Answer to comments by A. Lerchl on "Radiofrequency electromagnetic fields (UMTS, 1,950 MHz) induce genotoxic effects in vitro in human fibroblasts but not in lymphocytes" published by C. Schwarz et al. 2008.

Genotoxic effects induced in vitro by the third generation mobile communication standard UMTS have recently been described by Schwarz et al. (Int Arch Occup Environ Health 81:755-767, 2008). These findings which may have considerable significance for environmental health have been commented upon by Lerchl (Int Arch Occup Environ Health in press, 2008) (this issue). These comments which are invalid in part have to be set right. Although some of his minor points are correct the objected inconsistencies are largely based on the author's incomplete and superficial consideration of published data in the field. Moreover, the statistical points being made cannot cast doubts on the validity of the experimental data reported by Schwarz et al. and may not change the principal conclusion of in vitro genotoxic action of UMTS signals.

Longitudinal biomonitoring of nurses handling antineoplastic drugs.

AIMS AND OBJECTIVES: To assess a possible trend in the genotoxic risk of oncologic nurses during the working year, cytogenetic biomonitoring was performed. BACKGROUND: Exposure to cytostatic agents is a major occupational concern in oncologic personnel. In contrast to the controlled environment in oncology pharmacies, nurses may be subject to unexpected events of exposure due to the intensive contact with patients. DESIGN AND METHODS: The entire nursing staff of an oncology inpatient ward (n = 15) participated in a biomonitoring study over a period of nine months using the sister chromatid exchange test and the comet assay to detect DNA strand breaks. Blood samples were taken after a three-week summer break (base level), one, three, six and nine months thereafter. Airborne contaminations of cytotoxics were addressed by chromatographic methods. RESULTS: With regard to the single monitoring points, the comet assay revealed no significant alteration of the genotoxic burden within nine months. By contrast, the sister chromatid exchange levels were significantly increased after six and nine months when compared with base levels. A trend analysis covering the whole observation period revealed an increase in genotoxicity as shown by the sister chromatid exchange test and the alkaline but not the neutral comet assay. This increase, however, was small and reversible as shown by the trend analysis of sister chromatid exchange rates during the years of service. Air samples were negative for cytotoxic contaminants. CONCLUSIONS AND RELEVANCE TO CLINICAL PRACTICE: The small, but statistically significant genotoxic burden observed in oncologic nurses of an inpatient ward emphasises the need for a continuing effort to eliminate residual occupational risks. In comparison with historical controls, the current situation is characterised by beneficial safety improvements over the last years. Nevertheless, periodic training and awareness of the problems should be an integral part of advanced education.

Radiofrequency electromagnetic fields (UMTS, 1,950 MHz) induce genotoxic effects in vitro in human fibroblasts but not in lymphocytes.

OBJECTIVE: Universal Mobile Telecommunication System (UMTS) was recently introduced as the third generation mobile communication standard in Europe. This was done without any information on biological effects and genotoxic properties of these particular high-frequency electromagnetic fields. This is discomforting, because genotoxic effects of the second generation standard Global System for Mobile Communication have been reported after exposure of human cells in vitro. METHODS: Human cultured fibroblasts of three different donors and three different short-term human lymphocyte cultures were exposed to 1,950 MHz UMTS below the specific absorption rate (SAR) safety limit of 2 W/kg. The alkaline comet assay and the micronucleus assay were used to ascertain dose and time-dependent genotoxic effects. Five hundred cells per slide were visually evaluated in the comet assay and comet tail factor (CTF) was calculated. In the micronucleus assay 1,000 binucleated cells were evaluated per assay. The origin of the micronuclei was determined by fluorescence labeled anticentromere antibodies. All evaluations were performed under blinded conditions. RESULTS: UMTS exposure increased the CTF and induced centromere-negative micronuclei (MN) in human cultured fibroblasts in a dose and time-dependent way. Incubation for 24 h at a SAR of 0.05 W/kg generated a statistically significant rise in both CTF and MN (P = 0.02). At a SAR of 0.1 W/kg the CTF was significantly increased after 8 h of incubation (P = 0.02), the number of MN after 12 h (P = 0.02). No UMTS effect was obtained with lymphocytes, either unstimulated or stimulated with Phytohemagglutinin. CONCLUSION: UMTS exposure may cause genetic alterations in some but not in all human cells in vitro.

