There is a robust mechanistic basis for the role of oxidation damage to DNA in the aetiology of various major diseases (cardiovascular, neurodegenerative, cancer). Robust, validated biomarkers are needed to measure oxidative damage in the context of molecular epidemiology, to clarify risks associated with oxidative stress, to improve our understanding of its role in health and disease and to test intervention strategies to ameliorate it. Of the urinary biomarkers for DNA oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most studied. However, there are a number of factors which hamper our complete understanding of what meausrement of this lesion in urine actually represents. DNA repair is thought to be a major contributor to urinary 8-oxodG levels, although the precise pathway(s) has not been proven, plus possible contribution from cell turnover and diet are possible confounders. Most recently, evidence has arisen which suggests that nucleotide salvage of 8-oxodG and 8-oxoGua can contribute substantially to 8-oxoG levels in DNA and RNA, at least in rapidly dividing cells. This new observation may add an further confounder to the conclusion that 8-oxoGua or 8-oxodG, and its nucleobase equivalent 8-oxoguanine, concentrations in urine are simply a consequence of DNA repair. Further studies are required to define the relative contributions of metabolism, disease and diet to oxidised nucleic acids and their metabolites in urine in order to develop urinalyis as a better tool for understanding human disease.
D-Psicose is a rare sugar present in small quantities in natural products. In a previous study, we showed that D-psicose suppresses plasma glucose increases and reduces body fat accumulation in rats. Based on acute toxicity testing in rats, D-psicose is classified as an ordinary substance (LD50 = 16 g/kg). Elucidating the effects of long term feeding of D-psicose in rats will be essential prior to its utilization as a physiologically functional food. In this study, male Wistar rats (3 weeks old) were fed diets containing 3% D-psicose or sucrose for 12–18 months. The rats actually ingested 1.28 g/kg body weight per day D-psicose or 1.22 g/kg body weight per day of sucrose. Body weight gain and intra-abdominal adipose tissue weight in rats fed the D-psicose diet for 18 months were significantly lower than those in rats fed the sucrose diet. Relative weights of liver and kidney were significantly higher in the D-psicose group than in the sucrose group. However, no gross pathological findings were evident at dietary doses of 3% D-psicose or correlated with hypertrophy of liver and kidney. No clinical chemical test value was suggestive of overt D-psicose treatment-related toxicity. Therefore, the present study found no adverse effects at 3% D-psicose in the diet.
Paraoxonase-1 (PON-1), an HDL-associated enzyme has been shown to possess antioxidant/anti-inflammatory properties and protect against atherogenesis. The aim of the study was to explore the association of PON-1, with lipids, antioxidant-vitamins and lifestyle in male subjects with and without angiographically documented coronary artery disease (CAD) and in healthy volunteers. PON-1 activity was measured spectrophotometrically using phenyl acetate as substrate. PON-1 and HDL-Cholesterol were significantly lower and lipid peroxides and triglycerides were higher in CAD patients than in normal coronary and control subjects. PON-1 activity showed positive association with HDL-C and inverse relation with lipid peroxides and no association with antioxidant vitamins in healthy subjects. Smoking habit, alcohol intake and diabetes mellitus did not seem to influence PON-1 activity. Serum levels of PON-1 and HDL-C showed inverse association with the presence of CAD but are not related to the severity of disease in terms of number of diseased vessels. Interventional means by diet/drugs to enhance PON-1 activity may contribute to attenuation of atherosclerosis.
Our objective was to examine the effect of orange juice and hesperetin on serum total antioxidant capacity (TAC), lipid peroxidation (MDA), uric acid and hepatic xanthine oxidase (XO) and xanthine dehydrogenase (XDH) activity in hyperuricemic rats. Experimentally hyperuricemia in rats was induced by intraperitoneal injection of potassium oxonate (250 mg/kg). Orange juice (5 ml/kg) and hesperetin (5 mg/kg) was given by oral gavage to rats for 2 weeks and biochemical data was measured. Data showed that orange juice supplementation increased serum TAC and decreased MDA concentration (p≤0.05). Orange juice also inhibited hepatic XO and XDH activity and decreased serum uric acid levels. Hesperetin, which is the main flavanone constituent in orange juice, also exhibited antioxidative and antihyperuricemic properties, but its effect was weaker than that of orange juice. Although the hypouricemic effect of allopurinol (5 mg/kg), as a positive control, was much higher than that of orange juice and hesperetin, it could not significantly change biomarkers of oxidative stress. These features of orange juice and hesperetin make them an attractive candidate for the prophylactic treatment of hyperuricaemia, particularly if these compounds are to be taken on a long-term basis.
