Trimethylselenonium ion determination in human urine by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry optimization of the hydride generation step.

This work describes the intricacies of the determination of the trimethylselenonium ion (TMSe) in human urine via high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). By definition, this technique requires that the separated TMSe can be online converted into a volatile compound. Literature data for the determination of TMSe via the hydride generation technique are contradictory; i.e., some authors claim that direct formation of volatile compounds is possible under reduction with NaBH4, whereas others reported that a digestion step is mandatory prior to conversion. We studied and optimized the conditions for online conversion by varying the mobile phase composition (pyridine, phosphate, and acetate), testing different reaction coils, and optimizing the hydride generation conditions, although technically no hydride (H2Se) is formed but a dimethylselenide (DMSe). The optimized conditions were used for the analysis of 64 urine samples of 16 (unexposed) volunteers and the determination of low amounts of TMSe (LOD = 0.2 ng mL-1). Total (specific gravity-corrected) selenium concentrations in the urine samples ranged from 7.9 ± 0.7 to 29.7 ± 5.0 ng mL-1 for individual volunteers. Four volunteers were characterized as TMSe producers (hINMT genotype GA) and 12 were non-producers (hINMT genotype GG). Urine of TMSe producers contained 2.5 ± 1.7 ng mL-1 of TMSe, compared to 0.2 ± 0.2 ng mL-1 for non-producers.

ALAD and APOE polymorphisms are associated with lead and mercury levels in Italian pregnant women and their newborns with adequate nutritional status of zinc and selenium.

The impacts of single-nucleotide polymorphisms (SNPs) in ALAD and VDR genes on Pb health effects and/or kinetics are inconclusive at low exposure levels, while studies including APOE SNPs are rare. In this study, we examined the associations of ALAD, VDR and APOE SNPs with exposure biomarkers of Pb and other trace elements (TEs) in Italian pregnant women (N = 873, aged 18-44 years) and their newborns (N = 619) with low-level mixed-element exposure through diet, the environment or endogenously. DNA from maternal peripheral venous blood (mB), sampled during the second and third trimesters, was genotyped for ALAD (rs1800435, rs1805313, rs1139488, rs818708), VDR (rs2228570, rs1544410, rs7975232, rs731236) and APOE (rs429358, rs7421) using TaqMan SNP assays. Personal and lifestyle data and TE levels (mB, maternal plasma, hair and mixed umbilical cord blood [CB]) from the PHIME project were used. Multiple linear regression models, controlling for confounding variables, were performed to test the associations between SNPs and TEs. The geometric means of mB-Pb, mB-Hg, mB-As and mB-Cd (11.0 ng/g, 2.16 ng/g, 1.38 ng/g and 0.31 ng/g, respectively) indicated low exposure levels, whereas maternal plasma Zn and Se (0.72 µg/mL and 78.6 ng/g, respectively) indicated adequate micronutritional status. Variant alleles of ALAD rs1800435 and rs1805313 were negatively associated with mB-Pb levels, whereas a positive association was observed for rs1139488. None of the VDR SNPs or their haplotypes had any association with Pb levels. Regarding APOE, the ϵ4 allele was associated with lower mB-Hg and CB-Hg, while a positive association was found with the ϵ2 allele and CB-Pb when the model included only newborn girls. The observed associations indicate possible modification effects of ALAD and APOE SNPs on Pb or Hg kinetics in women and their newborns with low exposure to non-essential TEs, as well as an adequate nutritional status of Zn and Se.

Pilot study on the concentrations of organochlorine compounds and potentially toxic elements in pregnant women and local food items from the Finnish Lapland.