Effect of occupational safety measures on micronucleus frequency in semiconductor workers.

OBJECTIVES: To examine whether semiconductor workers exposed to complex mixtures of chemical waste show an increase in genotoxic effects, and, if so, whether occupational safety measures protect these workers. METHODS: To assess chemical exposure in the workplace, air monitoring of boron trifluoride and boron trichloride was performed and urinary concentrations of fluoride were measured. The cytokinesis-block micronucleus test on isolated lymphocytes was used for the detection of genotoxic effects. Two series of monitoring have been performed in order to assess the effect of implemented protection measures. RESULTS: We found a significantly higher mean frequency of micronuclei in exposed workers than in controls, whereas air monitoring and measurement of urinary fluoride failed to detect chemical exposure of these workers. Twelve years after implementation of protective measures, the mean level of micronuclei in exposed individuals was found to be as low as those from controls. CONCLUSIONS: These findings indicate that exposed workers in the semiconductor industry may have an increased risk of genotoxic effects from complex mixtures of chemical waste products. The decline of the mean level of micronuclei in exposed workers down to the base level of controls after implementation of protective measures points to the significance of adequate safety standards to protect against chromosomal damage in semiconductor personnel.

Influence of an insertion variant in the 5'UTR of the endothelin-1 gene on orthostatic intolerance.

BACKGROUND: Orthostatic intolerance is a multifactorial disease in which the genetic contribution is probably the result of a number of genes acting in combination. Recent work has shown that orthostatic intolerance is influenced by endothelial nitric oxide synthase gene polymorphisms. Since endothelin-1 (ET-1) is one of the most important vasoconstrictor peptides, a frequent adenine insertion polymorphism within the 5'-untranslated region (5'UTR), which is of functional importance for ET-1 expression, could influence orthostatic intolerance. The aim of this study was therefore to ascertain whether this frequent variant of the endothelin-1 gene influences the risk for orthostatic intolerance. METHODS: We studied 257 white patients (120 cases with orthostatic intolerance and 137 controls) for genotyping of the 5'UTR I variant. From this cohort, 111 patients and 99 control subjects underwent a tilt-table test or an upright posture study, including monitoring of blood pressure, heart rate, and plasma catecholamines, in the supine position and during 30 minutes of standing. Genotyping was performed in all participants. Chi tests of independence were used to test for associations between orthostatic intolerance and genotype. In addition, an association of the insertion polymorphism with hemodynamic variables (heart rate, supine and upright blood pressure) was ascertained using one-way analysis of variance. RESULTS: The 5'UTR I variant was significantly less common in patients with orthostatic intolerance (allele frequency 0.36 and 0.28, in controls and cases, respectively). Additionally, we found a significant decrease in the risk of orthostatic intolerance among people who were homozygous for the 5'UTR variant (I/I) compared with the wild-type variant (D/D) (odds ratio, 0.41; 95% confidence interval, 0.17 to 0.97; P = 0.04). No association between the 5'UTR variant and heart rate or blood pressure regardless of diagnosis was found. CONCLUSIONS: Our current results suggest that the hereditary adenine insertion variant in the 5'-UTR of the endothelin-1 gene is protective for orthostatic intolerance. The increased ET-1 protein expression that has been linked with the I variant might be associated with a more efficient hemodynamic response to standing. This is likely one of several common genetic loci that may represent modifiers of orthostatic intolerance phenotypes.

Schellong test in orthostatic dysregulation: a comparison with tilt-table testing.

