data suggest that 10 months of caloric restriction can increase the resistance of tissues to agents inducing oxidative stress. This may be an important factor in explaining how caloric restriction decreases cancer incidence.

Superoxide radical-initiated apoptotic signalling pathway in selenite-treated HepG(2) cells: mitochondria serve as the main target.

The exact role of superoxide radicals (O(2)(*)(-)) in apoptosis is still a matter of debate. The main objective of the present study is to evaluate the apoptotic signalling pathway initiated by O(2)(*)(-). The reductive reaction of sodium selenite with glutathione was used as the intracellular O(2)(*)(-)-generating system. When cells were exposed to 5 to 25 microM selenite, a temporal pattern of apoptotic events was observed following the elevation of O(2)(*)(-), in which cytochrome c release and mitochondrial depolarization preceded caspase-3 activation and DNA fragmentation. The simultaneous treatment with N-acetylcysteine and 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl markedly reduced O(2)(*)(-) level and suppressed the mitochondrial changes and the downstream apoptotic events. Moreover, pretreatment with cyclosporin A plus trifluoperazine, two mitochondrial permeability transition (MPT) inhibitors, was capable of attenuating O(2)(*)(-)-mediated cytochrome c release and mitochondrial depolarization, and subsequently inhibiting apoptosis. Thus, the present results provide convincing evidence that O(2)(*)(-) generated from the reductive reaction of selenite with GSH is capable of triggering a mitochondria-dependent apoptotic pathway. Such knowledge may not only help to obtain a better understanding of the apoptotic effect of selenite per se, but of the role of O(2)(*)(-) in initiation and execution of apoptosis.

Mitochondrial damage by the "pro-oxidant" peroxisomal proliferator clofibrate.

Clofibrate is a peroxisome proliferator that can cause hepatic cancer in rodents. It has been suggested that oxidative damage is involved in this hepatocarcinogenesis, although the data are conflicting. We confirmed that clofibrate causes oxidative damage in nuclei from the livers of mice treated with this substance, measured both as protein carbonyls and levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA. In addition, clofibrate also affects mitochondria, causing elevated levels of carbonyls and 8-OHdG, increased state 4 respiration and decreased adenosine triphosphatase (ATPase) activity. No evidence for clofibrate-induced lipid peroxidation in mitochondria was obtained. We propose that mitochondria may be a major target of injury and a source of oxidative stress in clofibrate-treated animals.

Genetic, dietary, and other lifestyle determinants of plasma homocysteine concentrations in middle-aged and older Chinese men and women in Singapore.

BACKGROUND: Epidemiologic studies have identified the plasma homocysteine concentration as a risk factor for atherothrombotic vascular disease. There is little information on the distributions and determinants of homocysteine concentrations in Asian populations. OBJECTIVE: The present study was designed to examine the relations between genetic and lifestyle factors and plasma homocysteine concentrations among Chinese in Singapore. DESIGN: Plasma total homocysteine, folate, vitamin B-12, and vitamin B-6 concentrations and genetic variation at the methylenetetrahydrofolate reductase (MTHFR) locus were measured in 486 Chinese men and women aged 45-74 y in Singapore. Data on dietary and other lifestyle factors were collected in face-to-face interviews. RESULTS: Men had higher plasma concentrations of total homocysteine than women (P = 0.0001). Age was positively associated with plasma homocysteine in both sexes (P for trend = 0.0001). Plasma concentrations of folate, vitamin B-12, and vitamin B-6 were inversely associated with homocysteine concentrations. Among individuals with low plasma folate, those possessing 2 copies of MTHFR mutant alleles had significantly higher homocysteine concentrations than did those with > or = 1 copy of the wild-type allele. Cigarette smoking, daily coffee consumption, and physical inactivity were positively related to plasma homocysteine concentrations in both sexes (P < 0.05). However, these associations disappeared after adjustment for plasma folate concentrations. CONCLUSIONS: Age, sex, plasma folate, vitamin B-12 and B-6 concentrations, and MTHFR genotype are independent determinants of plasma homocysteine in middle-aged and older Chinese in Singapore. These factors combined could account for up to 40% of the total variation in homocysteine concentrations in this Asian population.

