Diagnostic characterization of respiratory allergies by means of a multiplex immunoassay.

Allergic sensitization is commonly assessed in patients by performing the skin prick test (SPT) or determining specific immunoglobulin (IgE) levels in blood samples with the ImmunoCAP™ assay, which measures each allergen and sample separately. This paper explores the possibility to investigate respiratory allergies with a high throughput method, the Meso Scale Discovery (MSD) multiplex immunoassay, measuring IgE levels in low volumes of blood. The MSD multiplex immunoassay, developed and optimized with standards and allergens from Radim Diagnostics, was validated against the SPT and the ImmunoCAP assay. For 18 adults (15 respiratory allergy patients and three controls), blood collection and the SPT were performed within the same hour. Pearson correlations and Bland-Altman analysis showed high comparability of the MSD multiplex immunoassay with the SPT and the ImmunoCAP assay, except for house dust mite. The sensitivity of the MSD multiplexed assay was ≥78% for most allergens compared to the SPT and ImmunoCAP assay. Additionally, the specificity of the MSD multiplex immunoassay was ≥ 87% - the majority showing 100% specificity. Only the rye allergen had a low specificity when compared to the SPT, probably due to cross-reactivity. The reproducibility of the MSD multiplex immunoassay, assessed as intra- and interassay reproducibility and biological variability between different sampling moments, showed significantly high correlations (r = 0·943-1) for all tested subjects (apart from subject 13; r = 0·65-0·99). The MSD multiplex immunoassay is a reliable method to detect specific IgE levels against respiratory allergens in a multiplexed and high-throughput manner, using blood samples as small as from a finger prick.

Pooled analysis of genotoxicity markers in relation to exposure in the Flemish Environment and Health Studies (FLEHS) between 1999 and 2018.

BACKGROUND: The Flemish Environment and Health Studies (FLEHS) are human biomonitoring surveys running in Flanders since 1999. Additionally to biomarkers of exposure, markers of genotoxicity and oxidative stress have been measured, including the alkaline comet and micronucleus assay in peripheral whole blood cells, and urinary concentrations of 8-oxo-2'-deoxyguanosine (8-oxodG). AIM: Exposure-effect associations were explored in a pooled dataset of nine different cross-sectional FLEHS surveys. Data of adolescents collected in a time frame of about 20 years (1999-2018) were compiled. The aim of the study was to examine whether increased variation in exposure, lifestyle and environmental factors would lead to more powerful and robust exposure-effect associations. MATERIALS & METHODS: The biomarkers were measured in 2283 adolescents in the age range of 14-18 years. Exposure to polycyclic aromatic hydrocarbons [1-hydroxypyrene (1-OHP)], benzene (tt'-muconic acid), metals (arsenic, cadmium, copper, nickel, thallium, lead, chromium), persistent organochlorines and phthalates were assessed in blood or urine. Furthermore, outdoor air levels of particulate matter (PM10 and PM2.5) at the residences of the youngsters were calculated. Pooled statistical analysis was done using mixed models. Study-specific differences in the genotoxicity markers and in the strength/direction of the association were accounted for. This was done by incorporating the random factor 'study' and a random study slope (if possible). The exposure markers were centered around the study-specific mean in order to correct for protocol changes over time. RESULTS: A significant association was observed for the urinary oxidative stress marker 8-oxodG, which was positively associated with 1-OHP (5% increase for doubling of 1-OHP levels, p = 0.001), and with urinary copper (26% increase for doubling of copper levels, p = 0.001), a metal involved in the Fenton reaction in biological systems. 8-oxodG was also associated with the sum of the metabolites of the phthalate di(2-ethylhexyl) phthalate (DEHP) (3% increase for doubling of the DEHP levels, p = 0.02). For those associations, data pooling increased the statistical power. However, some of the associations in the individual surveys, were not confirmed in the pooled analysis (such as comet assay and 8-oxodG vs. atmospheric PM; and 8-oxodG vs. urinary nickel). This may be due to inconsistencies in exposure-effect relations and/or variations in the pollutant mix over time and regions. CONCLUSION: Pooled analysis including a large population of 2283 Flemish adolescents showed that 8-oxodG, a marker of oxidative DNA damage is a valuable marker to assess impact of daily life pollutants, such as PAHs, Cu and the phthalate DEHP.

Gender differences in cadmium and cotinine levels in prepubertal children.