OBJECTIVE: To evaluate the role of the Schellong test (ST) in forms of orthostatic dysregulation in comparison with the tilt-table test (TT). METHODS: 67 young males (mean age 22 +/- 4 years) from the military service, representing two different cohorts, were examined by ST and TT, which served as gold standard. 32 of the 67 subjects were asymptomatic while 35 had sought medical advice because of orthostatic complaints. The subjects subsequently were classified into four categories according to the TT: normal TT, orthostatic hypotension (OH), postural orthostatic tachycardia syndrome (POTS), and neurocardiogenic syncope (NCS). Chi-square test was used to calculate the sensitivity and specificity of ST in detecting forms of orthostatic dysregulation (OH, POTS and NCS). RESULTS: In total, TT detected 23 recruits with POTS, 16 with NCS and 2 with OH. Out of the 32 asymptomatic subjects only one was diagnosed having POTS by TT and ST, the rest had a normal ST and TT. For detecting POTS, ST sensitivity was 61% and specificity was 100% compared with TT. For detecting NCS, ST sensitivity was 31% and specificity 100% compared with the reference test, the TT. The data concerning OH could not be analyzed because of the small number of cases. CONCLUSIONS: In conclusion the results of our study indicate that ST can be used in first line in the diagnosis of patients with orthostatic symptoms by the medical practitioner. If the ST is normal, further examination by TT is indispensable, because sensitivity of ST concerning POTS and NCS is relatively low.

Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields.

The issue of adverse health effects of extremely low-frequency electromagnetic fields (ELF-EMFs) is highly controversial. Contradictory results regarding the genotoxic potential of ELF-EMF have been reported in the literature. To test whether this controversy might reflect differences between the cellular targets examined we exposed cultured cells derived from different tissues to an intermittent ELF-EMF (50 Hz sinusoidal, 1 mT) for 1-24h. The alkaline and neutral comet assays were used to assess ELF-EMF-induced DNA strand breaks. We could identify three responder (human fibroblasts, human melanocytes, rat granulosa cells) and three non-responder cell types (human lymphocytes, human monocytes, human skeletal muscle cells), which points to the significance of the cell system used when investigating genotoxic effects of ELF-EMF.

Endurance exercise training in orthostatic intolerance: a randomized, controlled trial.

Orthostatic intolerance is a syndrome characterized by chronic orthostatic symptoms of light-headedness, fatigue, nausea, orthostatic tachycardia, and aggravated norepinephrine levels while standing. The aim of this study was to assess the protective effect of exercise endurance training on orthostatic symptoms and to examine its usefulness in the treatment of orthostatic intolerance. 2768 military recruits were screened for orthostatic intolerance by questionnaire. Tilt-table testing identified 36 cases of orthostatic intolerance out of the 2768 soldiers. Subsequently, 31 of these subjects with orthostatic intolerance entered a randomized, controlled trial. The patients were allocated randomly to either a "training" (3 months jogging) or a "control" group. The influence of exercise training on orthostatic intolerance was assessed by determination of questionnaire scores and tilt-table testing before and after intervention. After training, only 6 individuals of 16 still had orthostatic intolerance compared with 10 of 11 in the control group. The Fisher exact test showed a highly significant difference in diagnosis between the 2 groups (P=0.008) at the end of the study. Analysis of the questionnaire-score showed significant interaction between time and group (P=0.001). The trained subjects showed an improvement in the average symptom score from 1.79+/-0.4 to 1.04+/-0.4, whereas the control subjects showed no significant change in average symptom score (2.09+/-0.6 and 2.14+/-0.5, respectively). Our data demonstrate that endurance exercise training leads to an improvement of symptoms in the majority of patients with orthostatic intolerance. Therefore, we suggest that endurance training should be considered in the treatment of orthostatic intolerance patients.

No cognitive deficits in men formerly exposed to lead.

