Low cadmium exposure in males and lactating females-estimation of biomarkers.

BACKGROUND: Urine cadmium (Cd) and renal function biomarkers, mostly analysed in urine spot samples, are well established biomarkers of occupational exposure. Their use and associations at low environmental level are common, but have recently been questioned, particularly in terms of physiological variability and normalisation bias in the case of urine spot samples. AIM: To determine the appropriateness of spot urine and/or blood Cd exposure biomarkers and their relationships with renal function biomarkers at low levels of exposure. To this end, we used data from Slovenian human biomonitoring program involving 1081 Slovenians (548 males, mean age 31 years; 533 lactating females, mean age 29 years; 2007-2015) who have not been exposed to Cd occupationally. RESULTS: Geometric means (GMs) of Cd in blood and spot urine samples were 0.27ng/mL (0.28 for males and 0.33 for females) and 0.19ng/mL (0.21 for males and 0.17 for females), respectively. Differing results were obtained when contrasting normalisation by urine creatinine with specific gravity. GMs of urine albumin (Alb), alpha-1-microglobulin (A1M), N-acetyl-beta-glucosaminidase (NAG), and immunoglobulin G (IgG) were far below their upper reference limits. Statistical analysis of unnormalised or normalised urine data often yielded inconsistent and conflicting results (or trends), so association analyses with unnormalised data were taken as more valid. Relatively weak positive associations were observed between urine Cd (ng/mL) and blood Cd (ß=0.11, p=0.002 for males and ß=0.33, p<0.001 for females) and for females between urine NAG and blood Cd (ß=0.14, p=0.04). No associations were found between other renal function biomarkers and blood Cd. Associations between Cd and renal function biomarkers in urine were stronger (p<0.05, ß=0.11-0.63). Mostly, all of the associations stayed significant but weakened after normalisation for diuresis. In the case of A1M, its associations with Cd were influenced by current smoking and blood Pb in males and by pre-pregnancy smoking and blood Se in females (ß up to 0.34, p<0.001). Statistical analysis of unnormalised or normalised urine data often yielded inconsistent and conflicting results (or trends), so association analyses data with unnormalised were taken as more valid. CONCLUSIONS: The observed uncertainties introduced by urine normalisation, particularly by creatinine, confirm blood Cd as a superior low-Cd exposure biomarker versus urine Cd in cases when 24h urine is unattainable. Evidence that A1M can be positively related to Cd, smoking (current or pre-pregnancy), Pb, and Se status, points to the versatile biological functions of A1M.

Exposure to mercury in susceptible population groups living in the former mercury mining town of Idrija, Slovenia.

500 years of mercury (Hg) mining in the town of Idrija has caused severe pollution in Idrija and its surroundings. Following the closure of the mine in 1995, the environment remains contaminated with Hg. Sources of elemental-, inorganic- and methyl Hg exposure were identified, potential environmental level of exposure to Hg was evaluated and actual internal exposure to Hg was assessed in selected susceptible population groups comprising school-age children and pregnant women living in Idrija and in control groups from rural and urban environments. The study of pregnant women (n=31) was conducted between 2003 and 2008, and the study of school-age children (n=176) in 2008. Potential interaction of Hg with selenium (Se) in plasma was assessed in both study populations, while in pregnant women antioxidative enzyme activity (glutathione peroxidase, superoxide dismutase and catalase) in erythrocytes of maternal and cord blood was also assessed. Actual exposure to Hg as indicated by levels of Hg in children's blood (geometric mean (GM) 0.92µg/L), mother's blood (GM 1.86µg/L), children's urine (GM 1.08µg/g crea.), mother's urine (GM 2.51µg/L), children's hair (GM 241ng/g) and mother's hair (GM 251ng/g) was higher in the two study groups from Idrija than in the control groups from rural areas, but was still at the level of a "normal" population and reflects mainly exposure to elemental Hg (Hg°) from dental amalgam and, to a certain extent atmospheric Hg°. Furthermore, the internal doses of Hg received during pregnancy did not decrease the bioavailability of Se. Based on observation in children, the increase in Se protein expression is suggested to be a consequence of moderately elevated exposure to Hg°. The observed changes in activity of antioxidative enzymes, as biomarkers of oxidative stress, appear to be mainly associated with pregnancy per se and not with an increased exposure to Hg. In view of the continuing increased potential for Hg exposure and the low number of pregnant women studied, the results warrant a further longitudinal study of a larger group of pregnant women residing in the area of the former mercury mine.