Cardiovascular risk factors in non-insulin-dependent diabetics compared to non-diabetic controls: a population-based survey among Asians in Singapore.

Cardiovascular risk factors were compared between 126 people with non-insulin-dependent diabetes mellitus (NIDDM) and 530 non-diabetics (controls), in a random sample of people (Chinese, Malays, and Asian Indians) aged 40-69 years from the general population of Singapore. Data were adjusted for age and ethnicity. For both genders, people with NIDDM had higher mean body mass indices, waist-hip ratios and abdominal diameters. They also had a higher prevalence of hypertension, higher mean levels of fasting serum triglyceride, slightly lower mean levels of serum high-density-lipoprotein cholesterol, and higher mean levels of plasma plasminogen activator inhibitor-1 and tissue plasminogen activator (antigen). These factors are components of syndrome X (metabolic syndrome) and increase the risk of atherosclerosis and thrombosis. In contrast, there were no important differences for cigarette smoking, serum total and low-density-lipoprotein cholesterol, serum apolipoproteins A1 and B, plasma factor VIIc and plasma prothrombin fragment 1 + 2. Females with NIDDM, but not males, had a higher mean serum fibrinogen level than non-diabetics, which could explain why NIDDM has a greater cardiovascular effect in females than males. Serum lipoprotein(a) concentrations were lower in people with NIDDM. Mean levels of serum ferritin, a pro-oxidant, were higher in people with NIDDM than controls, but there were no important differences for plasma vitamins A, C and E, and serum selenium, which are anti-oxidants.

Cardiovascular risk factors in relation to cigarette smoking: a population-based survey among Asians in Singapore.

To investigate how cigarette smoking increases the risk of cardiovascular disease, risk factors were compared between 166 cigarette smokers and 312 non-smokers, in a random sample of males (Chinese, Malays and Asian Indians) aged 30-69 years from the general population of Singapore. There was adjusted for age and ethnic group. The prevalence of hypertension was lower in cigarette smokers (15.2%) than non-smokers (21.9%), with the difference reduced by adjustment for body mass index (BMI). Smokers had: lower mean serum HDL-cholesterol (0.76 versus 0.81 mmol/l) and higher mean serum fasting triglyceride (1.92 versus 1.71 mmol/l), which will increase atherosclerosis; higher mean plasma fibrinogen (2.75 versus 2.67 g/l) and plasminogen activator inhibitor 1 [PAI-1] (24.9 versus 22.2 ng/ml), which will increase thrombosis; and lower mean plasma vitamin C (4.4 versus 6.4 mg/l) and serum selenium (118 versus 123 microg/l), which may increase atherosclerosis. Adjustment for BMI slightly increased the differences for HDL-cholesterol, fasting triglyceride, fibrinogen and PAI-1, indicating that less generalised obesity among smokers reduces their increased cardiovascular disease risk. Smoking was not found to be related to: diabetes mellitus; serum total cholesterol, LDL-cholesterol, apolipoproteins A1 and B and lipoprotein(a); plasma factor VIIc and prothrombin fragment 1 + 2; and plasma vitamins A and E and serum ferritin. There was no evidence of increased insulin resistance in smokers, as measured by mean fasting serum insulin.

Salvia miltiorrhiza inhibits cell growth and induces apoptosis in human hepatoma HepG(2) cells.

Salvia miltiorrhiza (SM) is a traditional Chinese herbal medicine, commonly used to treat liver diseases in China for centuries. Several earlier studies have indicated that SM exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we evaluated the molecular mechanism of SM in a human hepatoma cell line, HepG(2). Our results show that SM exerted clear cytotoxic effects, and strongly inhibited the proliferation of HepG(2) cells. It was also observed that SM treatment caused apoptotic cell death as evaluated by: (a), morphological changes by using acridine orange/ethidium bromide staining; (b), DNA fragmentation by TdT-mediated dUTP nick end labeling (TUNEL); and (c), sub-G(1) cell analysis. Furthermore, depletion of intracellular glutathione (GSH) and reduction of mitochondrial membrane potential were found to be involved in the initiation of apoptosis by SM.