OBJECTIVES: The objective of the study was to investigate long-term cognitive effects resulting from low to moderate lead exposure below current threshold values. Executive functions, attention, visuospatial and visuomotor functioning in workers formerly exposed to lead were investigated. METHODS: 48 men formerly exposed to lead and with a mean current blood level (PbB) of 5.4 microg Pb/100 ml were investigated, together with 48 matched controls (PbB, 4.7 microg Pb/100 ml) out of a pool of 61 males. The two groups were matched for age, years spent in education, verbal intelligence and gram alcohol consumption per week. The following neuropsychological tests were used: modified Wisconsin card sorting test, block design test, visual recognition test, simple reaction time, choice reaction and digit-symbol substitution. Lead exposure was assessed using both current and cumulative measurements. RESULTS: There were no significant differences in cognitive parameters between the two groups. When analyzing dose-response relationships, negative correlations were found between PbB and performance in the block design test, and between PbB and scores in the visual recognition and digit-symbol substitution tests. High cumulative exposure (IBL, >5000; duration of exposure, >5 years) correlated only with wrong reactions in the choice reaction test. CONCLUSIONS: The results of our study indicate that cognitive deficits resulting from low-level exposure to lead are reversible. The study was limited to low-level long-term exposure (all PbB values were always below 55 microg Pb/100 ml), and extrapolation of these results to persons heavily exposed to lead is not possible.

No effects of intermittent 50 Hz EMF on cytoplasmic free calcium and on the mitochondrial membrane potential in human diploid fibroblasts.

The recently described increase in DNA strand breaks of cultured human diploid fibroblasts after intermittent exposure to extremely-low-frequency electromagnetic fields (ELF-EMF) of more than about 70 microT ELF-EMF is difficult to explain by a direct induction of covalent bond disruption. Therefore the hypothesis has been tested that ELF-EMF-induced DNA strand breaks might be mediated by cellular processes that cause alteration of the intracellular concentration of free calcium ([Ca2+]i) and/or the membrane potential (DeltaPsi(m)). [Ca2+]i was determined by the ratiometric fura-2 technique. Changes in DeltaPsi(m) were assessed by using the potential-dependent lipophilic cationic probe JC-1. Human fibroblasts were exposed to intermittent ELF-EMF (50 Hz, 1000 microT). Although exposure of fiboblasts to ELF-EMF resulted in a highly significant increase in DNA strand breaks as determined by the comet assay, no effect on JC-1 fluorescence emission or on [Ca2+]i has been observed when comparing exposed with sham-exposed cells. Therefore, it is suggested that ELF-EMF-induced DNA strand breaks are unlikely to be caused by intracellular changes that affect [Ca2+]i and/or DeltaPsi(m).

High prevalence of chronic venous disease in hospital employees.

OBJECTIVES: Chronic venous disease (CVD), which comprises primary/idiopathic abnormalities of the venous system, and secondary sequels after deep venous thrombosis are major health issues in Western countries. The present study was conducted to prove the hypothesis that the development of CVD might be triggered by exogenous, occupational risk factors. METHODS: We determined the prevalence and social relationship of CVD in a wide cross-section of hospital employees (n = 209; medical doctors and nurses, medical technician assistants, secretaries, scientific staff, cleaners and utility workers) without predocumented CVD. Prevalence, known endogenous risk factors for CVD and occupational and environmental risk factors (family history of venous disease, history of deep venous thrombosis, current oral contraceptive therapy, obesity, regular participation in sports or frequent use of saunas or sun-baths, and long periods of standing during work) were investigated. The restriction in quality of life due to symptoms of CVD was also evaluated. CVD was classified according to the CEAP criteria. RESULTS: CVD was present in a total of 70 employees (34%), predominantly in women. Standing at work was a predisposing factor. We found the highest prevalence of CVD in utility workers and cleaners and the lowest prevalence in medical technician assistants, secretaries and scientific staff. CONCLUSION: The study demonstrated that within a representative cross-section of hospital employees in a University hospital the prevalence of CVD was highest in women, especially in those working in a standing position or under conditions of high temperature and humidity. The results warrant regular evaluation of risk factors with subsequent primary prophylaxis of CVD.

Age-related effects on induction of DNA strand breaks by intermittent exposure to electromagnetic fields.

