Enhanced expression of the LDH-A gene after gravity-changing stress in human RSa cells.

A major issue in radiation and space biology is whether gene expression levels are altered in cells exposed to gravity-changing stress. In the present study, genes up- or down-regulated in radiation-sensitive human RSa cells cultured under gravity-changing conditions, were identified using a PCR-based mRNA differential display method. Exposure of cells to gravity-changing stress was performed by free-fall with a drop-shaft facility or by an airplane-conducted parabolic flight. Among the candidates for gravity-changing stress-responsive genes obtained by the differential display analysis, the lactate dehydrogenase A gene (LDH-A) was confirmed by Northern blotting analysis to exhibit increased expression levels. The gravity-changing stress consisted of a combination of microgravity and hypergravity. However, exposure of the cells to hypergravity produced by centrifuge only slightly affected the LDH-A mRNA expression. Thus, LDH-A was found to be a candidate for the genes which play a role in the cellular response to gravity-changing stress, and mainly to microgravity.     

Urinary 8-OHdG elevations in a partial lesion rat model of Parkinson's disease correlate with behavioral symptoms and nigrostriatal dopaminergic depletion

Increased oxidative stress contributes to pathogenesis of Parkinson's disease (PD). 8-hydroxy-2'-deoxyguanosine (8-OHdG) is the oxidation product most frequently measured as an indicator of oxidative DNA damage. Several studies have shown increased 8-OHdG in PD patients. There are few basic laboratory data examining 8-OHdG levels in animal models of PD. In this study, we utilized hemiparkinsonian model of rats induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). The urinary 8-OHdG level was measured in relation to behavioral and pathological deficits arising from 6-OHDA-induced neurotoxic effects on the nigrostriatal dopaminergic pathway. All rats were subjected to a series of behavioral tests for 42 days after 6-OHDA injection. We collected urine samples with subsequent measurement of 8-OHdG level using ELISA kits. For immunohistochemical evaluation, tyrosine hydroxylase (TH) staining was performed. Significant increments in urinary 8-OHdG level were observed continuously from day 7 until day 35 compared to control group, which showed a trend of elevation as early as day 3. Such elevated urinary 8-OHdG level significantly correlated with all of the behavioral deficits measured here, suggesting that urinary 8-OHdG level provides a good index of severity of parkinsonism. Urinary 8-OHdG level also had a significant positive correlation with the survival rate of dopaminergic fibers or neurons, advancing the concept that oxidative stress during the early phase of 6-OHDA neurotoxicity may correspond to disease progression closely approximating neuronal degeneration in the nigrostriatal dopaminergic system. The present results demonstrate that alterations in urinary 8-OHdG level closely approximate onset and disease progression in PD.     

Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract Background. Measurement of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p 相似文献   

Increased oxidative stress in rats with chronic nitric oxide depletion: measurement of urinary 8-hydroxy-2'-deoxyguanosine excretion

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative stress. We examined the effect of chronic blockade of nitric oxide (NO) on urinary excretion of 8-OHdG in rats. Two types of NO synthase inhibitor were used: N(G)-nitro-L-arginine methyl ester (L-NAME) as a non-selective inhibitor and aminoguanidine (AG) as a selective inhibitor of the inducible isoform. Oral administration of L-NAME (20, 50 and 80 mg/dl of drinking water), but not AG (400 mg/dl), for 4 weeks induced systemic hypertension and a significant reduction in urinary excretion of NO2-/NO3-. Rats treated with L-NAME also showed a significant increase in urinary 8-OHdG excretion compared with the control animals. The effects of L-NAME (50 mg/dl) on blood pressure and urinary excretion of NO2/NO3- and 8-OHdG were restored by a large dose of L-arginine (2.0 g/dl). Chronic AG administration did not significantly alter urinary 8-OHdG excretion. On combining all the data, there was a significant negative correlation between urinary NO2-/NO,- and 8-OHdG. These observations suggest the importance of constitutive NO synthase activity in the maintenance of oxidant buffering capacity in rats. Oral administration of L-NAME may serve as a model of hypertension due to chronic NO deficiency with increased oxidative stress.     