We treated patients with C-viral chronic hepatitis (CH) and liver cirrhosis (LC) with polaprezinc and determined prospectively the effect on long-term outcome. 62 patients were enrolled. Of these, 32 were administered 1.0 g polaprezinc and the remainder were not administered polaprezinc. We measured the serum zinc concentrations using conventional atomic absorption spectrometry and conducted a prospective study to determine the long-term outcome of the polaprezinc therapy. Changes of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the polaprezinc administration group were significantly lower than those of the untreated group. The decrease in platelet count was clearly less than that of the untreated group. The factors that inhibited increases in serum zinc concentrations following administration of polaprezinc included low serum zinc concentration states. Furthermore, the reductions of AST and ALT levels in the low zinc group were significantly greater than those of the high zinc group. When the patients who were administered polaprezinc were divided into two groups whose zinc concentrations increased (zinc responders) or remained stable or decreased (zinc non-responders), the zinc responders had a clearly lower cumulative incidence of HCC than the zinc non-responders. We conclude zinc supplementation improved the long-term outcome in C-viral CH and LC patients.
The effects of topical application of Rafflesia hasseltii buds and flowers extract on the rate of wound healing and histology of healed wound were assessed. Four groups of adult male Sprague Dawley rats were experimentally wounded in the posterior neck area. A thin layer of blank placebo was applied topically to wounds of Group 1 rats. Wounds of experimental animals (Group 2 and 3) were treated with placebo containing 5% and 10% R. hasseltii buds extract, respectively. A thin layer of Intrasite gel was applied topically to wounds of Group 4 animals as reference. Macroscopically, wounds treated with placebo containing 5% and 10% R. hasseltii buds extract or Intrasite gel have been significantly accelerated the rate of wound healing compared to placebo-treated wounds. Histological analysis of healed wounds has confirmed this effect. Wounds treated with placebo containing 5%, 10% R. hasseltii buds extract or Intrasite gel showed markedly less scar width at wound enclosure and granulating tissue contained markedly more collagen and proliferating fibroblasts, but with the absence of inflammatory cells compared to wounds treated with blank placebo. In conclusion, the findings of increased rate of wound closure and contraction together with the histological findingssuggest that Rafflesia hasseltii buds extract is very effective in accelerating the wound healing process.
Myristic acid (MyA), which is a saturated fatty acid (C14:0) and a side chain of phorbol 12-myristate 13-acetate (PMA), was examined if MyA stimulates human polymorphonuclear leukocytes (PMNs) to release oxygen radicals comparable to PMA by applying electron paramagnetic resonance (EPR)-spin-trapping method. When MyA was added to isolated human PMNs, spin adducts of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH and DMPO-OOH were time-dependently observed. The amounts of these spin adducts were larger than those of PMNs stimulated by PMA. These results clearly show that MyA is more potent agent to prime human PMNs than PMA, in a point of view of not only O2·− but also ·OH production. This fact calls attention that too much intake of MyA that is known to be contained vegetable oils can lead to crippling effect through uncontrolled production of reactive oxygen species.
The standardised Ginkgo biloba extract EGb761 is known for its potential beneficial effects in the prevention and therapy of neurodegenerative disorders including Alzheimer’s disease (AD). However, the molecular mechanisms and the specific role of its constituents are largely unknown. The aim of the present feeding trial was to investigate the effects of EGb761 and its major constituents on the expression of genes encoding for proteins involved in the pathogenesis of AD in mouse brain. Six month old C57B6 mice were fed semi synthetic diets enriched with either EGb761 or one of its main fractions, flavonols and terpenelactones, respectively, over a period of 4 weeks. Thereafter, mRNA of α-secretase, neprilysin, amyloid precursor protein (App), App binding protein-1 and acetylcholine esterase was quantified in hippocampus and cortex. EGb761 and its flavonol fraction had no effects on relative mRNA levels of the respective genes in mouse brain. However, the terpenelactone fraction significantly decreased the mRNA levels of App in the hippocampus. Taken together, a 4 week dietary treatment with EGb761 or its main fractions had only moderate effects on mRNA levels of AD related genes in cortex and hippocampus of mice.