Several studies indicating a decline of DNA repair efficiency with age raise the question, if senescence per se leads to a higher susceptibility to DNA damage upon environmental exposures. Cultured fibroblasts of six healthy donors of different age exposed to intermittent ELF-EMF (50 Hz sinus, 1 mT) for 1-24 h exhibited different basal DNA strand break levels correlating with age. The cells revealed a maximum response at 15-19 h of exposure. This response was clearly more pronounced in cells from older donors, which could point to an age-related decrease of DNA repair efficiency of ELF-EMF induced DNA strand breaks.

Intermittent extremely low frequency electromagnetic fields cause DNA damage in a dose-dependent way.

OBJECTIVES: Epidemiological studies have reported an association between exposure to extremely low frequency electromagnetic fields (ELF-EMFs) and increased risk of cancerous diseases, albeit without dose-effect relationships. The validity of such findings can be corroborated only by demonstration of dose-dependent DNA-damaging effects of ELF-EMFs in cells of human origin in vitro. METHODS: Cultured human diploid fibroblasts were exposed to intermittent ELF electromagnetic fields. DNA damage was determined by alkaline and neutral comet assay. RESULTS: ELF-EMF exposure (50 Hz, sinusoidal, 1-24 h, 20-1,000 mu T, 5 min on/10 min off) induced dose-dependent and time-dependent DNA single-strand and double-strand breaks. Effects occurred at a magnetic flux density as low as 35 mu T, being well below proposed International Commission of Non-Ionising Radiation Protection (ICNIRP) guidelines. After termination of exposure the induced comet tail factors returned to normal within 9 h. CONCLUSION: The induced DNA damage is not based on thermal effects and arouses concern about environmental threshold limit values for ELF exposure.

Orthostatic intolerance is not necessarily related to a specific mutation (Ala457Pro) in the human norepinephrine transporter gene.

BACKGROUND: Orthostatic intolerance (OI) is a syndrome characterized by lightheadedness, palpitations, fatigue, blurred vision, dizziness, chest discomfort, cognitive impairment, and occasionally syncope. These symptoms usually occur after upright posture and are associated with tachycardia and high plasma concentrations of norepinephrine. It has been proposed that a mutation in exon 9 of the norepinephrine transporter gene (Ala457Pro), resulting in more than 98% loss of function compared with the wild type, might provide a pathogenetic mechanism to explain the clinical symptoms of patients with OI. METHODS: We studied 46 young men from military service who had sought medical advice because of dizziness while standing. Every patient underwent a tilt-table test, with monitoring of blood pressure, heart rate, and plasma catecholamines in supine position and during 30 minutes of standing. Fourteen patients showing the full-blown OI syndrome (30 bpm increase in heart rate and 600 pg/mL plasma norepinephrine levels while standing) underwent direct DNA sequencing of exon 9 of the norepinephrine-transporter gene. RESULTS AND CONCLUSIONS: The specific mutation (Ala457Pro) was not detected in any of the 14 OI patients. Based on these findings, we doubt that this specific genetic transport defect is a frequent cause of the impaired uptake of norepinephrine in OI patients. Its routine determination will therefore not be helpful to establish the clinical diagnosis of OI.

Induction of DNA strand breaks by intermittent exposure to extremely-low-frequency electromagnetic fields in human diploid fibroblasts.

Results of epidemiological research show low association of electromagnetic field (EMF) with increased risk of cancerous diseases and missing dose-effect relations. An important component in assessing potential cancer risk is knowledge concerning any genotoxic effects of extremely-low-frequency-EMF (ELF-EMF). Human diploid fibroblasts were exposed to continuous or intermittent ELF-EMF (50Hz, sinusoidal, 24h, 1000microT). For evaluation of genotoxic effects in form of DNA single- (SSB) and double-strand breaks (DSB), the alkaline and the neutral comet assay were used. In contrast to continuous ELF-EMF exposure, the application of intermittent fields reproducibly resulted in a significant increase of DNA strand break levels, mainly DSBs, as compared to non-exposed controls. The conditions of intermittence showed an impact on the induction of DNA strand breaks, producing the highest levels at 5min field-on/10min field-off. We also found individual differences in response to ELF-EMF as well as an evident exposure-response relationship between magnetic flux density and DNA migration in the comet assay. Our data strongly indicate a genotoxic potential of intermittent EMF. This points to the need of further studies in vivo and consideration about environmental threshold values for ELF exposure.