Inhibition of aflatoxin B1-DNA binding and adduct formation by selenium in rats.

The effect of selenium on aflatoxin B1-DNA binding and adduct formation was studied. Male Fischer 344 rats, fed with up to 8 ppm of sodium selenite in drinking water for 8 weeks, were given a single i.p. dose of aflatoxin B1. The rats were killed 24 h later and the amount of AFB1 bound to hepatic DNA and the amount of DNA adducts formed were determined. Selenium pretreatment resulted in a dose-dependent inhibition of AFB1-DNA binding as well as adduct formation. This was accompanied by an increase of reduced glutathione (GSH) in the liver of selenium-treated animals. These results suggest that selenium could effectively inhibit AFB1-induced DNA damage, which may be partially responsible for its anticarcinogenic effect against AFB1.

Protective effect of ebselen against hydrogen peroxide-induced cytotoxicity and DNA damage in HepG2 cells.

The protective effect of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound, against hydrogen peroxide (H2O2)-induced cytotoxicity and DNA damage was investigated in a human hepatoma cell line, HepG2. The inhibitory effect of H2O2 on cell growth was determined using the tetrazolium dye colorimetric test (MTT test), and the cytotoxicity and lipid peroxidation were estimated by lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation, respectively. DNA damage was detected using single cell gel electrophoresis (comet assay), and intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). The results showed that H2O2 suppressed the growth of HepG2 cells and the addition of ebselen significantly reduced the suppression. Furthermore, ebselen also displayed a dose-dependent reduction of LDH leakage and MDA formation in H2O2-treated cells. The results also demonstrate that ebselen was able to reduce the ROS formation and DNA damaging effect caused by H2O2 in a dose-dependent manner. These findings suggest that ebselen has a strong protective ability against the cytotoxicity and DNA damaging effect caused by reactive oxygen species.

Effects of chronic exposure to low doses of trichloroethylene on steroid hormone and insulin levels in normal men.

The aim of this study was to examine the serum levels of insulin and some adrenal steroid hormones in men chronically exposed to low doses of trichloroethylene (TCE). A total of 85 workers participated in this study. Each worker had urine collected and analyzed for trichloroacetic acids (UTCA) on the same day that a blood sample was taken for analyses of serum testosterone, sex hormone-binding globulin (SHBG), androstenedione, cortisol, aldosterone, and insulin. The mean concentration of environmental TCE was 29.6 ppm and the mean UTCA was 22.4 mg/g creatinine (range 0.8-136.4). TCE exposure did not cause any significant changes to the adrenal steroid hormone productions. The results showed that UTCA was significantly correlated to serum insulin levels. Insulin and SHBG responded in tandem, with the highest levels found in workers exposed to TCE for less than 2 years; levels of both parameters were significantly lowered in those exposed for more than 2 years. A triphasic response in insulin levels to TCE, which depended on the duration of exposure, was noted. Initial exposure caused an acute rise in insulin levels. This was followed by a fall to normal levels in those exposed 2-4 years and then a slight rise in those exposed for more than 6 years. The mechanism for this pattern of response to TCE exposure is yet unknown.

Microcystic cyanobacteria extract induces cytoskeletal disruption and intracellular glutathione alteration in hepatocytes.