Association of hepatitis virus infection,alcohol consumption and plasma vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical workers

Urinary 8-hydroxydeoxyguanosine (8-OHdG) DNA adduct has been used as a biomarker in epidemiological studies. However, the determinants for urinary 8-OHdG have not been clearly identified. We tested urinary 8-OHdG levels in 205 male workers who had been exposed to vinyl chloride monomer (VCM). Epidemiological information was obtained by an interviewer-administered questionnaire. Hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV) were also determined by immunoassay. Plasma antioxidants including Vitamins A and E, alpha- and beta-carotenes were assayed by high performance liquid chromatography. Median of urinary 8-OHdG level was 9.8 ng/mg creatinine (range, 1.4-60.1). Multiple linear regression analysis showed that alcohol drinkers had higher urinary 8-OHdG than those who did not, but there was no dose-response between the amount of alcohol consumption and urinary 8-OHdG. Workers with positive HBsAg, anti-HCV and elevated plasma Vitamin A level were independently associated with higher levels of urinary 8-OHdG, whereas age, smoking, body mass index, plasma alpha- and beta-carotenes, Vitamin E levels, or VCM exposure did not show such an association. The results suggest that active inflammation of hepatitis B and C, alcohol consumption and higher Vitamin A level can induce oxidative stress. Thus, we conclude that potential determinants need to be considered in epidemiological studies when urinary 8-OHdG is used as a biomarker.     

Enhancement of protease activity involved in modulation of cell mutability by microgravity stress in UVC-irradiated human cells

It is an intriguing question whether gravity-changing stress modulates human cell mutability. To resolve this problem, it is necessary to determine the cellular events leading to modulation. We previously detected protease activation just after UV (UVC, principally 254 nm wavelength) irradiation followed by hypomutability in cultured human cells. We here investigated whether UV-activated protease activity is affected in human UVAP-1 cells exposed to gravity-changing stress prior to UV irradiation.     

Urinary excretion of 8-hydroxydeoxyguanosine and malondialdehyde after high dose radiochemotherapy preceding stem cell transplantation

The urinary excretion of the hydroxylated DNA base 8-hydroxydeoxyguanosine (8-OHdG) and the lipid peroxidation product malondialdehyde (MDA) was monitored in 11 patients with hematological malignancies undergoing total body irradiation and high-dose chemotherapy preceding bone marrow transplantation. Nine patients showed a prompt increase in urinary 8-OHdG (8-25 times the initial baseline level) on days 0-7 after irradiation onset; the excretion then decreased during the aplastic period and increased again when engraftment took place (in 7 patients). A significant positive correlation was found between urinary 8-OHdG and whole blood leukocyte count, both on day 5 (p =.04, r =.72) and on day 22 (p =.009, r =.80) after irradiation onset. One patient who lacked the first peak of 8-OHdG excretion showed low blood leukocyte counts (less than 2 x 10(9)/l) before therapy onset; this patient, however, later had a successful engraftment and then also showed considerable increases in both 8-OHdG excretion and leukocyte count. These observations suggest leukocytes play a part in the excretion of 8-OHdG after conditioning therapy preceding bone marrow transplantation. As opposed to the biphasic 8-OHdG excretion, the excretion of MDA showed a single peak appearing on days 11-19 after radiochemotherapy onset, i.e., during the period in which the patients suffered from cytopenia, mucositis, and other side effects of the treatment. It is suggested, therefore, that these clinical manifestations are associated with increased lipid peroxidation. Altogether, these findings illustrate the utility of serial urinary samples for monitoring oxidative stress due to conditioning therapy in clinical practice. They also demonstrate that different oxidative stress markers may behave quite differently regarding their appearance in the urine after whole-body oxidative stress.     

