Earthworms and soil pollutants.

Although the toxicity of metal contaminated soils has been assessed with various bioassays, more information is needed about the biochemical responses, which may help to elucidate the mechanisms involved in metal toxicity. We previously reported that the earthworm, Eisenia fetida, accumulates cadmium in its seminal vesicles. The bio-accumulative ability of earthworms is well known, and thus the earthworm could be a useful living organism for the bio-monitoring of soil pollution. In this short review, we describe recent studies concerning the relationship between earthworms and soil pollutants, and discuss the possibility of using the earthworm as a bio-monitoring organism for soil pollution.

Effect of age, smoking and other lifestyle factors on urinary 7-methylguanine and 8-hydroxydeoxyguanosine.

Urinary 8-hydroxydeoxyguanosine (8-OH-dG) and 7-methylguanine (m7Gua) were measured by a column-switching high performance liquid chromatography method as markers of oxidative and methylating DNA damage, respectively. We investigated the associations between urinary 8-OH-dG or m7Gua and various lifestyle and demographic factors, such as age and sex. The urinary 8-OH-dG excretion level was positively correlated with cigarette smoking, but inversely correlated with fruit consumption, physical activity and total energy consumed per day. A multiple regression analysis revealed that daily physical activity and healthy meal combinations decreased the urinary 8-OH-dG level, whereas alcohol consumption increased it. In terms of the urinary m7Gua measurement, cigarette smoking, age and consumption of meat, fish, egg, soybean, etc. were positively correlated with the urinary m7Gua level, whereas body weight, BMI, physical activity, and dietary index score, which indicates good nutritional balance, were negatively correlated with the amount of m7Gua. Based on a multiple regression analysis, cigarette smoking and age correlated with the m7Gua level, while high BMI and healthy meal combinations have significant reducing effects on m7Gua level. Therefore, the urinary m7Gua level is considered to be a useful marker of DNA methylation, not only from smoking, but also from aging and unhealthy dietary habits.

Evaluation of oxidative stress in a group of adolescents exposed to a high level of aflatoxin B1--a multi-center and multi-biomarker study.

The association between aflatoxin B1 (AFB1) exposure and oxidative stress was extensively examined in 84 adolescents from an area at high risk for hepatocellular carcinoma in China. Plasma level of aflatoxin B1-albumin adducts (AAAs) was associated with AFB1 excretion in urine (r = 0.394, P < 0.001). Urinary AFB1 was also associated with both the urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG) (r > or = 0.479, P < 0.001) and 8-OHdG and hOGG1 levels in peripheral leukocytes (r > or = 0.308, P < or = 0.005). Similarly, AAA was significantly associated with both the urinary excretion of 8-OHdG (r > or = 0.259, P < or = 0.018) and the 8-OHdG and hOGG1 levels in peripheral leukocytes (r > or = 0.313, P < or = 0.004). In addition, urinary 8-OHdG was correlated with both the level of DNA 8-OHdG (r > or = 0.24, P < or = 0.05) and the expression of hOGG1 in peripheral leukocytes (r > or = 0.429, P < 0.001). Protein carbonyl content (PCC) level was significantly associated with not only the level of DNA 8-OHdG (r > or = 0.366, P < 0.001) and the urinary 8-OHdG (r > or = 0.258, P < or = 0.018) but also the expression of hOGG1 in peripheral leukocytes (r = 0.485, P < 0.001). A significant but weak association was found between high-performance liquid chromatograph-electrochemical detection (HPLC-ECD) and enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG (r = 0.334, P = 0.002) and between HPLC-ECD and flow cytometry assays for 8-OHdG in leucocytes (r = 0.395, P < 0.001). Significant associations were observed between AAA and PCC and liver function indices (alanine aminotransferase and aspartate aminotransferase). These findings suggest significant contribution from AFB1 exposure to oxidative stress and subsequent repair among adolescents that may impose substantial risk for hepatocarcinogenesis in adulthood in this region.