The effect of cardioprotective diet rich with natural antioxidants on chronic inflammation and oxidized LDL during cardiac rehabilitation in patients after acute myocardial infarction.

BACKGROUND: Chronic inflammation, the fundamental pathogenetic process of atherosclerosis, can be modified by pharmacological and non-pharmacological measures as a part of secondary prevention after acute myocardial infarction (AMI). The aim of our study was to determine the effect of diet, rich with natural antioxidants, added to physical activity (as a part of cardiac rehabilitation (CR) program) on inflammatory markers and ox-LDL, a marker of oxidative stress, closely involved in the process of chronic inflammation. METHODS: 41 male patients after AMI undergoing CR were divided into a diet group (supervised cardioprotective diet throughout the CR), and control group (CR without diet). We measured hsCRP, leucocytes, neutrophils, IL-6, oxLDL, exercise capacity and classic risk factors before and after CR program. RESULTS: Patients from the diet group presented with a significant decline in classic risk factors (BMI, waist circumference, waist to hip ratio, systolic blood pressure, heart rate, blood glucose, total cholesterol, LDL, TAG) and inflammatory markers (hsCRP, leucocytes, neutrophils) compared to control group. Furthermore, when studying nonsmokers, we observed significant decline of oxLDL in the diet group. CONCLUSIONS: The addition of cardioprotective diet, rich with natural antioxidants, to physical activity as a part of a CR program, positively modifies not just classic risk factors and exercise capacity, but also diminishes chronic inflammation markers. These effects, and oxLDL decline were most prominent in nonsmoking patients.

A combination of low-dose fluvastatin and valsartan decreases inflammation and oxidative stress in apparently healthy middle-aged males.

PURPOSE: We have previously shown that a "short-term, low-dose" treatment approach with statins, angiotensin receptor blockers, and especially their low-dose combination, is effective in improving arterial wall properties in apparently healthy middle-age men. This study was performed to expand investigation of its effects on inflammation and oxidative stress. METHODS: The study was performed supplementary to 3 previous studies, overall 65 treated participants (25 received fluvastatin 10 mg, 20 valsartan 20 mg, 20 their combination) and 65 participants placebo. The stored blood samples (collected at inclusion and after 30 days of treatment) were used to measure high-sensitivity CRP, interleukin-6, vascular cell adhesion molecule-1, total antioxidant status, glutathione peroxidase, and selenium concentration. RESULTS: A low-dose combination decreased inflammation parameters (high-sensitivity CRP: from 1.2 ± 0.1 to 0.7 ± 0.1 mg/L; P < .001; vascular cell adhesion molecule-1: from 523 ± 21 to 482 ± 12 pg/mL; P < .05; while interleukin-6 did not reach the level of significance). It also increased antioxidant defenses, as measured by total antioxidant status and glutathione peroxidase (from 1.4 ± 0.04 to 1.5 ± 0.04 mmol/L; P < .01, and from 1.2 ± 0.06 to 1.4 ± 0.06 µkat/g hemoglobin; P < .05, respectively), accompanied by decreased selenium levels. Low-dose valsartan was separately less effective than the combination. No changes were observed in the control groups. CONCLUSIONS: Low-dose combination of fluvastatin and valsartan and, to a lesser extent low-dose valsartan alone, produced important anti-inflammatory and anti-oxidative effects. These results confirm and extend the potential of the "short-term, low-dose" preventive strategy.

Influence of short-term cardiac rehabilitation on oxidative stress in men after myocardial infarction depends upon smoking status.

PURPOSE: Oxidative stress is an important nonclassical risk factor for myocardial infarction (MI), and thus, it seems extremely important to recognize factors that effectively reduce it. The aim of our study was to explore possible influences of short-term cardiac rehabilitation (CR) of only 2 weeks in duration on oxidative stress in men after MI. METHODS: Male patients (N = 21; aged 41-88 years, median 56 years), 6 to 8 weeks after acute MI, were included in our observational study using a pretest/posttest design. We investigated markers of oxidative stress and antioxidant enzymes before and after CR of only 2 weeks in duration and influence of smoking status on these differences. RESULTS: We found significant decrease in isoprostanes in urine in nonsmokers (n = 9) (P = .036) but not in smokers (n = 12) (not significant) during CR. After CR, nonsmokers had lower isoprostanes in urine (P = .039), lower non-transferrin-bound iron (P = .020), and higher erythrocyte catalase (P = .023) than smokers. Of classical risk factors, only low-density lipoprotein cholesterol was lower in nonsmokers before (P = .041) and after CR (P = .015) than in smokers. No other significant differences were seen at the beginning or at the end of CR. CONCLUSIONS: To our knowledge, the results of our study indicate for the first time that short-term CR of only 2 weeks in duration already has a positive effect on reduction of oxidative stress in the body. However, this positive effect is seen only in nonsmokers and not in smokers.