Nonalcoholic steatohepatitis (NASH) can develop into end-stage disease such as cryptogenic cirrhosis and hepatocellular carcinoma. Hence, it is important to understand the pathogenesis of NASH. In general, the “two-hit theory” has prevailed as a pathogenic mechanism of NASH. According to this theory, lipopolysaccharides (LPS) contained in normal portal blood are the “second hit,” but their role is not completely understood. Based on this theory, we evaluated the role of LPS in NASH pathogenesis. For the first hit to develop metabolic abnormalities, a synthetic diet rich in disaccharide (synthetic diet: 12.1 cal% disaccharide) was fed to Zucker (fa/fa) rats for 12 weeks. For the second hit, 100 μg/kg LPS was injected intraperitoneally once daily for 2 weeks. Synthetic diet-fed rats treated with LPS showed an increase in the triglyceride content and higher expression of profibrogenic mRNAs in the liver. Plasma alanine aminotransferase levels were significantly elevated using this protocol. Furthermore, histological examination demonstrated that this protocol induced mild hepatic fibrosis and focal necrosis in the livers of all rats. Synthetic diet-fed Zucker (fa/fa) rats treated with LPS could be useful for understanding the development of hepatic fibrosis in the two-hit theory.
Low caloric diet (LCD) is used for weight loss. Paraoxonase 1 (PON-1) is associated with the antioxidant functions of high-density lipoprotein (HDL). Among limited data on the relationships between obesity and PON-1, there has been no study on the effects of a stand-alone LCD on the physiological lactonase activity of PON-1. We investigated the prospective effects of LCD intervention (2 months) for weight loss on serum PON-1 activities (lactonase, arylesterase [mono-esterase] and tri-esterase) and HDL cholesterol (HDL-C), and their association with low-density lipoprotein cholesterol (LDL-C) in overweight and non-morbidly obese but otherwise healthy women (n = 30; mean age, 50.3 years; mean body mass index [BMI], 28.5 kg/m2). In addition to the data such as BMI, blood pressure, blood glucose and lipids, PON-1 activities were examined between pre- and post-intervention. The intervention reduced all metabolic outcomes, and PON-1 lactonase activity (determined with 5-[thiobutyl]butyrolactone) significantly decreased by 6.1%, paralleled by arylesterase (by 7.3%) and tri-esterase (by 7.8%). In multiple regression analysis, the percent change of PON-1 lactonase was significantly, positively and independently correlated to that of LDL-C (β = 0.51), HDL-C (β = 0.40), and BMI (β = 0.37). Our results showed that the solo diet treatment on weight loss might reduce serum PON-1 lactonase activity with reduced HDL-C and LDL-C. The relationship between the lactonase and LDL-C may be adaptive, plausibly hypothesizing less need for PON-1 activity as an antioxidant property to protect lipoproteins. Further research is needed to confirm this prediction.
Recent reports described a high incidence of nonalcoholic steatohepatitis (NASH) in patients with obstructive sleep apnea. Accordingly, we hypothesized that recurrent and intermittent hypoxemia plays an important role in the pathogenesis of NASH. Our objective was construction of a practical and accurate experimental model to reproduce the key features of NASH in humans. Chemical hypoxemia through methemoglobinemia was induced by daily intraperitoneal injection of sodium nitrite (40 mg/kg) for 4 weeks in rats with fatty liver. The later was induced by 4-week feeding a choline-deficient high-fat diet (CDHF). Besides, the normal chow diets feeding groups were prepared with in the same manner except for CDHF feeding. The animal experiment was performed in four groups; Normal control, Hypoxemia, CDHF, and CDHF + hypoxemia. Nitrite was given for the later 4 weeks to each rat of Hypoxemia and CDHF + hypoxemia. CDHF + hypoxemia rats were confirmed to develop histological changes that resemble those of patients with NASH, together with biochemical liver dysfunction, while CDHF group was limited in mild steatosis, and Hypoxemia group liver was normal. Present study established a reproducible and useful NASH model resembling the main features of NASH in humans, and showed first that recurrent and intermittent hypoxemia aggravate fatty liver to steatohepatitis and liver fibrosis.
There are some evidences that the increased oxidative stress and thus increased oxidizability of lipoproteins and DNA can contribute to the development of certain human diseases, such as cardiovascular disease. To confirm the association of DNA damage with cardiovascular disease, we investigated susceptibility of DNA to oxidation in lymphocytes and oxidative stress related parameters in blood of patients with coronary artery disease (CAD). Subjects were consisted of 42 patients (27 men, 15 women) with documented CAD and 49 apparently healthy subjects (33 men, 16 women) as controls. Cellular DNA damage induced by 100 μM H2O2 was measured using Comet assay and quantified by TL. There were no differences in age (61.4 ± 1.7 years vs 62.0 ± 2.2 years) between the two groups. All the findings were shown to be independent of either sex or smoking habit. The patients showed significantly higher TL (87.3 ± 1.6 μm) compared to the control (79.3 ± 1.7 μm, p<0.01). Plasma TRAP, vitamin C, γ-tocopherol, and α-carotene levels in patients group were lower than those of control groups, while erythrocytic catalase activity increased in patients group. In conclusion, we observed that reduced overall antioxidant status was closely connected to higher susceptibility of DNA damage in CAD patients.