In the Arctic, main sources of persistent organic pollutants and potentially toxic elements are industry and agriculture in the lower latitudes. However, there are also local sources of pollution. Our study was focused on possible pollution in the Finnish Lapland, transferred from the Pechenganikel industrial complex located in the borders of Russia, Finland and Norway. Local food items and blood samples of pregnant women from the Inari municipality were collected and organochlorine compounds (OCs) and metal(oid)s analyzed. Most of the examined food samples showed detectable levels of these compounds. The mean concentrations of DDTs and polychlorobiphenyls (PCBs) were higher in fish (0.18-0.32 ng/g and 0.34-0.64 ng/g, respectively), than in the other food groups (0.027-0.047 ng/g and 0.11-0.20 ng/g, respectively). PCBs were found at the highest concentrations in blood samples of the pregnant women, and congeners 153 and 118 were dominant. The mean concentration of PCB153, 0.29 µg/kg serum lipid, was lower than those described in many other studies. Concerning DDTs, the 4,4'-DDT/4,4'-DDE ratio, 0.092, in the blood samples was lower than that observed in the food items, 0.25-0.71, reflecting old uses of the DDT pesticide. None of the observed levels of selected potentially toxic elements in blood samples and in food items exceeded the known safe limits. Higher concentrations of PCB52 and γ-HCH were observed in the serum of pregnant women who consumed greater amounts of meat, and berries and mushrooms, respectively. The OC concentrations from the pregnant women currently studied were lower than those observed fourteen years ago with pregnant women from the same municipality. Compounds whose occurrence is likely related to a long-distance transport showed clear decreases, e.g., 63% for PCBs, and for those from pesticides, decreases were 93% and 97% for 4,4'-DDE and ß-HCH, respectively. No obvious influence from the Pechenganikel complex is observed from the results.

Organohalogens: A persisting burden in Slovenia?

Persistent organic pollutants (POPs) represent a concern for the environment and human health due to their persistence and toxicity. Exposure in Slovenia is geographically differentiated because the country, as part of former Yugoslavia, has a history of industry and regional contamination and is - at the same time - known for its clean nature. The PCB pollution of the Krupa River drew the public's attention to the chemical burden of Slovenians, and the demand for studies has been rising since. We assessed the exposure of men (n = 548) and primiparous women (n = 536) to POPs in 12 regions of Slovenia as well as exposure pathways via questionnaires. Most PCDD/Fs, PCBs, and PBDEs could be determined in pooled samples of maternal milk at low concentrations (1.57 pg/gTEQ, 1.47 pg/gTEQ, and 1076 pg/g fat, respectively), but a much lower number of compounds could be measured above the LOQ in pooled men's plasma samples (PCDD/Fs 0.08 pg/gTEQ, PCBs 0.007 pg/gTEQ, ΣPBDE 920 pg/g), and only HCB, p,p'-DDE, ΣDDT, and the non-dioxin-like PCB congeners 138, 153, and 180 could be determined in individual samples of milk (concentration range 5-60 ng/g fat). In individual samples of men's serum, only p,p'-DDE and ΣPCB were detected at concentrations of 0.25 ng/g and 0.3 ng/g, respectively. Nonetheless, we were able to differentiate between polluted and unpolluted areas on a national level, with higher exposure levels in the PCB polluted region of Bela Krajina, the industrial region Zasavje, and the capital, Ljubljana. Despite low concentrations, determinants of exposure, such as age, proximity to roads, old building materials, private water supplies, and consumption of alcohol, fish, meat, and eggs that have previously been observed only at higher levels could still be identified. Furthermore, levels of PCBs and PBDEs were highly correlated suggesting common exposure sources and pathways, whereas PCDD/Fs were correlated to a lesser extent. The calculated ratio between DDT and DDE in maternal milk samples was decreasing with the year of sampling, suggesting no ongoing exposure to DDT. The study findings suggest low exposure of men and lactating women to legacy pollutants in Slovenia, which gave rise to the hypothesis that Slovenia's geographical location might provide shelter from the long-range transport of POPs via Westerly winds. This hypothesis remains to be confirmed within future studies.

Urinary phthalate concentrations in the slovenian population: An attempt to exposure assessment of family units.

Phthalates are widespread contaminants with differing chemical characteristics, which largely determine their product applications, and they can leach into the environment. Due to their endocrine disruptive properties at long-term low-level exposure, they propose a health threat to people that has been associated with several adverse health effects such as: decreased male fertility and impacts on neurological development. People are exposed to different phthalates on a daily basis. Accordingly, this study aims to determine urinary concentrations of seven phthalate metabolites in Slovenian mothers (n = 155), fathers (n = 77), and children (n = 155) within the European project DEMOCOPHES and to identify potential sources of exposure using questionnaire data on sociodemographic characteristics. Furthermore, the appropriateness of two adjustment methods (creatinine and specific gravity) has been evaluated. First morning urine samples were obtained from one urban and one rural location in 2011. Samples were analysed with Ultra Performance Liquid Chromatography Tandem Mass Spectrometry according to the COPHES SOP protocol by VITO NV laboratory in Belgium. All investigated metabolites were detected in all populations. Children's urinary concentrations exceeded those of adults for most metabolites. We observed variations in concentrations depending on sociodemographic and geographic characteristics, such as food and product sources (e.g. plastic packaging, tins, personal care products, PVC) as well as lifestyle and habits (e.g. living space, time spent outside). We observed geographic and sociodemographic differences in our populations that could be confirmed for the three populations separately and for family units. Concentrations are significantly higher at the rural sampling location as well as in households with a lower level of education. We found both the urinary concentrations and the intake doses to be within the European range as presented in the literature. Between creatinine and specific gravity, we found specific gravity the more appropriate option for phthalates. To our knowledge, this is the first study investigating exposure to phthalates in the Slovenian population while considering the common exposure of family units.