Automated assay for non-transferrin-bound iron in serum samples.

BACKGROUND: Non-transferrin-bound iron (NTBI) is a powerful promoter of free radical damage and highly toxic to biological systems, resulting in oxidative damage to proteins, lipids and DNA. METHODS: This assay is based on the binding of serum NTBI by the chelator nitrilotriacetic acid (NTA) and measurement of the ultrafiltrated Fe-NTA complex with the ferrozine reagent kit by a biochemical analyzer. To determine NTBI at extremely low concentrations, the program parameters for serum iron measurement were modified. RESULTS: Linearity was up to 15 µmol/L with analytical recovery of 93%-103%. The limit of detection was 0.076 µmol/L. The within-run coefficient of variation was 2.37%, 1.23%, and 0.812% at concentrations of 0.338, 1.717, and 5.916 µmol/L, respectively. NTBI concentrations measured after exercise in samples obtained from 14 rowers, divided into two groups, were substantially higher in all samples. The median NTBI concentrations (range) before and after exercise were 0.197 (-0.11 to 0.58), and 3.353 (2.39-8.97) µmol/L, respectively, in older rowers and 0.197 (-0.18 to 1.17), and 1.360 (0.47-2.49) µmol/L, respectively, in younger rowers. CONCLUSIONS: With the described modification for serum iron determination, NTBI can be measured with high sensitivity and specificity. The data presented are illustrative examples of the applicability of this assay.

Antioxidant enzymes show adaptation to oxidative stress in athletes and increased stress in hemodialysis patients.

The aim of the study is to compare oxidative stress in hemodialysis patients in controls and in rowers. The patients are a model of decreased antioxidant capacity, and the athletes (rowers) are a model of the highest antioxidant capacity due to their chronic adaptation to demanding training. Thirty-five subjects participated in the study, 9 patients with end-stage renal disease treated by hemodialysis, 12 healthy young subjects from the normal population, and 14 rowers. The antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase, as well as non-transferrin-bound iron as a promoter of free radical damage, were determined. Blood analysis was taken in dialysis patients in the morning, before the dialysis procedure. There was significantly higher activity of catalase in dialysis patients (catalase 4.26 +/- 0.35 mkat/g Hb) compared to the controls (catalase 2.73 +/- 0.38 mkat/g Hb) and rowers (catalase 1.71 +/- 0.30 mkat/g Hb). Superoxide dismutase activity was significantly lower (10.42 +/- 1.46 microkat/g Hb) than in the controls (11.94 +/- 1.18 microkat/g Hb) and rowers (14.09 +/- 0.92 microkat/g Hb). There was no significant differences between glutathione peroxidase activities in the three groups. Superoxide dismutase and Se were higher in rowers than in dialysis patients (P < 0.05). The concentrations of both non-transferrin-bound iron and ferritin were significantly higher in dialysis patients. Hemodialysis patients might have increased oxidative stress, which is characterized by significantly higher erythrocyte enzyme activity of catalase and lower activity of superoxide dismutase. Top rowers had increased superoxide dismutase and glutathione peroxidase, perhaps because of adaptation during training, which was not the case in dialysis patients and controls.

Glutathione level after long-term occupational elemental mercury exposure.