Microcystins are a group of highly liver-specific toxins, although their exact mechanisms of action remain unclear. We examined the effects of microcystic cyanobacteria extract (MCE) collected from a contaminated water source on the organization of cellular microtubules (MTs) and microfilaments (MFs) in hepatocytes. We also investigated the effects on lactate dehydrogenase (LDH) leakage and intracellular glutathione (GSH). Primary cultured rat hepatocytes exposed to MCE (equivalent to 125 microg/mL lyophilized algae cells) showed a characteristic disruption of MTs and MFs in a time-dependent manner. Under these conditions, MCE caused aggregation of MTs and MFs and a severe loss of MTs in some cells. Moreover, MCE-induced cytoskeletal alterations preceded the LDH leakage. On the other hand, the treatment of cells with MCE led to a dose-dependent increase of intracellular GSH. However, time-course study showed a biphasic change of intracellular GSH levels with a significant increase in the initial stage followed by a decrease after prolonged treatment. Furthermore, pretreatment with N-acetylcystein (NAC), a GSH precursor, significantly enhanced the intracellular GSH level and decreased the MCE-induced cytotoxicity as well as cytoskeleton changes. In contrast, buthionine-(S, R)-sulfoximine, a specific GSH synthesis inhibitor, increased the cell susceptibility to MCE-induced cytotoxicity by depleting the intracellular GSH level. These findings suggest that intracellular GSH plays an important role in MCE-induced cytotoxicity and cytoskeleton changes in primary cultured rat hepatocytes. Increasing intracellular GSH levels protect cells from MCE-induced cytotoxicity and cytoskeleton changes.

Cadmium-induced oxidative cellular damage in human fetal lung fibroblasts (MRC-5 cells).

Epidemiological evidence suggests that cadmium (Cd) exposure causes pulmonary damage such as emphysema and lung cancer. However, relatively little is known about the mechanisms involved in Cd pulmonary toxicity. In the present study, the effects of Cd exposure on human fetal lung fibroblasts (MRC-5 cells) were evaluated by determination of lipid peroxidation, intra-cellular production of reactive oxygen species (ROS), and changes of mitochondrial membrane potential. A time- and dose-dependent increase of both lactate dehydrogenase leakage and malondialdehyde formation was observed in Cd-treated cells. A close correlation between these two events suggests that lipid peroxidation may be one of the main pathways causing its cytotoxicity. It was also noted that Cd-induced cell injury and lipid peroxidation were inhibited by catalase and superoxide dismutase, two antioxidant enzymes. By using the fluorescent probe 2',7'-dichlorofluorescin diacetate, a significant increase of ROS production in Cd-treated MRC-5 cells was detected. The inhibition of dichlorofluorescein fluorescence by catalase, not superoxide dismutase, suggests that hydrogen peroxide is the main ROS involved. Moreover, the significant dose-dependent changes of mitochondrial membrane potential in Cd-treated MRC-5 cells, demonstrated by increased fluorescence of rhodamine 123 examined using a laser-scanning confocal microscope, also indicate the involvement of mitochondrial damage in Cd cytotoxicity. These findings provide in vitro evidence that Cd causes oxidative cellular damage in human fetal lung fibroblasts, which may be closely associated with the pulmonary toxicity of Cd.

Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive oxygen species formation in primary cultured rat hepatocytes.

Cyanobacteria contamination of water has become a growing public health problem worldwide. Microcystis aeruginosa is one of the most common toxic cyanobacteria. It is capable of producing microcystins, a group of cyclic heptapeptide compounds with potent hepatotoxicity and tumor promotion activity. The present study investigated the effect of microcystic cyanobacteria on primary cultured rat hepatocytes by examining mitochondrial membrane potential (MMP) changes and intracellular reactive oxygen species (ROS) formation in cells treated with lyophilized freshwater microcystic cyanobacteria extract (MCE). Rhodamine 123 (Rh-123) was used as a fluorescent probe for changes in mitochondrial fluorescence intensity. The mitochondrial Rh-123 fluorescence intensity in MCE-treated hepatocytes, examined using a laser confocal microscope, responded in a dose- and time-dependent manner. The results thus indicate that the alteration of MMP might be an important event in the hepatotoxicity caused by cyanobacteria. Moreover, the parallel increase of ROS formation detected using another fluorescent probe, 2',7'-dichlorofluorescin diacetate also suggests the involvement of oxidative stress in the hepatotoxicity caused by cyanobacteria. The fact that MMP changes precede other cytotoxic parameters such as nuclear staining by propidium iodide and cell morphological changes suggests that mitochondrial damage is closely associated with MCE-induced cell injury in cultured rat hepatocytes.