The plasma or serum levels of various enzymes and components are known to increase in rats with water-immersion restraint stress (WIRS). We examined whether oxidative stress is involved in increases in the serum levels of various enzymes and components in rats with WIRS. Rats were exposed to WIRS for 6 h after oral administration of vitamin E (VE) (50 or 250 mg/kg). Rats with WIRS had increased serum alanine aminotransferase, aspartate aminotranseferase, lactate dehydrogenase, creatine kinase, urea nitrogen, creatinine, glucose, corticosterone, adrenocorticotropic hormone and lipid peroxide (LPO) levels, increased kidney and heart VE levels, decreased skeletal muscle VE level, and increased LPO levels in all tissues studied. Pre-administered VE (50 or 250 mg/kg) attenuated the increased serum alanine aminotransferase, aspartate aminotranseferase, lactate dehydrogenase, creatine kinase, urea nitrogen, creatinine, and LPO levels, the decreased skeletal muscle VE level, and the increased LPO levels in all tissues studied more effectively at its higher dose than at its lower dose. However, either dose of the pre-administered VE did not affect the increased serum glucose, corticosterone, and adrenocorticotropic hormone levels. These results suggest that oxidative stress is involved in increases in the serum levels of various enzymes and components in rats with WIRS.
Iron may induce oxidative stress via production of reactive oxygen species, facilitating mammary carcinogenesis. This study investigated the role of iron in relation to oxidative stress as a potential risk factor in the development of breast cancer (BC). BC patients (n = 121) and healthy age-matched controls (n = 149) were entered into the study. Iron and antioxidant vitamins intakes were estimated using a quantitative food frequency questionnaire. Thirty one subjects from each group provided blood samples for measurement of serum iron, plasma malondialdehyde (MDA) and ferric reducing ability of plasma (FRAP). Total and non-heme iron intake of BC patients were lower than those of the controls. However, the serum iron level was significantly higher in BC patients. Plasma MDA levels were also significantly higher in BC patients whereas no significant difference in FRAP values were observed between the two groups. Log-transformed serum iron concentration showed no significant correlation with MDA or FRAP. These results suggest that serum iron overload may be a breast cancer risk factor possibly due to increased oxidative stress.
Many researchers have hypothesized that differences in reactive oxygen species levels can trigger the cellular decision between hypertrophy and cell death in cardiomyocytes. In the present study, we examined the relationship between reactive oxygen species levels and hypertrophy or cell death in H9c2 cardiomyocytes after the addition of hydrogen peroxide. Following addition of hydrogen peroxide, we observed a slight increase in fluorescence intensity of 2',7'-dichlorofluorescein, a probe of intracellular reactive oxygen species, and cell hypertrophy in H9c2 cells (normal cells). In contrast, a dramatic increase in fluorescence intensity was followed by cell death in glutathione-depleted H9c2 cells. In the presence of the antioxidant Trolox or the iron chelator deferoxamine, both normal and glutathione-depleted cells developed hypertrophy without a concomitant increase in levels of reactive oxygen species. An inhibitor of p53, pifithrin-alpha, prevented cell death after the addition of hydrogen peroxide; instead a substantial increase in levels of reactive oxygen species and hypertrophy were observed. These results suggest that H9c2 cells exhibit differential sensitivity to intracellular reactive oxygen species generation with regard to their hypertrophic versus death responses to exogenously added hydrogen peroxide.
Nutritional and immunological status of patients with obstructive jaundice is usually severely altered, with high mortality rates. The n-3 polyunsaturate fatty acids (PUFA), particularly eicosapentaenoic acid (EPA, 20:5 n-3), posess potent immunomodulatory activities. Thus, our aim was to compare the plasma phospholipid fatty acid (FA) composition of these patients with healthy subjects, as well as before and after 7 days preoperative supplementation with high doses of EPA (0.9 g per day) and docosahexaenoic acid (DHA, 22:6 n-3, 0.6 g per day). We found impaired FA status in obstructive jaundice patients, especially EPA, DHA and PUFA, but significantly increased content of total n-3 FA, 22:5 n-3 FA and particularly EPA, which increased more than 3 fold, after 7 days supplementation. In addition, the n6/n3 ratio significantly decreased from 14.24 to 10.24, demonstrating severely improved plasma phospholipid profile in these patients after the intervention.