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure.

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.

Reduced cognitive abilities in lead-exposed men.

OBJECTIVES: Since it is still controversial whether-low-to moderate long-term lead exposure below current threshold values causes neurobehavioural deficits in adults, we investigated executive functions of the prefrontal cortex, attention, and visuospatial and visuomotor functioning in lead-exposed subjects. METHODS: Forty-seven lead-exposed subjects with a mean blood lead (PbB) level of 30.8 microg/100 ml and 53 non-lead-exposed aged-matched subjects (PbB: 4.32 microg/100 ml) with the same socio-economic background were investigated. Both groups were also matched on verbal intelligence. Neuropsychological tests were done by the modified Wisconsin card sorting test, block design test, visual recognition test, simple reaction time, choice reaction and digit symbol substitution. Lead exposure was assessed by current and cumulative measures. RESULTS: While there were significant differences in the results of the Wisconsin, block design and visual recognition tests, no differences were found in simple reaction time, choice reaction and digit symbol substitution. Significant correlations existed between current exposure and cognitive deficits. No correlation was found between cumulative exposure measures and cognitive parameters. CONCLUSIONS: Our results show that PbB below 70 microg/100 ml reduce neurobehavioural abilities, particularly visuospatial abilities and executive functions referring to the prefrontal cortex. As neurobiological substrate of the prefrontal dysfunction, glutamatergic system disturbances are discussed.

Basal levels of DNA strand breaks in human leukocytes determined by comet assay.

In order to determine background levels of DNA strand breaks, we examined 80 healthy individuals by comet assay considering age, sex, and smoking as confounding factors. Only age was found to have a significant effect on basal levels. One thousand cells of each donor were graded by eye into 5 categories according to the amount of DNA in the tail: classification group A (no damage) <5%, B (low damage) 5-20%, C (medium damage) 20-40%, D (high damage) 40-95%, and group E (total damage) >95%. The interpretation of the comet assay was modified to achieve a tail factor, which represents the DNA damage of 1000 scored cells as a single number, without the need of an image analysis software package. Hydrogen peroxide and bleomycin used for in vitro exposure of lymphocytes, produced clear dose-related responses in the comet assay. Our data encourage the application of the used classification model for a sensitive and fast quantification of DNA damage. Results in this study are in agreement with most of the earlier investigations.

Urinary 8-hydroxydeoxyguanosine and sister chromatid exchanges in patients with total hip replacements.

Ion release from metal implants has been suspected to increase the risk of genotoxic effects in patients wearing orthopedic metal devices. In this study we used urinary 8-hydroxydeoxyguanosine (8-OHdG) as marker of oxidative DNA damage and the frequency of sister chromatid exchanges in lymphocytes to test a possible relationship between the concentrations of chromium or cobalt and the induction of cytogenetic modifications in 46 patients with total hip replacements. A broad range of individual levels of metals has been observed in these patients: chromium in blood, 1.59-14.11 microg/L; chromium in urine, 0.79-93.80 microg/24 h; cobalt in blood, 0.77-37.80 microg/L; cobalt in urine, 2.59-166.94 microg/24 h. By linear regression analysis, no significant correlation between urinary 8OHdG or sister chromatid exchange (SCE) and the concentrations of metals was found. However, cobalt in blood as well as 8-OHdG in urine were higher in patients with implants 3-4 yr old as compared to patients with implants 1-2 yr old. Smoking significantly increased the frequency of SCE. Our data do not indicate a dependence of 8-OHdG in urine or SCE on the levels of chromium or cobalt in patients with total hip replacements.