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.

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.

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.

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.

Quantification of PCDD/Fs and dioxin-like PCBs in small amounts of human serum using the sensitive H1L7.5c1 mouse hepatoma cell line: optimization and analysis of human serum samples from adolescents of the Flemish human biomonitoring program FLEHS II.

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the throughput analysis 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 method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in human serum with the new sensitive H1L7.5c1 mouse hepatoma cell line was optimized. Sample dilution factors of 5 and 2.4 were selected for routine analysis of respectively the PCDD/Fs and dl-PCBs. The validation studies showed that repeatability and within-lab reproducibility for the quality control (QC) standard were within the in-house criteria. A long-term within-lab reproducibility of 25% for the PCDD/F fraction and 41% for the dl-PCB fraction for the analysis of pooled serum samples, expressed as pg BEQ/g fat, was determined. CALUX recoveries of the spiked procedural blanks were within the acceptable in-house limits of 80-120% for both fractions and the LOQ was 30.3 pg BEQ/g fat for the PCDD/Fs and 14.5 pg BEQ/g fat for the dl-PCBs. The GC-HRMS recovery of a C13-spiked pooled serum sample was between 60 and 90% for all PCDD/F congeners and between 67 and 82% for the non-ortho PCBs. An adequate separation between both fractions was found. The CALUX/GC-HRMS ratio for a pooled serum sample was respectively 2.0 and 1.4 for the PCDD/Fs and the dl-PCBs, indicating the presence of additional AhR active compounds. As expected, a correlation was found between human serum samples analyzed with both the new H1L7.5c1 cell line and the more established H1L6.1c3 cell line. The geometric mean CALUX-BEQ values, reported for the adolescents of the second Flemish Environment and Health Study (FLEHS II) recruited in 2009-2010, were 108 (95% CI: 101-114) pg CALUX-BEQ/g fat for the PCDD/Fs and 32.1 (30.1-34.2) pg CALUX-BEQ/g fat for the dioxin-like PCBs.

Hormone levels and sexual development in Flemish adolescents residing in areas differing in pollution pressure.

In 2002, the Centre for Environment and Health in Flanders, Belgium started a human biomonitoring program. For 1679 adolescents, residing in nine study areas with differing pollution pressure, hormone levels and the degree of sexual maturation were measured. Possible confounding effects of lifestyle and personal characteristics were taken into account. Participants from the nine different study areas had significantly different levels of sex hormones (total and free testosterone, oestradiol, aromatase, luteinizing hormone) and the thyroid hormone free triiodothyronine, after correction for confounders. Significantly higher hormone concentrations were measured in samples from participants residing in the area around the waste incinerators, while significantly lower values were found in participants residing in the Albert Canal zone with chemical industry. Sexual maturation of boys as well as girls tended to be somewhat slower in the industrial city of Antwerp and in the Antwerp harbour compared to the other areas in Flanders. Even within the same study area, significant differences in hormone levels could be observed between sub-areas. Data on the internal exposure of the same adolescents to lead, cadmium, PCBs, p,p'-DDE, HCB, 1-hydroxypyrene and t,t'-muconic acid have already been published. The observed differences in hormone levels and in sexual maturation could however only in part be explained by the measured differences in internal exposure to pollutants, suggesting that also other pollutants and other factors that vary in function of the area of residence could play a role. Nevertheless, our results also suggest that local (environmental) factors, acting within a short distance, might influence the measured hormone levels and degree of sexual maturation.

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.

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.

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'.