Susceptibility to environmental stressors has been described for fetal and early childhood development. However, the possible susceptibility of the prepubertal period, characterized by the orchestration of the organism towards sexual maturation and adulthood has been poorly investigated and exposure data are scarce. In the current study levels of cadmium (Cd), cotinine and creatinine in urine were analyzed in a subsample 216 children from 12 European countries within the DEMOCOPHES project. The children were divided into six age-sex groups: boys (6-8 years, 9-10 years and 11 years old), and girls (6-7 years, 8-9 years, 10-11 years). The number of subjects per group was between 23 and 53. The cut off values were set at 0.1 µg/L for Cd, and 0.8 µg/L for cotinine defined according to the highest limit of quantification. The levels of Cd and cotinine were adjusted for creatinine level. In the total subsample group, the median level of Cd was 0.180 µg/L (range 0.10-0.69 µg/L), and for cotinine the median wet weight value was 1.50 µg/L (range 0.80-39.91 µg/L). There was no significant difference in creatinine and cotinine levels between genders and age groups. There was a significant correlation between levels of cadmium and creatinine in all children of both genders. This shows that even at such low levels the possible effect of cadmium on kidney function was present and measurable. An increase in Cd levels was evident with age. Cadmium levels were significantly different between 6-7 year old girls, 11 year old boys and 10-11 year old girls. As there was a balanced distribution in the number of subjects from countries included in the study, bias due to data clustering was not probable. The impact of low Cd levels on kidney function and gender differences in Cd levels needs further investigation.

A pilot study on the feasibility of European harmonized human biomonitoring: Strategies towards a common approach, challenges and opportunities.

In 2004 the European Commission and Member States initiated activities towards a harmonized approach for Human Biomonitoring surveys throughout Europe. The main objective was to sustain environmental health policy by building a coherent and sustainable framework and by increasing the comparability of data across countries. A pilot study to test common guidelines for setting up surveys was considered a key step in this process. Through a bottom-up approach that included all stakeholders, a joint study protocol was elaborated. From September 2011 till February 2012, 17 European countries collected data from 1844 mother-child pairs in the frame of DEMOnstration of a study to COordinate and Perform Human Biomonitoring on a European Scale (DEMOCOPHES).(1) Mercury in hair and urinary cadmium and cotinine were selected as biomarkers of exposure covered by sufficient analytical experience. Phthalate metabolites and Bisphenol A in urine were added to take into account increasing public and political awareness for emerging types of contaminants and to test less advanced markers/markers covered by less analytical experience. Extensive efforts towards chemo-analytical comparability were included. The pilot study showed that common approaches can be found in a context of considerable differences with respect to experience and expertize, socio-cultural background, economic situation and national priorities. It also evidenced that comparable Human Biomonitoring results can be obtained in such context. A European network was built, exchanging information, expertize and experiences, and providing training on all aspects of a survey. A key challenge was finding the right balance between a rigid structure allowing maximal comparability and a flexible approach increasing feasibility and capacity building. Next steps in European harmonization in Human Biomonitoring surveys include the establishment of a joint process for prioritization of substances to cover and biomarkers to develop, linking biomonitoring surveys with health examination surveys and with research, and coping with the diverse implementations of EU regulations and international guidelines with respect to ethics and privacy.

Interpreting biomarker data from the COPHES/DEMOCOPHES twin projects: Using external exposure data to understand biomarker differences among countries.

In 2011 and 2012, the COPHES/DEMOCOPHES twin projects performed the first ever harmonized human biomonitoring survey in 17 European countries. In more than 1800 mother-child pairs, individual lifestyle data were collected and cadmium, cotinine and certain phthalate metabolites were measured in urine. Total mercury was determined in hair samples. While the main goal of the COPHES/DEMOCOPHES twin projects was to develop and test harmonized protocols and procedures, the goal of the current paper is to investigate whether the observed differences in biomarker values among the countries implementing DEMOCOPHES can be interpreted using information from external databases on environmental quality and lifestyle. In general, 13 countries having implemented DEMOCOPHES provided high-quality data from external sources that were relevant for interpretation purposes. However, some data were not available for reporting or were not in line with predefined specifications. Therefore, only part of the external information could be included in the statistical analyses. Nonetheless, there was a highly significant correlation between national levels of fish consumption and mercury in hair, the strength of antismoking legislation was significantly related to urinary cotinine levels, and we were able to show indications that also urinary cadmium levels were associated with environmental quality and food quality. These results again show the potential of biomonitoring data to provide added value for (the evaluation of) evidence-informed policy making.