Suppression of hyperemia and DNA oxidation by indomethacin in cerebral ischemia.

We investigated antioxidative activity and the effect of indomethacin, an agent that inhibits cyclooxygenase, on extracellular glutamate and cerebral blood flow in cerebral ischemia in gerbils. Pre-ischemic administration of indomethacin (5 mg/kg, i.p.) significantly rescued hippocampal CA1 neurons (9+/-6 cells/mm in the ischemia, 87+/-43 cells/mm in the indomethacin group, P<0.001). DNA fragmentation induced by ischemia was also examined using the terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) method and indomethacin reduced TUNEL positive cells (140+/-21 in the ischemia, 99+/-31 in the indomethacin group, P<0.01). In addition, indomethacin attenuated the increase in hippocampal blood flow during reperfusion, but not increased extracellular glutamate by ischemia. Eight-hydroxydeoxyguanosine (8-OH-dG), a highly sensitive marker of DNA oxidation, was measured 90 min following ischemia using high-pressure liquid chromatography. Indomethacin significantly decreased the level of ischemia-induced 8-OH-dG in the hippocampus (P<0.05). These results suggest that indomethacin may protect neurons by attenuating oxidative stress and reperfusion injury in ischemic insult.

Analysis of 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) as a reliable marker of cellular oxidative DNA damage after gamma-irradiation.

In order to improve 8-hydroxyguanine (8-OH-Gua) detection in DNA, we digested isolated DNA with nuclease P1 and analyzed for 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The amount of 8-OH-Gua in the DNA was expressed as the ratio of 8-OH-dGMP to deoxycytidine monophosphate (dCMP). Using this analysis, the background level of 8-OH-Gua in DNA from human lung carcinoma cells (A549) was several-fold lower than that obtained by a previous method. A549 cells were exposed to 20-60 Gy of gamma-radiation and an increase in 8-OH-Gua concentration was observed with increasing gamma-ray dose (0.3 residues per 10(7) dCMP per Gy). Moreover, by an immunohistochemical procedure using a commercial FITC-kit, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of gamma-irradiation. Using an endonuclease nicking assay, we also found that gamma-rays decreased 8-OH-Gua repair activity. The results indicate that 8-OH-dGMP is a useful and sensitive marker for estimating oxidative damage in DNA.

Analyses of associations between reactive oxygen metabolites and antioxidant capacity and related factors among healthy adolescents.

Evidence based on epidemiologic investigations using biochemical parameter is meaningful for health promotion and administration among adolescents. We conducted Reactive Oxygen Metabolites (ROM) and Biological Antioxidant Potentials (BAP) tests, along with a questionnaire survey, for a sample of 74 high school students (16.51±0.11 years of aged mean±SE), to investigate the associations between ROM, BAP, and related factors, including BMI and blood biochemical data. Venous blood samples (approximately 7cc) were collected. At the same time, each individual's information was obtained from the questionnaire. The mental health status was investigated using the Center for Epidemiologic Study Depression scale (CES-D) included in the same questionnaire. The mean values and standard errors of all variables were calculated. In addition, the relationships between ROM and BAP with these factors were analyzed. The results revealed the preferred levels of ROM (261.95 ± 9.52 U.CARR) and, BAP (2429.89±53.39 µmol/L) and blood biochemical data. Few significant relationships between two markers and related factors were found. So, we detected a cluster with an imbalance between ROM and BAP, which means low antioxidant ability, whereas the other clusters had conditions with moderate balance or good balance between them. Moreover, we determined the Oxidative stress-Antioxidant capacity ratio (OAR), using the ROM and BAP values, in order to clarify the characteristic of the detected clusters.However, comparative analyses across the three clusters did not yield significant differences in all related factors. No correlations between ROM, BAP and related factors were indicated, although significant association between ROM and BAP was observed (R2=0.1156, R=0.340, P=0.013). The reason for these results can be explained by the influences of good health and young age. On the other hand, present study suggests that some latent problems among adolescents may be related to unhealthy conditions in the future. Also, this study indicated that ROM and BAP may be useful as markers of the oxidative stress status. After this, further investigations using biomarkers based on epidemiologic design should be conducted, to reveal the reliability of the present results.