Many in vitro and in vivo studies have elucidated the interaction of inorganic mercury (Hg) and glutathione. However, human studies are limited. In this study, we investigated the potential effects of remote long-term intermittent occupational elemental Hg vapour (Hg degrees ) exposure on erythrocyte glutathione levels and some antioxidative enzyme activities in ex-mercury miners in the period after exposure. The study included 49 ex-mercury miners divided into subgroups of 28 still active, Hg degrees -not-exposed miners and 21 elderly retired miners, and 41 controls, age-matched to the miners subgroup. The control workers were taken from "mercury-free works". Reduced glutathione (GSH) and oxidized disulphide glutathione (GSSG) concentrations in haemolysed erythrocytes were determined by capillary electrophoresis, while total glutathione (total GSH) and the GSH/GSSG ratio were calculated from the determined values. Catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities in erythrocytes were measured using commercially available reagent kits, while urine Hg (U-Hg) concentrations were determined by cold vapour atomic absorption (CVAAS). No correlation of present U-Hg levels, GSH, GSSG, and antioxidative enzymes with remote occupational biological exposure indices were found. The mean CAT activity in miners and retired miners was significantly higher (p<0.05) than in the controls. No differences in mean GPx activity among the three groups were found, whereas the mean GR activity was significantly higher (p<0.05) in miners than in retired miners. The mean concentrations of GSH (mmol/g Hb) in miners (13.03+/-3.71) were significantly higher (p<0.05) than in the control group (11.68+/-2.66). No differences in mean total GSH, GSSG levels, and GSH/GSSG ratio between miners and controls were found. A positive correlation between GSSG and present U-Hg excretion (r=0.41, p=0.001) in the whole group of ex-mercury miners was observed. The significantly lower GSH level (p<0.05) determined in the group of retired miners (9.64+/-1.45) seems to be age-related (r= -0.39, p=0.001). Thus, the moderate but significantly increased GSH level, GR and CAT activity in erythrocytes in the subgroup of miners observed in the period after exposure to Hg degrees could be an inductive and additive response to maintain the balance between GSH and antioxidative enzymes in interaction with the Hg body burden accumulated during remote occupational exposure, which does not represent a severely increased oxidative stress.

The impact of long-term past exposure to elemental mercury on antioxidative capacity and lipid peroxidation in mercury miners.

Limited information is available on the effects of chronic mercury exposure in relation to the risk of cardiovascular disease (CVD). It is known from in vitro and in vivo studies that Hg can promote lipid peroxidation through promotion of free radical generation, and interaction with antioxidative enzymes and reduction of bioavailable selenium. The objective of the study was to test the hypothesis that long-term past occupational exposure to elemental Hg (Hg0) can modify antioxidative capacity and promote lipid peroxidation in miners. The study population comprised 54 mercury miners and 58 workers as the control group. The miners were examined in the post-exposure period. We evaluated their previous exposure to Hg0, the putative appearance of certain nonspecific symptoms and signs of micromercurialism, as well as the main behavioural and biological risk factors for CVD, and determined: 1) Hg and Se levels in blood and urine, 2) antioxidative enzymes, Cu/Zn superoxide dismutase (CuZn-SOD), catalase (CAT), and selenoenzyme glutathione peroxidase (GSH-Px) activity in erythrocytes as indirect indices of free radical activity, 3) pineal hormone melatonin (MEL) in blood and urine, and 4) lipid hydroperoxides (LOOHs) and malondialdehyde (MDA) as lipid peroxidation products. The mercury miners were intermittently exposed to Hg0 for periods of 7 to 31 years. The total number of exposure periods varied from 13 to 119. The cumulative U-Hg peak level varied from 794-11,365 microg/L. The current blood and urine Hg concentrations were practically on the same level in miners and controls. Miners showed some neurotoxic and nephrotoxic sequels of micromercurialism. No significant differences in behavioural and biological risk factors for CVD were found between miners and controls. A weak correlation (r = 0.36, p < 0.01) between systolic blood pressure and average past exposure U-Hg level was found. The mean P-Se in miners (71.4 microg/L) was significantly lower (p < 0.05) than in the controls (77.3 microg/L), while the mean U-Se tended to be higher (p < 0.05) in miners (16.5 microg/g creatinine) than in the controls (14.0 microg/g creatinine). Among antioxidative enzyme activities, only CAT in erythrocytes was significantly higher (p < 0.01) in miners (3.14 MU/g Hb) than in the controls (2.65 MU/g Hb). The mean concentration of B-MEL in miners (44.3 ng/L) was significantly higher (p < 0.01) than in the controls (14.9 ng/L). The mean value of U-MEL sulphate (31.8 microg/L) in miners was significantly lower (p < 0.01) than in the control group (46.9 microg/L). Among the observed lipid peroxidative products, the mean concentration of U-MDA was statistically higher (p < 0.01) in miners (0.21 micromol/mmol creatinine) than in the controls (0.17 micromol/mmol creatinine). In the group of miners with high mercury accumulation and the presence of some nonspecific symptoms and signs of micromercurialism, the results of our study partly support the assumption that long-term occupational exposure to Hg0 enhances the formation of free radicals even several years after termination of occupational exposure. Therefore, long-term occupational exposure to Hg0 could be one of the risk factors for increased lipid peroxidation and increased mortality due to ischaemic heart disease (ICH) found among the mercury miners of the Idrija Mine.