Real-time selected ion flow tube mass spectrometry to assess short- and long-term variability in oral and nasal breath.

Breath-based non-invasive diagnostics have the potential to provide valuable information about a person's health status. However, they are not yet widely used in clinical practice due to multiple factors causing variability and the lack of standardized procedures. This study focuses on the comparison of oral and nasal breathing, and on the variability of volatile metabolites over the short and long term. Selected ion flow tube mass spectrometry (SIFT-MS) was used for online analysis of selected volatile metabolites in oral and nasal breath of 10 healthy individuals five times in one day (short-term) and six times spread over three weeks (long-term), resulting in nearly 100 breath samplings. Intra-class correlation coefficients (ICCs) were used to assess short- and long-term biological variability. Additionally, the composition of ambient air was analyzed at different samplings. The selected volatiles common in exhaled breath were propanol, 2,3-butanedione, acetaldehyde, acetone, ammonia, dimethyl sulfide, isoprene, pentane, and propanal. Additionally, environmental compounds benzene and styrene were analyzed as well. Volatile metabolite concentrations in ambient air were not correlated with those in exhaled breath and were significantly lower than in breath samples. All volatiles showed significant correlation between oral and nasal breath. Five were significantly higher in oral breath compared to nasal breath, while for acetone, propanal, dimethyl sulfide, and ammonia, concentrations were similar in both matrices. Variability depended on the volatile metabolite. Most physiologically relevant volatiles (acetone, isoprene, propanol, acetaldehyde) showed good to very good biological reproducibility (ICC > 0.61) mainly in oral breath and over a short-term period of one day. Both breathing routes showed relatively similar patterns; however, bigger differences were expected. Therefore, since sampling from the mouth is practically more easy, the latter might be preferred.

Translating biomonitoring data into risk management and policy implementation options for a European Network on Human Biomonitoring.

BACKGROUND: The "European Environment & Health Action Plan 2004-2010" originates from the concern of the European Commission on the well-being of individuals and the general population. Through this plan, the Commission has set the objectives to improve the information chain for a better understanding of the link between sources of pollution and health effects, to better identify existing knowledge gaps, and improve policy making and communication strategies. Human biomonitoring (HBM) has been included as one of the tools to achieve these objectives. As HBM directly measures the amount of a chemical substance in a person's body, taking into account often poorly understood processes such as bioaccumulation, excretion, metabolism and the integrative uptake variability through different exposure pathways, HBM data are much more relevant for risk assessment than extrapolations from chemical concentrations in soil, air, and water alone. However, HBM primarily is a stepping stone between environmental and health data, and the final aim should be an integrated and holistic systematic risk assessment paradigm where HBM serves as a pivotal point between environment and health, on the one hand leaning on environmental data to provide detailed information on the sources and pathways of pollutants that enter the human body, and on the other hand clarifying new and existing hypotheses on the relationship between environmental pollutants and the prevalence of diseases. With the large amount of data that is being gathered in the different national survey projects, and which is expected to become available in Europe in the near future through the expected European Pilot Project on HBM, a framework to optimize data interpretation from such survey projects may greatly enhance the usefulness of HBM data for risk managers and policy makers. RESULTS: This paper outlines an hierarchic approach, based on the stepwise formulation of 4 subsequent steps, that will eventually lead to the formulation of a variety of policy relevant risk reduction options. CONCLUSION: Although the usefulness of this approach still needs to be tested, and potential fine-tuning of the procedure may be necessary, approaching the policy implications of HBM in an objective framework will prove to be essential.

Internal exposure to pollutants measured in blood and urine of Flemish adolescents in function of area of residence.