Selected elements and fatty acid composition in human milk as indicators of seafood dietary habits.

The maternal diet and living environment can affect levels of chemical elements and fatty acid (FA) composition and their stable isotopes (δ13CFA) in human milk. Information obtained from questionnaires is frequently imprecise, thus limiting proper associations between external and internal exposures as well as health effects. In this study, we focused on seafood as a source of potentially toxic and essential elements and nutritional FAs. Concentrations of selected elements in human milk (As, Cd, Cu, Mn, Pb, Se and Zn) were determined using inductively coupled plasma mass spectrometry (ICP-MS) and Hg using cold vapour atomic-absorption spectrometry (CV-AAS). The identification and quantification of FAs in maternal milk were performed by an in-situ trans-esterification method (FAMEs), and the characterization of FAMEs was performed by gas chromatography with a flame ionisation detector (GC-FID). δ13CFA was determined by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Seventy-four lactating Slovenian women from the coastal area of Koper (KP), with more frequent consumption of seafood, and the inland area of Pomurje (MS), with less frequent seafood consumption, were included in this study. Along with basic statistical analyses, data mining approaches (classification and clustering) were applied to investigate whether FA composition and δ13CFA could improve the information regarding dietary sources of potentially toxic elements. As and Hg levels in milk were found to be statistically higher in populations from KP than in those from MS, and 71% of individual FAs and 30% of individual δ13CFA values in milk differed statistically between the studied areas. In 19 cases, the levels of FAs in milk were higher in KP than in MS; these FAs include C20:5ω3 and C22:6ω3/C24:1ω9, which are typically contained in fish. In 16 cases, the mean percentage of FAs was higher in MS than in KP; these FAs include the PUFAs C18:2ω6, C18:3ω3, and C20:4ω6 which are important for human and infant growth. The difference in δ13C levels of C10:0, C12:0, C14:0, C16:1, C16:0, C18:1ω9c, C22:6ω3, and δ13C 18:0-16:0 in the study groups was statistically significant. In all seven cases where δ13C of FA significantly differed between KP and MS, δ13C was higher in KP, indicating a higher proportion of a marine-based diet. The data mining approaches confirmed that the percentage of selected FAs (iC17:0, C4:0, C18:2ω6t, aC17:0, CLA, and C22:4ω6) and δ13CFA of C18:1ω9c in human milk could be used to distinguish between high and low frequency of fresh seafood consumption.

Pregnancy exposome and child psychomotor development in three European birth cohorts.

Characterization of the exposome, the totality of all environmental factors that one is exposed to from conception onwards, has been recommended to better evaluate the role of environmental influences on developmental programming and life-course vulnerability to major chronic diseases. In the framework of the Health and Environment-wide Associations based on Large population Surveys (HEALS) project we considered the pregnancy exposome exploiting two databases (PHIME and REPRO_PL) that include birth cohorts from three EU countries (Croatia, Slovenia and Poland). The databases contained information on several chemical exposures, socio-demographic, lifestyle and health related factors from conception to child birth, and neuropsychological scores assessed by the Bayley Scales of Infant and Toddler Development in the first two years of life. Our main goal was to assess consistency of environmental influences on neurodevelopment, if any, across European countries differing for geographical, socio-demographic characteristics and levels of chemical exposures to metals such as lead (Pb), mercury (Hg), cadmium (Cd) and trace elements, including micronutrients such as zinc (Zn) and selenium (Se). To this aim, we first selected variables common to the different databases, then applied univariate and multivariate regression analyses to identify factors linked to neurodevelopment, and finally performed meta-analysis to detect potential heterogeneity among cohorts and pooled estimates. Significant differences in exposure levels among the three sub-cohorts were observed as for Hg and Se; exposure levels under study were relatively low and within the range described in existing EU biomonitoring studies. The univariate analyses did not show any common pattern of association as only in the Polish cohort chemical exposure had an impact on neuropsychological outcome. In the meta-analysis, some consistent trends were evident, relative to the adverse influence of Pb on children's language and cognition and the positive influence of Se on language abilities. The effects of the neurotoxic metal Hg positively influenced the motor scores in the Polish cohorts, while it decreased the motor scores in the Slovenia and Croatian sub-cohorts. The only socio-demographic factor consistently associated to the outcome among cohorts was child's sex, with females performing better than males on cognitive and language scores. These findings point to the need of harmonizing existing cohorts or creating prospective study designs that facilitate comparisons in the exposome over time, places and kind of environmental exposures.

