8-Hydroxy-2'-deoxyguanosine as a marker of oxidative DNA damage related to occupational and environmental exposures.

Oxidative DNA damage is considered to play an important role in pathophysiological processes, ageing and cancer. So far major interest has been on measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG), the preferred methods relying on HPLC or GC-mass spectrometry. The high biological relevance of 8-OHdG is due to its ability to induce G-->T transversions, which are among the most frequent somatic mutations found in human cancers. Effects of workplace exposures on the level of white blood cell 8-OHdG or urinary 8-OHdG have been reported with controversial results. Exposures examined include asbestos, azo-dyes, benzene, fine particulate matter (PM(2.5)), glassworks, polycyclic aromatic hydrocarbons (PAHs), rubber manufacturing, silica, metals, styrene, toluene and xylenes. The available data indicate that there is still a lack of well established dose-response relations between occupational or environmental exposures and the induction of 8-OHdG. Smoking has been most consistently identified as a confounder for 8-OHdG, but various occupational studies did not reveal higher levels of 8-OHdG in smokers. Despite the conflicting results, the reported studies show promise for 8-OHdG as a biomarker of oxidative stress associated with chemical exposure. However, there are critical aspects related to the analytical challenge, artifactual production of 8-OHdG, inter- and intra-individual variation, confounding factors and inter-laboratory differences, implying that further work is needed to reach a consensus on the background level of 8-OHdG.

Functional adrenergic receptor polymorphisms and idiopathic orthostatic intolerance.

OBJECTIVES: Idiopathic orthostatic intolerance (IOI) is a common disorder that is characterized by chronic orthostatic symptoms and substantial increases in heart rate and plasma norepinephrine concentrations that are disproportionately high while standing. Several features of the syndrome, including the tachycardia, tremulousness, and exaggerated norepinephrine have been considered potentially due to hypoactive or hyperactive states of adrenergic receptors of the sympathetic nervous system. The aim of this study was therefore to ascertain whether genotypes at eight polymorphic loci within five relevant adrenergic receptor genes (alpha2A, alpha2B, alpha2C, beta1 and beta2) influence the risk for IOI. METHODS: We studied 80 young men in military service (20 patients with IOI and 60 age-matched controls). All participants underwent a tilt table test including monitoring of blood pressure, heart rate and plasma catecholamines, in the supine position and during 30 min of standing. Genotyping at the eight loci (alpha2ALys251, alpha2BDel301-303, alpha2CDel322-325, beta1Gly49, beta1Arg389, beta2Arg16, beta2Glu27, beta2Ile164) was performed in all participants. Chi-square tests of independence were used to test for associations between IOI and genotype. In addition, an association of the polymorphisms with haemodynamic variables (heart rate, supine and upright blood pressure) was ascertained using one-way variance analysis. RESULTS: For the beta1Gly49 polymorphism we found a decrease in the risk of IOI among persons who were homozygous (odds ratio, 0.88; 95% confidence interval, 0.81-0.97). In addition, we found an association between beta1Gly49 and decreased heart rate in the upright position, regardless of IOI diagnosis. There were no associations with the other studied polymorphisms and IOI. CONCLUSIONS: Our current results suggest that the beta1Gly49 polymorphism is protective for IOI. This is likely one of several common genetic loci that may represent modifiers of IOI phenotypes.

Diagnostic management of orthostatic intolerance in the workplace.

OBJECTIVE: Orthostatic intolerance (OI) is a syndrome that is characterised by headache, concentration difficulties, palpitation of the heart, dizziness associated with postural tachycardia and plasma norepinephrine concentrations that are disproportionately high when the sufferer is in the upright posture. In contrast to other forms of orthostatic dysregulation - orthostatic hypotension (OH) and postural orthostatic tachycardia syndrome (POTS) - OI, hitherto, could be diagnosed only by a tilt table examination, with high expenditure. In this paper we examine the reliability and validity of a questionnaire as a screening instrument for OI. METHODS: We studied 138 young men (mean age 21.6 years) who were undergoing military service. After a medical check and filling in the questionnaire, the participants underwent a tilt table test including monitoring of blood pressure, heart rate and plasma catecholamines, in the supine position and during 30 min of standing. The questionnaire consisted of ten items registering presence and frequency of typical OI symptoms. RESULTS: Probands (104) showed normal tilt table test results. OI was diagnosed in 14 probands, OH in 6 and POTS in 14. The OI participants scored significantly higher in the questionnaire than the healthy subjects did: the mean score of the OI group was 22.6, the healthy participants had a mean score of 3.9. Participants with POTS had a mean score of 13.5 and subjects with OH had a mean score of 17.0. Reliability analysis showed a Cronbach's alpha of 0.888. Validity analysis showed that 93.5% of the probands with any kind of orthostatic dysregulation can be detected. CONCLUSIONS: We were able to establish a short questionnaire as a reliable and valid screening instrument for OI. Usage of this questionnaire can simplify enormously the diagnostic management of patients with suspected OI.