The Centre for Environment and Health in Flanders, the Northern part of Belgium, started a biomonitoring program on adolescents in 2003. 1679 adolescents residing in nine areas with different patterns of pollution participated in the study. Possible confounding effects of lifestyle and personal characteristics were taken into account. The geometric mean levels of cadmium and lead in whole blood amounted to 0.36 and 21.7 microg l(-1), those of PCBs, DDE and HCB in serum to 68, 94 and 20.9 ng g(-1) fat, and those of 1-hydroxypyrene and t,t'-muconic acid in urine to 88 ng g(-1) creatinine and 72 microg g(-1) creatinine. Significant regional differences in internal lead, cadmium, PCBs, DDE and HCB exposure were observed in function of area of residence, even after adjustment for age, sex, smoking (and body mass index for the chlorinated compounds). Compared to a reference mean, internal exposure was significantly higher in one or more of the areas: Cd and Pb in the Antwerp agglomeration, Cd in the Antwerp harbour, PCBs in the Ghent agglomeration, PCBs, DDE and HCB in the Ghent harbour, Cd, PCBs, DDE and HCB in the rural area, DDE in Olen and in the Albert canal areas. Adolescents living in an area with intensive fruit cultivation (showing overall the lowest values) and, surprisingly, in areas around household waste incinerators (average of six areas), had no significantly increased internal exposures. Subjects from separate areas around waste incinerators showed significant differences in body load of various environmental contaminants.

Automated detection of irradiated food with the comet assay.

Food irradiation is the process of exposing food to ionising radiation in order to disinfect, sanitise, sterilise and preserve food or to provide insect disinfestation. Irradiated food should be adequately labelled according to international and national guidelines. In many countries, there are furthermore restrictions to the product-specific maximal dose that can be administered. Therefore, there is a need for methods that allow detection of irradiated food, as well as for methods that provide a reliable dose estimate. In recent years, the comet assay was proposed as a simple, rapid and inexpensive method to fulfil these goals, but further research is required to explore the full potential of this method. In this paper we describe the use of an automated image analysing system to measure DNA comets which allow the discrimination between irradiated and non-irradiated food as well as the set-up of standard dose-response curves, and hence a sufficiently accurate dose estimation.

The effect of paternal methyl-group donor intake on offspring DNA methylation and birth weight.

Most nutritional studies on the development of children focus on mother-infant interactions. Maternal nutrition is critically involved in the growth and development of the fetus, but what about the father? The aim is to investigate the effects of paternal methyl-group donor intake (methionine, folate, betaine, choline) on paternal and offspring global DNA (hydroxy)methylation, offspring IGF2 DMR DNA methylation, and birth weight. Questionnaires, 7-day estimated dietary records, whole blood samples, and anthropometric measurements from 74 fathers were obtained. A total of 51 cord blood samples were collected and birth weight was obtained. DNA methylation status was measured using liquid chromatography-tandem mass spectrometry (global DNA (hydroxy)methylation) and pyrosequencing (IGF2 DMR methylation). Paternal betaine intake was positively associated with paternal global DNA hydroxymethylation (0.028% per 100 mg betaine increase, 95% CI: 0.003, 0.053, P=0.03) and cord blood global DNA methylation (0.679% per 100 mg betaine increase, 95% CI: 0.057, 1.302, P=0.03). Paternal methionine intake was positively associated with CpG1 (0.336% per 100 mg methionine increase, 95% CI: 0.103, 0.569, P=0.006), and mean CpG (0.201% per 100 mg methionine increase, 95% CI: 0.001, 0.402, P=0.049) methylation of the IGF2 DMR in cord blood. Further, a negative association between birth weight/birth weight-for-gestational age z-score and paternal betaine/methionine intake was found. In addition, a positive association between choline and birth weight/birth weight-for-gestational age z-score was also observed. Our data indicate a potential impact of paternal methyl-group donor intake on paternal global DNA hydroxymethylation, offspring global and IGF2 DMR DNA methylation, and prenatal growth.

Oxidative stress reactions induced in beans (Phaseolus vulgaris) following exposure to uranium.

The present study aimed to analyze the biological effects induced by bioaccumulation of uranium in Phaseolus vulgaris. Ten-day-old seedlings were exposed to 0, 0.1, 1, 10, 100 and 1000 microM U in diluted Hoagland solution. Following 1, 2, 4 and 7 days' exposure, plants were monitored for uranium uptake, biometric parameters, capacities of enzymes involved in the anti-oxidative defense mechanisms (GPOD, SPOD, GLUR, SOD, ICDH, G-6P-DH), glutathione (GSH) pool and DNA integrity. Uranium contents were up to 900-fold higher in roots (31-14,916 mg kg(-1) FW following 7 days' exposure to 0.1 and 1000 microM U, respectively) as compared to primary leaves (1-16 mg kg(-1) FW following 7 days' exposure to 0.1 and 1000 microM U, respectively). Uranium exposure did not significantly affect plant growth compared to the control. For all enzymes studied, except SOD, enzyme capacities in roots were slightly stimulated with increasing contaminant concentrations (though not significantly). For roots exposed to 1000 microM U, enzyme capacities were significantly reduced. Enzyme capacities in leaves were not affected by uranium treatment. Total and reduced GSH levels were higher in primary leaves of uranium (

The comet assay: a tool to study alteration of DNA integrity in developing plant leaves.