8-Hydroxyguanine levels and repair capacity during mouse embryonic stem cell differentiation.

To evaluate the defence capacities of embryonic stem (ES) cells against gene impairment, this study measured the levels of 8-hydroxyguanine (8-OH-Gua), a well-known marker of oxidative stress in DNA, and its repair capacity during differentiation. Undifferentiated ES cells (EB3) were cultured without leukaemia inhibitory factor (LIF) for 0, 4 and 7 days and are referred to as ES-D0, ES-D4 and ES-D7, respectively. These three cell lines were treated with 300 µM hydrogen peroxide (H(2)O(2)) for 48 and 72 h. After treatment, the amounts of 8-OH-Gua in the cells were determined by the high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) method. The levels of 8-OH-Gua in ES-D7 treated with H(2)O(2) were higher than those in ES-D0 and ES-D4, suggesting that the DNA in the undifferentiated cells was protected against gene impairment, as compared to that in the differentiated cells. To examine the repair capacity for 8-OH-Gua, this study analysed the expression of 8-OH-Gua repair-associated genes, 8-oxoguanine DNA glycosylase 1 (OGG1), MutY homolog (MUTYH) and Mut T homolog 1 (MTH1), in ES-D0, ES-D4 and ES-D7. The mRNA levels of MUTYH and MTH1 showed no significant change, whereas OGG1 mRNA was significantly decreased in ES-D7 treated with H(2)O(2). Moreover, it was observed that ES-D7 treated with H(2)O(2) readily underwent apoptosis, in comparison to its undifferentiated counterparts, ES-D0 and ES-D4. Taken together, ES cells are more resistant to DNA oxidative stresses than differentiated cells.

Enhancement of adipogenesis induction by conditioned media obtained from cancer cells.

White adipose tissue is a multifunctional endocrine organ that synthesizes and secretes cytokine-like proteins termed adipokines. In the present study, the effects of cancer-derived medium on adipogenesis were examined. We prepared conditioned media from cancer cell lines, and cultured preadipocytes in the conditioned media. After 10 days of culture, intracellular lipid droplet accumulation was measured. We observed that the conditioned media significantly induced adipogenesis or enhanced the adipogenesis induced by adipogenesis inducers. Although we could not define the factors, these undefined factors derived from cancer cells may induce adipogenesis, and the resulting adipogenesis may affect cancer development.

Occupational and lifestyle factors and urinary 8-hydroxydeoxyguanosine.

The amount of 8-hydroxydeoxyguanosine (8-OH-dG) excreted in urine can be used not only as an indicator of DNA repair capacity, but also as a potential marker of oxidative DNA damage. To clarify the oxidation-related factors, in consideration of cancer risk, this study investigated how urinary 8-OH-dG was associated with occupational and lifestyle factors in 372 healthy workers. The creatinine-adjusted urinary 8-OH-dG level was significantly higher in male subjects, smokers and drinkers compared with their counterparts. There were significant positive correlations of the 8-OH-dG level with average number of working hours, involvement in work, average number of cigarettes smoked, average volume of alcohol consumed and serum cortisol level, and there were significant negative correlations of the 8-OH-dG level with body mass index (BMI) and consumption of soybean products, rice and light-colored vegetables. Multiple regression analysis showed that average number of working hours and average number of cigarettes smoked were significant predictors of increased 8-OH-dG levels, whereas being female and BMI were significant predictors of decreased 8-OH-dG levels. Working hours, BMI and smoking were significant predictors of urinary 8-OH-dG in male subjects, whereas age and BMI were significant predictors in female subjects. We suggest that several occupational and lifestyle factors, particularly long working hours and cigarette smoking, are linked to the formation of 8-OH-dG in workers.