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.

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 intra-individual coefficient of variation (CV) of urinary 8-OHdG/24 h ranged from 0.18 to 1.06 (mean CV = 0.48). Women excreted significantly lower amounts of 8-OHdG/24 h 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/24 h excretion and urinary creatinine (rp = 0.61), and cotinine (rp = 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 mumol/mol) opposed to 45 non-smoking probands (1.62 +/- 0.50 mumol/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 intra-individual 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.

Long-term monitoring of sister chromatid exchanges and micronucleus frequencies in pharmacy personnel occupationally exposed to cytostatic drugs.

OBJECTIVES: Many antineoplastic drugs were found to have carcinogenic, mutagenic and teratogenic potential. The aim of this study was to carry out cytogenetic and internal dose monitoring of hospital pharmacy personnel regularly involved in the preparation of cytostatic agents, in order to test possible cytostatics-induced genotoxic effects due to occupational exposure under routine working conditions, and in cases of accidental contamination. METHODS: Platinum in whole blood and anthracyclines in plasma were measured to assess internal exposure to cytostatics. The level of cytogenetic damage was determined in peripheral blood lymphocytes with the micronucleus test and the sister chromatid exchange assay. Five series of monitoring were performed over a period of 2 years. RESULTS: No significant differences in the mean frequencies of sister chromatid exchanges (SCE) and micronuclei (MN) were found between occupationally exposed probands and controls (9.9 +/- 1.4 vs 10.1 +/- 1.2 SCEs/cell and 21.2 +/- 7.2 vs 23.3 +/- 7.5 MN/2000 binucleated (BN) cells, n = 16). Significant elevations of SCE or MN were detected in seven out of 12 cases of accidental contamination at the workplace, whereas no increase in platinum in blood and anthracyclines in plasma was observed in these probands. Two cases of non-reported contamination were identified by measurement of epirubicin in plasma. Smoking was found to increase the SCE significantly. No correlation between individual SCE scores and MN scores was observed. CONCLUSIONS: Our findings support a transient increase in SCE or MN after relevant exposure to cytostatic drugs in cases of accidental contamination. The lack of significant differences in SCE and MN between hospital pharmacy personnel and unexposed controls, points to high standards of safety at the corresponding workplaces.

8-Hydroxydeoxyguanosine in leukocyte DNA and urine of quartz-exposed workers and patients with silicosis.

OBJECTIVE: To examine radical-induced DNA damage and its elimination in workers exposed to quartz and in patients with silicosis, and to assess the relationship of these effects to lung function. METHODS: Blood and spontaneous urine samples were obtained from active, quartz-exposed workers without silicosis (n = 63), and from retired workers with silicosis (n = 42). Levels of 8-hydroxydeoxyguanosine (8-OHdG) were determined in peripheral blood leukocyte DNA and urine, by the use of high-performance liquid chromatography coupled with ultra violet- (UV) and electrochemical detection. RESULTS: No significant differences in the mean levels of 8-OHdG in leukocyte DNA and of urinary excretion of 8-OHdG were found between silicosis patients and quartz-exposed healthy workers. However, in the group of silicosis patients with increased oxidative DNA damage the urinary excretion of 8-OHdG was lower than in the corresponding group of active workers without silicosis. In the case of silicosis, urinary 8-OHdG correlated positively, and 8-OHdG in DNA correlated negatively, with forced expiratory volume in one second (FEV1) and forced vital capacity (FVC). Healthy workers with a personally estimated high dust exposure in the workplace showed higher levels of 8-OHdG in DNA than did workers with moderate dust exposure. No association of 8-OHdG formation and/or elimination with duration of employment, field of activity, smoking or age was found. CONCLUSION: Our findings suggest that a less effective repair of 8-OHdG is associated with a higher degree of pulmonary airway obstruction in patients with silicosis.

Radiation synovectomy using 165Dy ferric-hydroxide and oxidative DNA damage in patients with different types of arthritis.