Increased levels of UV-induced protease activity in human UVAP-1 cells exposed to gravity-changing stress: involvement of E-64-sensitive proteases in suppression of UV mutagenicity

Under the 1G condition, the increase in antipain-sensitive protease activity promptly after UV (mainly 254 nm wavelength) irradiation in cultured human cells is detected and found to be one of the intriguing events involved in suppression of cell mutability. It was found that two cell lines, RSa and its variant UVAP-1 cells are applicable; the former is hypermutable and not susceptible to protease activation, while the latter is hypomutable and susceptible. In the present study it was investigated whether the increase in protease activity by UV irradiation is also observed in hypomutable human UVAP-1 cells exposed to gravity-changing stress and whether the increase is involved in suppression of UV mutagenicity. Exposure of human UVAP-1 cells to gravity-changing stress such as free-fall and parabolic flight prior to UV irradiation resulted in a pronounced increase in protease activity, but not to hypergravity conditions (2 and 10G) prior to UV irradiation. To characterize the proteases, components of lysates from the cells exposed to free-fall prior to UV irradiation were fractionated by high performance liquid chromatography, indicating two separate fractions with highly increased levels of E-64-sensitive protease activity. In the cells treated with E-64 during their exposure to free-fall, K-ras codon 12 base substitution mutation was detected after UV irradiation, although the mutation was not detected after UV irradiation alone. Thus, the increase in E-64-sensitive protease activity may be involved in the suppression of UV mutagenicity in UVAP-1 cells exposed to free-fall.     

Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection

There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high-performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8-OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used.     

Quantitative determination of urinary 8-hydroxy-2'-deoxyguanosine level in healthy Japanese volunteers

8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.     

Phase 1 study of multiple biomarkers for metabolism and oxidative stress after one-week intake of broccoli sprouts

Little is known about the direct effect of broccoli sprouts on human health. So we investigated the effect of broccoli sprouts on the induction of various biochemical oxidative stress markers. Twelve healthy subjects (6 males and 6 females) consumed fresh broccoli sprouts (100 g/day) for 1 week for a phase 1 study. Before and after the treatment, biochemical examination was conducted and natural killer cell activity, plasma amino acids, plasma PCOOH (phosphatidylcholine hydroperoxide), the serum coenzyme Q(10), urinary 8-isoprostane, and urinary 8-OHdG (8-hydroxydeoxyguanosine) were measured. With treatment, total cholesterol and LDL cholesterol decreased, and HDL cholesterol increased significantly. Plasma cystine decreased significantly. All subjects showed reduced PCOOH, 8-isoprostane and 8-OHdG, and increased CoQ(10)H(2)/CoQ(10) ratio. Only one week intake of broccoli sprouts improved cholesterol metabolism and decreased oxidative stress markers.     

Comparison of 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract

Background. Measurement of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p <?0.001). According to ROC analysis, the AUC value of HbA1c was higher than the value of the AUC of 8-OHdG (0.882 and 0.771, respectively). Conclusions. This study shows that the urine 8-OHdG levels increase in diabetic patients. However, urinary 8-OHdG is not a useful clinical marker, compared with UACR, to predict the development of diabetic nephropathy in diabetic patients.     

Changes in urine 8-hydroxydeoxyguanosine levels of super-marathon runners during a four-day race period

We have determined the urinary 8-hydroxydeoxyguanosine (8-OHdG) levels of five well trained supra-marathon runners during a four-day race. The daily running distances of the four-day race were the following; 93 km, 120 km, 56 km and 59 km, respectively. Pre-race and post-race urine samples were collected on each day and analyzed by a monoclonal antibody technique. The urinary 8-OHdG content increased significantly on the first day and tended to decrease from the third day. By the fourth day 8-OHdG content was significantly less than measured on the first three days. The serum creatine kinase activity changed in a similar fashion, showing a large increase (P<0.001) up to the third day when it decreased significantly from the peak value (P<0.05). We conclude that extreme physical exercise causes oxidative DNA damage to well trained athletes. However, repeated extreme exercise-induced oxidative stress does not propagate on increase of urinary 8-OHdG, but rather causes an adaptation leading to normalization of oxidative DNA damage.     