DNA integrity of Nicotiana tabacum L. and Vicia faba L. leaves in different stages of growth was analysed with the single cell gel electrophoresis (comet) assay. With this test DNA of individual cells is stretched by electrophoresis and the migration is measured, which gives an image of the nuclear DNA organisation. Nuclei were sampled when the plants had developed an apical bud, five true leaves and cotyledons. To get an idea of the kind of lesions observed, three different comet protocols were used. The neutral protocol with electrophoresis in a neutral buffer and the semi-alkaline or alkaline assay with alkaline unwinding followed by electrophoresis in neutral alkaline buffer, respectively. For V. faba there was a successive increased cellular DNA mobility with age of the leaves. The percentage DNA migration in control cells of fully developed leaves from N. tabacum almost reached the same level than after irradiation of not fully developed leaves with 50 Gy X-rays. The increased stretching of DNA with leaf age was most obvious if the DNA duplex was converted to single strands by alkali treatment before electrophoresis. Therefore, it could be concluded that with the ageing of leaves there is a decrease in DNA integrity, which could be the result of rising amounts of DNA single-strand breaks and 'alkali-vulnerable sites'.

Repair of X-ray induced DNA damage measured by the comet assay in roots of Vicia faba.

The comet assay was used to measure DNA damage and repair in nuclei released from 1 cm root ends of Vicia faba after X-ray irradiation. Irradiation induced a linear increase of DNA content in comet tail with doses under various denaturation and electrophoretic conditions. The pH of the electrophoresis solution played the most important role in the detection of DNA damage. After irradiation with 30 Gy of X-rays, most of the DNA damage was removed during the first 20 min, even in the presence of DNA repair inhibitors. This first, rapid phase of DNA repair was not affected by incubation on ice, but was partially blocked by 3-aminobenzamide. When DNA was exposed to alkali (0.3 M NaOH) and electrophoresed at neutral pH, all DNA damage was removed in 2 hr, even in the presence of 3-aminobenzamide. Complete repair was inhibited by incubation on ice (30% of DNA remaining in tail) and partially by aphidicolin (13% DNA remaining in tail). Under alkaline (0.3 M NaOH) pretreatment and electrophoresis, more than 20% of detected DNA damage remained unrepaired after 2 hr of postirradiation incubation with and without 3-aminobenzamide at room temperature. Aphidicolin and incubation on ice inhibited the removal of DNA damage to 33% and 39% DNA, respectively. Moreover, aphidicolin treatment attenuated the first phase of damage removal.

Comparison of CALUX-TEQ values with PCB and PCDD/F measurements in human serum of the Flanders Environmental and Health Study (FLEHS).

In 1999, a campaign of the Flemish Ministry of Health, Belgium was set up to assess pollutant concentrations and related health effect biomarkers in humans living in two regions of Flanders. The study was called the 'Flemish Environment and Health Study' (FLEHS). Concentrations of selected organochiorine pesticides, polychlorinated biphenyls (PCB) and polychlorinated dibenzo-p-dioxins (PCDD) and flirans (PCDF) were measured by gas chromatography-mass spectrometry in 47 pooled human serum samples originating from 200 individual women between 50 and 65 years living in two Flemish regions. The CALUX (Chemical-Activated Luciferase gene eXpression) bioassay was assessed on the same pools. The correlation between CALUX-TEQ and total TEQ (sum of PCDD/PCDF, non- and mono-ortho PCBs) varied from 0.43 to 0.73 for the rural and urban region, respectively. The mean value for the total TBQ (75 pg WHO-TEQ/g fat) was two times higher than the mean TEQ value determined with the CALUX bioassay (36 pg TEQ/g fat). This shows that the assessment of dioxin-like exposure by these two measurements was different. However, regional differences in concentrations were observed for neither total TEQs, nor CALUX-TEQs. It was concluded that the CALUX can be an alternative screening tool for biomonitoring purposes, especially when the objective is to compare different groups of people (e.g. living in different regions).

The alkaline comet test on plant cells: a new genotoxicity test for DNA strand breaks in Vicia faba root cells.