UNLABELLED: Radiation synovectomy is an effective treatment for chronic synovitis refractory to pharmacological treatment in patients with rheumatoid or seronegative arthritis. Concerns persist about possible radiation-induced cytogenetic damage after radiation synovectomy leading to recommendations to use this technique only in the elderly. Micronucleus (MN) frequency in lymphocytes and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8OHdG) as an indicator of cellular oxidative DNA base damage are biomarkers of radiation-induced cytogenetic damage. The course of both biomarkers was studied in patients with different types of chronic synovitis undergoing radiation synovectomy with very short-lived 165Dy-ferric-hydroxide (DFH). METHODS: Radiation synovectomy of the knee was performed in 13 men and 12 women (mean age, 44+/-15 y) using a mean activity of 9.48+/-1.65 GBq 165Dy-DFH in 27 consecutive treatments. MN frequency in lymphocytes and urinary excretion of 8OHdG, measured by high-performance liquid chromatography, were assessed before and 4 (MN only) and 20 h after radiation synovectomy. RESULTS: Urinary excretion of 8OHdG in patients (in micromol/mol creatinine; pretreatment mean, 3.1+/-3.4; median, 2.27) was not significantly different from that in healthy volunteers (mean, 2.0+/-1.2; median, 1.87) and not altered by radiation synovectomy (post-treatment mean, 2.5+/-1.5; median, 2.04, NS). An increase in 8OHdG levels after radiation synovectomy of more than 1 SD was found in only 1 patient, who experienced leakage to the lymph nodes but who already had elevated urinary 8OHdG levels before treatment. The frequency of MN/500 binucleated cells (BNCs) was slightly lower in patients (pretreatment mean, 4.3+/-2.6; median, 4.25) than in healthy volunteers (mean, 5.4+/-2.3; median, 5.3) and did not significantly change after therapy, either (4-h post-treatment mean, 3.9+/-2.1, median, 3.8; 20-h post-treatment mean, 4.1+/-2, median 3.8 MN/500 BNC). In 22 of 27 treatments, no leakage to nontarget organs could be monitored, whereas leakage to the local lymph nodes and the liver was detected after 5 treatments. CONCLUSION: Radiation synovectomy using 165Dy-DFH causes no significant radiation burden to most patients as indicated by the absence of adverse changes in levels of biomarkers of cytogenetic damage and a low incidence of leakage. These data suggest that the risk of malignancy may not be elevated.

Increased blood cobalt and chromium after total hip replacement.

OBJECTIVE: To determine metal concentrations in blood and urine of patients who received cobalt-chromium-alloy metal on metal hip implants. METHODS: Cobalt and chromium were determined in blood and urine of 76 patients and 26 controls by electrothermal atomic absorption spectroscopy. RESULTS: A significant postoperative elevation of the metal concentrations was observed for total hip replacement patients in contrast to the control group. Twenty-nine patients exceeded the EKA (Expositionäquivalente für Krebserzeugende Arbeitsstoffe) threshold limits for cobalt in blood and for cobalt and chromium in urine. We obtained a significant correlation between cobalt in blood and cobalt in urine (r = 0.79; p < 0.005), chromium in blood and chromium in urine (r = 0.79; p < 0.005), cobalt in blood and chromium in blood (r = 0.69; p = 0.008), and cobalt in urine and chromium in urine (r = 0.95; p = 0.004). CONCLUSION: Our findings suggest that in total hip replacements using metal-metal pairings, metal ions of the alloys are released. This release may lead to significantly elevated metal concentrations in biological fluids. Long-term studies are needed to determine the risk of metal-metal implants as a potential cause of cobalt and chromium toxicity.

Exercise-induced DNA effects in human leukocytes are not accompanied by increased formation of 8-hydroxy-2'-deoxyguanosine or induction of micronuclei.

The present study examined the effects of a short-distance triathlon on the induction of DNA effects in peripheral leukocytes, urinary excretion of oxidized DNA bases, and frequency of micronuclei in lymphocytes of human volunteers. Induction of DNA effects was measured as increased DNA migration using the alkaline comet assay. Increased DNA migration was found in leukocytes of all individuals at different time points after exercise and revealed a biphasic pattern. Twenty-four hours postexercise, elevated DNA migration was found, whereas lower values were detected 48 h after exercise. Seventy-two hours postexercise, the maximum increase in DNA migration was found and baseline values were still elevated after 120 h. A modified protocol of the comet assay for the detection of oxidized DNA bases revealed no differences in leukocytes before and directly after the triathlon. Urinary excretion of 8-hydroxy-2'-deoxyguanosine remained unaltered during the 5 consecutive days sampled. No differences were found in the micronucleus-frequency in lymphocytes before or 48 and 96 h after exercise. Our data suggest that DNA effects detected with the comet assay in leukocytes of humans after exercise are secondary effects that do not originate from oxidized DNA bases and do not result in chromosome damage.