Urinary bisphenol A in children, mothers and fathers from Slovenia: Overall results and determinants of exposure.

In the present study, urinary bisphenol A (BPA) levels were reported for the first time in the Slovenian general population and were evaluated with regard to dietary and non-dietary exposure sources, and compared according to age, gender and area of residence. First morning urine was collected from children (6-11 years), their mothers (30-52 years) and fathers (30-53 years), living in urban and rural areas of Slovenia. Besides basic questionnaire data on general population characteristics, socio-economic status and dietary habits, BPA-specific data was also collected, including consumption of food and beverages from plastic and canned containers, presence of white dental fillings, the use of specific consumer products and hormonal treatments. Urine samples were analysed for both free and conjugated BPA using GC-MS/MS. The urinary levels of total BPA in children, mothers and fathers were low, with geometric means of 1.51, 0.79, and 0.20 µg/g creatinine, respectively. The levels were comparable with the levels reported for other European countries and were all below the current health-based guidance values. In line with large-scale surveys, the data revealed age-dependant BPA urinary levels, with the highest levels in the youngest age group. In mothers, urinary levels of BPA were determined by hormonal interactions more than dietary sources, while a positive association between urinary BPA and diet was apparent in children (canned food/drink and food from plastic material) and fathers (canned food/drink). The study clearly shows that physiological and behavioural differences account for differences in levels of urinary BPA among study groups, a finding that sets the priorities for future research.

Arsenic metabolites; selenium; and AS3MT, MTHFR, AQP4, AQP9, SELENOP, INMT, and MT2A polymorphisms in Croatian-Slovenian population from PHIME-CROME study.

The relationships between inorganic arsenic (iAs) metabolism, selenium (Se) status, and genetic polymorphisms of various genes, commonly studied in populations exposed to high levels of iAs from drinking water, were studied in a Croatian-Slovenian population from the wider PHIME-CROME project. Population consisted of 136 pregnant women in the 3rd trimester and 176 non-pregnant women with their children (n = 176, 8-9 years old). Their exposure to iAs, defined by As (speciation) analyses of biological samples, was low. The sums of biologically active metabolites (arsenite + arsenate + methylated As forms) for pregnant women, non-pregnant women, and children, respectively were: 3.23 (2.84-3.68), 1.83 (1.54-2.16) and 2.18 (1.86-2.54) ng/mLSG; GM (95 CI). Corresponding plasma Se levels were: 54.8 (52.8-56.9), 82.3 (80.4-84.0) and 65.8 (64.3-67.3) ng/mL; GM (95 CI). As methylation efficiency indexes confirmed the relationship between pregnancy/childhood and better methylation efficiency. Archived blood and/or saliva samples were used for single nucleotide polymorphism (SNP) genotyping of arsenic(3+) methyltransferase - AS3MT (rs7085104, rs3740400, rs3740393, rs3740390, rs11191439, rs10748835, rs1046778 and the corresponding AS3MT haplotype); methylene tetrahydrofolate reductase - MTHFR (rs1801131, rs1801133); aquaporin - AQP 4 and 9 (rs9951307 and rs2414539); selenoprotein P1 - SELENOP (rs7579, rs3877899); indolethylamine N-methyltransferase - INMT (rs6970396); and metallothionein 2A - MT2A (rs28366003). Associations of SNPs with As parameters and urine Se were determined through multiple regression analyses adjusted using appropriate confounders (blood As, plasma Se, ever smoking, etc.). SNPs' influence on As methylation, defined particularly by the secondary methylation index (SMI), confirmed the 'protective' role of minor alleles of six AS3MT SNPs and their haplotype only among non-pregnant women. Among the other investigated genes, the carriers of AQP9 (rs2414539) were associated with more efficient As methylation and higher urine concentration of As and Se among non-pregnant women; poorer methylation was observed for carriers of AQP4 (rs9951307) among pregnant women and SELENOP (rs7579) among non-pregnant women; MT2A (rs28366003) was associated with higher urine concentration of AsIII regardless of the pregnancy status; and INMT (rs6970396) was associated with higher As and Se concentration in non-pregnant women. Among confounders, the strongest influence was observed for plasma Se; it reduced urine AsIII concentration during pregnancy and increased secondary methylation index among non-pregnant women. In the present study of populations with low As exposure, we observed a few new As-gene associations (particularly with AQPs). More reliable interpretations will be possible after their confirmation in larger populations with higher As exposure levels.