The alkaline comet assay (single cell gel electrophoresis assay) is a sensitive method for the detection of DNA damage. This paper describes the first application of this assay to plant cells for genotoxicological assessment. Germinating Vicia faba (field bean) seedlings were kept in water with either methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), mitomycin C (MMC), cycloheximide (CH), cadmium chloride (CdCl2), potassium dichromate (K2Cr2O7), or chromium trichloride (CrCl3). Nuclei were isolated from the root cells and evaluated for the extent of DNA migration. With the exception of cycloheximide, all agents induced a significant increase in DNA migration. These results indicate that the comet assay may be a valuable tool for monitoring DNA damage in plant systems. However, there was a significant heterogeneity in the extent of DNA migration within and between seedlings, which may be intrinsic to the assay or indicative of sampling problems.

Persistent organochlorine pollutants in human serum of 50-65 years old women in the Flanders Environmental and Health Study (FLEHS). Part 1: Concentrations and regional differences.

In 1999, a campaign of the Flemish Ministry of Health, Belgium was set up to assess pollutant concentrations and related health effect biomarkers in humans living in two regions of Flanders. The study was called the 'Flemish Environment and Health Study' (FLEHS). One of the goals was to measure present concentrations of persistent organochlorine pollutants in a Flemish population and to compare values obtained from pooled and individual serum samples. Concentrations of selected organochlorine pesticides, polychlorinated biphenyls (PCB) and polychlorinated dibenzo-p-dioxins (PCDD) and furans (PCDF) were measured by gas chromatography-mass spectrometry. TEQ values were also assessed by Chemical-Activated LUciferase gene eXpression (CALUX) bioassay. The study population consisted of 200 women between 50 and 65 years living in two areas of Flanders, Belgium. Because of the large volumes serum needed for all measurements, the concentrations of organochlorines were measured in 47 pooled serum samples originating from these women. The concentrations of the indicator PCBs (359.8 ng/g fat) and organochlorine pesticides (hexachlorobenzene, p,p'-dichlorodiphenyldichloroethylene, p,p'-dichlorodiphenyl-trichloroethane, lindane and pentachlorophenol), were comparable to those found in other European countries. The concentrations of PCDD/PCDFs showed another picture. With a median value of 48 pg WHO-TEQ/g fat, the women had 2-fold higher levels than a comparable age group from Germany examined in 1996. The mean total WHO-TEQ including PCDD/F, non-ortho and mono-ortho PCBs was 72.7 pg WHO-TEQ/g fat, whereas the CALUX-TEQ mean value was only 35.0 pg TEQ/g fat. In order to assess the pooling procedure, indicator PCBs and CALUX-TEQs were measured in all 200 individuals that were integrated in the pools. The measured values were comparable to the pool results: 390.0 ng/g fat and 41.6 pg TEQ/g fat respectively. It was concluded that pooling of serum samples offers the possibility to measure exposure in the whole study population on a more cost-effective way. However, because of statistical power loss and no possibility of confounder adjustment, pooling is not the most effective way to study regional differences.

The alkaline single-cell gel electrophoresis/comet assay: a way to study DNA repair in radicle cells of germinating Vicia faba.

Dry seeds are known to accumulate DNA damage with time of storage. Repair of DNA lesions during germination of Vicia faba seeds was followed in the radicles using the alkaline single-cell gel electrophoresis/comet assay. In this assay nuclei were liberated, mixed with agarose and spread out over a microscope slide. After lysis of the nuclear membrane and unwinding of the DNA duplex, DNA was stretched during electrophoresis, giving a comet-like migration pattern. The more DNA was damaged, the higher its mobility. DNA repair took place rapidly the first hours of imbibition and more slowly until ca 33 h after onset of germination. A small amount of heavily damaged cells remained present. Labelling with BrdU provided the possibility to localize repair patches and replicated sites in the comet migration pattern. At 15 h of germination, incorporation of BrdU in radicle DNA was situated at random over the entire comet. At 33 h, DNA repair was more or less accomplished and BrdU was mainly localized in the 'heads' of most comets.

Computer-assisted fluoroscopy-based reduction of femoral fractures and antetorsion correction.