Impact of modernization on oxidative stress among indigenous populations in northern Laos

Objectives

To explore the impact of modernization on oxidative stress during a momentous health transition process, we investigated differences in oxidative stress among the indigenous populations of villages in northern Laos with different levels of modernization.

Methods

We conducted a cross-sectional study of 380 adults in three villages with different levels of modernization. Three biomarkers related to oxidative stress were measured: urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-isoprostane concentrations (both measured by liquid chromatography–tandem mass spectrometry), and blood telomere length (measured with qPCR). We examined associations between village-level modernization and oxidative stress-related biomarkers in a multilevel analysis including a random effect and covariates.

Results

The geometric means of urinary 8-OHdG and 8-isoprostane concentrations were 2.92 and 0.700 μg/g creatinine, respectively, in our study population. Higher urinary 8-OHdG concentrations and shorter telomeres were observed in participants from the more modernized villages, whereas urinary 8-isoprostane concentrations did not differ significantly among villages.

Conclusions

Our findings imply that modernization-induced changes in lifestyle may increase oxidative DNA damage. Baseline levels of oxidative lipid damage are expected to be high in the indigenous populations of northern Laos. Assessments of oxidative stress may provide valuable insights into the mechanisms of health transition in specific populations.     

Oxidative DNA damage (8-hydroxydeoxyguanosine) and body iron status: a study on 2507 healthy people

To clarify the relationship of oxidative stress and body iron status, we detected urinary 8-hydroxydeoxyguanosine (8-OHdG) as a biomarker of oxidative DNA damage, and measured serum ferritin and total iron-binding capacity (TIBC), both reflecting body iron store, on 2507 healthy people aged between 22 and 89 years (males, 1253; females, 1254). The urinary 8-OHdG excretion of males showed almost no change with age, but the excretion of premenopausal females was lower than that of males, whereas postmenopausal females excreted significantly more than males. The values of serum ferritin showed no remarkable change with age in males, but increased gradually in postmenopausal females without iron loss due to bleeding, although the males' values remained higher than those of females at all ages (p<.05). On the other hand, the values of TIBC remained within the narrow limits in males, regardless of age, whereas those of females always stayed at a higher level than the males (p<.05). Conclusively, urinary 8-OHdG correlated with serum ferritin positively and with TIBC inversely, which suggested that body iron status would control the generation of 8-OHdG in vivo. After all, the increase of urinary 8-OHdG excretion in postmenopausal females may be caused by the decrease of body iron loss.     

Effect of a Single Bout of Exercise and β-Carotene Supplementation on the Urinary Excretion of 8-Hydroxy-deoxyguanosine in Humans

We investigated the effects of acute exhaustive exercise and β-carotene supplementation on urinary 8-hydroxy-deoxyguanosine (8-OHdG) excretion in healthy nonsmoking men. Fourteen untrained male (19-22 years old) volunteers participated in a double blind design. The subjects were randomly assigned to either the β-carotene or placebo supplement group. Eight subjects were given 30 mg of β-carotene per day for 1 month, while six subjects were given a placebo for the same period. All subjects performed incremental exercise to exhaustion on a bicycle ergometer both before and after the 1-month β-carotene supplementation period. The blood lactate and pyruvate concentrations significantly increased immediately after exercise in both groups. The baseline plasma p-carotene concentration was significantly 17-fold higher after β-carotene supplementation. The plasma β-carotene decreased immediately after both trials of exercise, suggesting that β-carotene may contribute to the protection of the increasing oxidative stress during exercise. Both plasma hypoxanthine and xanthine increased immediately after exercise before and after supplementation. This thus suggests that both trials of exercise might enhance the oxidative stress. The 24-h urinary excretion of 8-OHdG was unchanged for 3 days after exercise before and after supplementation in both groups. However, the baseline urinary excretion of 8-OHdG before exercise tended to be lower after β-carotene supplementation. These results thus suggest that a single bout of incremental exercise does not induce the oxidative DNA damage, while β-carotene supplementation may attenuate it.     