Assay for the determination of urinary 8-hydroxy-2'-deoxyguanosine by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic procedure with electrochemical detection is described for the determination of urinary 8-hydroxy-2'-deoxyguanosine, a major oxidative DNA lesion induced by radical forming agents. A two-step solid-phase extraction procedure was followed for extracting 8-hydroxy-2'-deoxyguanosine from human urine and the analysis was performed on a RP-18 analytical column under isocratic conditions. The limit of detection of 8-hydroxy-2'-deoxyguanosine in urine was found to be 0.9 nM. The non-invasive assay provides an indirect measurement of oxidative DNA damage.

Habitual long-distance running does not enhance urinary excretion of 8-hydroxydeoxyguanosine.

The energy demand during physical exercise causes an increased oxygen uptake and supply to active tissues, which may increase the rate of free oxygen radical production and thereby affect the capacity of endogenous cellular defense systems. This could result in DNA base modifications, among which 8-hydroxydeoxyguanosine (8OHdG) is one of the most important and has widely been used as a biomarker of in vivo oxidative lesions. Therefore, we examined the effect of regular running exercise on the urinary levels of 8OHdG in 32 long-distance runners and in a group of untrained healthy subjects. The range of 8OHdG in urine was 0.12-6.45 mumol/mol creatinine in both groups, and no significant difference in the mean excretion levels between runners and control probands was observed. This gives no reason to believe that physical exercise in trained individuals may induce a disturbance of the oxidant-to-antioxidant balance.

Measurement of intravesicular volumes by salt entrapment.

Internal volume is a very sensitive parameter of vesicle morphology. Measurement of captured volumes by solute entrapment is legitimate for most types of vesicles (Perkin, W.R. et al. (1993) Chem. Phys. Lipids 64, 197-217). In this study chloride was selected as the most convenient marker ion because the ubiquity of Cl- in physiological buffers eliminates prelabeling with exogenous markers and because minute concentrations of trapped chloride are well detectable in the presence of large extravesicular nitrate concentrations. Perfect exchange of external chloride for nitrate was shown to be accomplished by gel filtration, dialysis, or sucrose gradient flotation-but only after significant technical improvements and/or elimination of experimental pitfalls. Reliability was cross-checked by simultaneous entrapment of Cl- and K+. Diafiltration and ion exchange chromatography appeared inapplicable for exchange of extravesicular salt. When a representative variety of vesicle preparations was analyzed for internal volume (as well as for external surface and size) unexpected features of vesicle morphology were discovered. This emphasizes the genuine role of macroscopic vesicle characterization in complementing information from electron microscopy.

[Flurbiprofen and indomethacin in chronic polyarthritis and gonarthrosis - a comparative multicentric double-blind cross-over study]. / Flurbiprofen und Indometacin bei chronischer Polyarthritis und Gonarthrose --eine vergleichende multizentrische Doppelblind-cross-over-Studie.

In two multicenter double-blind cross-over studies efficacy and safety of flurbiprofen and indomethacin were compared. Nineteen patients with rheumatoid arthritis and twenty with osteoarthrosis of the knee were treated. Both drugs were effective and almost equal. Also safety was good, however three patients on indomethacin and one on flurbiprofen had to be withdrawn from the trial because of side-effects.

[Case report of a primary pericollagen amyloidosis (author's transl)]. / Zur klinischen Diagnose der primär perikollagenen Amyloidose

The authors report about a female patient, 59 years old, who was suffering from a stenosis of the cardia. A malignant process was suspected and an operative treatment was planed. During the preoperative internal examinations a low voltage in the ECG was found so that an amyloidosis was suspected. This was proved by biopsy of the mucosa of the rectum. A chronic suppurative disease could not be found, it was then assumed that the patient is suffering from a "primary pericollagen amyloidosis". Because a splitting of the stenosis through the esophagoscope the biopsy showed no malignancy, but congo-red positive material around collagen tissue. This seldom disease, the differential-diagnosis and the possibilities of treatment are discussed. Under symptomatic therapy the patient is doing well 2 years since.