OBJECTIVE: Intra-operative fluoroscopy is a valuable tool for visualizing underlying bone, implant, and surgical tool positions in orthopedics. It has brought about the minimally invasive surgical technique of intramedullar nailing to fix femoral shaft fractures. However, the limited field of view and two-dimensional property of fluoroscopic images aggravate intra-operative control of surgical parameters. The purpose of this article is to introduce a surgical navigation system based on fluoroscopy that provides missing information for the procedure of femoral fracture fixation. MATERIALS AND METHODS: Optoelectronic markers are placed on a surgical drill, involved bone fragments, the femoral nail, and the fluoroscope to track their positions. Projection properties of the fluoroscope are acquired through an initial precalibration. The relative positions of bone fragments, implants, and surgical tools are displayed superimposed simultaneously and in real time on multi-planar intra-operative fluoroscopic images. This is achieved by computer simulation of X-ray projections that have taken place with acquisition of the fluoroscopic images. In addition, a method has been developed that allows contactless measurement of three-dimensional anatomic landmarks, based on their representation in fluoroscopic images. In combination with optoelectronic tracking, this enables dynamic calculation of important surgical parameters such as femoral antetorsion. RESULTS: A pilot surgery showed that fracture reduction can benefit from the developed computer-assisted method. An in-vitro study on computer-assisted measurement of femoral antetorsion demonstrated the high degree of precision of this technique.

Determination of PCDD/Fs, PBDD/Fs and dioxin-like PCBs in human milk from mothers residing in the rural areas in Flanders, using the CALUX bioassay and GC-HRMS.

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the determination of dioxin-like compounds in a large number of samples and requires only small sample volumes, the use of this technique in human biomonitoring programs provides a good alternative to GC-HRMS. In this study, a new CALUX method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in small amounts of human milk samples with the new sensitive H1L7.5c1 cell line was used to analyze 84 human milk samples, collected from mothers residing in the Flemish rural communities. The geometric mean CALUX-Bioanalytical Equivalent (CALUX-BEQ) values, reported for the 84 mothers from the study area were 10.4 (95% CI: 9.4-11.4) pg CALUX-BEQ per gram lipid or 0.41 (95% CI: 0.37-0.45) pg CALUX-BEQ per gram milk for the PCDD/Fs and 1.73 (1.57-1.91) pg CALUX-BEQ per gram lipid or 0.07 (95% CI: 0.06-0.08) pg CALUX-BEQ per gram milk for the dioxin-like PCBs. Multiple regression analysis showed significant associations between PCDD/Fs and weight change after pregnancy, smoking and consumption of local eggs. One pooled human milk sample was analyzed with both CALUX and GC-HRMS. The ratio of CALUX and GC-HRMS results for this sample were respectively 1.60, 0.58 and 1.23 for the PCDD/Fs, the dl-PCBs and the sum of both fractions, when using the 2005-TEF values. Additionally, also low levels of certain brominated dioxins and furans were detected in the pooled sample with GC-HRMS.

Persistent organic pollutants (POPs) in human milk: a biomonitoring study in rural areas of Flanders (Belgium).

To collect information on the concentrations of persistent organic pollutants (POPs) in the rural areas in Flanders (Belgium), 84 breastfeeding mothers were recruited in rural communities in East and West Flanders and Flemish Brabant in 2009-2010. Polychlorinated biphenyl (PCB) congeners, organochlorine pesticides, brominated flame retardants, perfluorinated compounds, polychlorinated dibenzodioxines and dibenzofurans, and dioxin-like PCBs were measured in individual milk samples and in a pooled milk sample, while some additional pollutants were only measured in the pooled sample. For most pollutants, the concentrations in this study were lower or comparable to the concentrations measured in the pooled Belgian sample of the WHO human milk study of 2006, except for the pesticides dichlorodiphenyltrichloroethane DDT (+25% for ΣDDT and metabolites) and trans-nonachlor (+94%), and for the brominated flame retardant hexachlorocyclododecane HBCD (+153%). Perfluorinated compounds were for the first time determined in human milk samples from Belgium and the concentrations were comparable to those from other European countries. Also, interesting associations were found between the concentrations of POPs measured in human milk and personal characteristics as well as dietary habits of the study population. PFOS en PFOA concentrations were significantly higher in milk of primiparous participants compared to mothers who gave birth to their second child. Lower brominated PBDE congeners increased with increasing BMI of the mothers (p=0.01 for BDE 47, p=0.02 for BDE 99 and p=0.02 for BDE 100). Participants consuming milk or dairy products daily had significant higher concentrations of ΣDDTs (p=0.03) and oxychlordane (p=0.047) in their human milk samples.