Longitudinal study of urinary 8-hydroxy-2'-deoxyguanosine excretion in healthy adults

Numerous studies have investigated the urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for the assessment of oxidative DNA damage in humans. In this study, we performed six consecutive series of measurement of urinary levels of 8-OHdG in 68 healthy probands, in order to provide information on the intra- and inter-individual variability of 8-OHdG and to estimate the influence of smoking, age, sex, body weight and body mass index (BMI) on the excretion of 8-OHdG. The intraindividual coefficient of variation (CV) of urinary 8-OHdG/24h ranged from 0.18 to 1.06 (mean CV=0.48). Women excreted significantly lower amounts of 8-OHdG/24h than men, but the difference lost its significance when the body weight or urinary creatinine were used as covariates. By multiple linear regression analysis significant correlations between the mean individual levels of 8-OHdG/24h excretion and urinary creatinine (r_p = 0.61), and cotinine (r_p = 0.27) have been observed, whereas no statistically significant effect of age, body weight and BMI was found. The 8-OHdG/creatinine ratio was found to be significantly increased in 23 smokers (1.95 ± 0.40 μmol/mol) opposed to 45 non-smoking probands (1.62 ± 0.50 μmol/mol), which is in good agreement with previously published data. No effect of passive smoking on the excretion of 8-OHdG was found. From our data we conclude that the intraindividual variability of urinary 8-OHdG excretion has been underestimated so far, indicating that values of 8-OHdG measured by single spot monitoring are not representative for individual base levels.     

Oxidative stress early in pregnancy and pregnancy outcome

The objectives of this study were to determine whether oxidative stress early in pregnancy influenced pregnancy outcome. A combination of assays were used for exogenous and endogenous anti-oxidants together with two well accepted biomarkers for oxidative stress, the urinary excretion of 8-iso-PGF(2alpha) (a biomarker marker for lipid oxidation, n=508) and 8-oxo-7,8 dihydro-2 deoxyguanosine (8-OHdG, a biomarker for DNA oxidation, n=487). The two biomarkers tracked different pregnancy outcomes. Isoprostanes were associated with an increased risk of pre-eclampsia and a decreased proportion of female births. In contrast, 8-OHdG tracked lower infant birthweight and shortened gestation duration. Birth defects were associated with low levels of 8-OHdG.     

Association of serum arginase I with oxidative stress in a healthy population

The association of serum arginase I with oxidative stress was evaluated cross-sectionally in a healthy population. The mean levels of serum arginase I in healthy people (n = 278) were 32.6 ± 22.3 ng/ml. Significant correlations of arginase I were observed with age, WBC, RBC, alanine aminotransferase (ALT), high-sensitivity C-reactive protein (hs-CRP), uric acid, body mass index (BMI) and urinary 8-isoprostane. Multiple regression analysis showed significant associations of arginase I with WBC, RBC, urinary 8-hydroxydeoxyguanosine (8-OHdG), age, HbA1c and urinary 8-isoprostane. In the associations of arginase I with 8-OHdG, 8-isoprostane and HbA1c, confounding factors and lifestyle factors such as sex, old age, smoking and alcohol consumption were involved. It was concluded that serum arginase I was associated with oxidative stress and HbA1c in addition to age, WBC and RBC in healthy Japanese people and may become a new biomarker for early prediction of diabetes mellitus and other oxidative stress-related diseases.     

Oxidative stress early in pregnancy and pregnancy outcome

The objectives of this study were to determine whether oxidative stress early in pregnancy influenced pregnancy outcome. A combination of assays were used for exogenous and endogenous anti-oxidants together with two well accepted biomarkers for oxidative stress, the urinary excretion of 8-iso-PGF_2α (a biomarker marker for lipid oxidation, n=508) and 8-oxo-7,8 dihydro-2 deoxyguanosine (8-OHdG, a biomarker for DNA oxidation, n=487). The two biomarkers tracked different pregnancy outcomes. Isoprostanes were associated with an increased risk of pre-eclampsia and a decreased proportion of female births. In contrast, 8-OHdG tracked lower infant birthweight and shortened gestation duration. Birth defects were associated with low levels of 8-OHdG.