Risk characterization of bisphenol-A in the Slovenian population starting from human biomonitoring data.

The current study aims to characterize exposure and risk associated to bisphenol-A (BPA) exposure in Slovenia, starting from biomonitoring data. Based on the urinary data, daily intake for the individuals was back-calculated using a physiology based biokinetic (PBBK) model properly parameterized for BPA, coupled with an exposure reconstruction algorithm. Re-running the PBBK model in forward mode allowed the estimation of biologically effective dose (free plasma BPA) and the respective daily area under the curve (AUC). Finally, risk characterization ratio was derived using both external and internal dose metrics. The urinary BPA levels were found low, with GM of 0.79, 1.51 and 0.20 µg/g creatinine for mothers, children and fathers respectively, similar to the levels of other European countries. Based on the above and accounting for the dynamics of exposure and biokinetics, daily intake was estimated, median exposure levels have been estimated equal to 0.019, 0.035 and 0.005 µg/kg_bw/d for mothers, fathers and children respectively. The highest estimated intake level was found in a child, equal to 0.87 µg/kg_bw/d, while the maximum intake for mothers and fathers were 0.7 and 0.8 µg/kg_bw/d respectively. The respective RCR levels using the EFSA t-TDI of 4 µg/kg_bw/d were 2 magnitudes of order lower below 1, independently of the selected method. It has to be noted that had daily intake been estimated solely based on the urinary concentrations mass balance, the estimated intake would be lower, as a result of the oversimplification on exposure and elimination time dynamics. This highlights the importance for using PBBK modelling based exposure reconstruction schemes for rapidly metabolized and excreted compounds such as BPA, as well as the study design of efficient sampling for rapidly metabolized compounds.

Combined prenatal exposure to mercury and LCPUFA on newborn's brain measures and neurodevelopment at the age of 18 months.

OBJECTIVES: Prenatal exposure of long chain polyunsaturated fatty acids (LCPUFA) are essential for normal fetal growth and neurodevelopment. Availability of LCPUFA depends mostly on maternal fish consumption. Fish consumption also exposes the fetus to mercury which is well known neurotoxicant. We analyzed the associations of combined LCPUFA and mercury from fish consumption during pregnancy on newborn's brain measures and child neurodevelopment in a northern Adriatic coastal area. PATIENTS AND METHODS: The prospective cohort study included 257 mother - infant pairs enrolled in a susceptible population of the Public Health Impact on long-term, low-level, Mixed Element exposure (PHIME) EU Sixth Framework Programme from 2 recruitment areas of the northern part of the Adriatic coast. Umbilical cord blood taken at delivery was used for measuring concentration of total mercury (THg) and specific LCPUFA - docosahexaenoic acid (DHA) and arachidonic acid (ARA). Neonatal cranial sonography was performed at the age of 3 days in 57 newborns. Neurodevelopmental assessment of cognitive, motor and language skills were conducted at 257 children at the age of 18 months using the Bayley Scales of Infant and Toddler Development, Third Edition. The participants were divided into two groups depending on the THg concentration in the umbilical cord blood (exposed > 5.8 µg/L and unexposed < 5.8 µg/L). Dietary habits and exposures to environmental and social factors were assessed through questionnaires. RESULTS: There is a statistically significant difference in the cerebellum length (p = 0.032) and the superior frontal gyrus width (p = 0.023) between the exposed and the unexposed group. In combined analysis, including possible protective variables as DHA and ARA (R2 = 0.22, p = 0.001), the negative contribution of THg on cerebellum length (beta = -0.16, p = 0.001) persisted. We found no correlation between THg concentration in umbilical cord blood and child neurodevelopment scores at the age of 18 months. Language score with receptive and expressive subscores was significantly associated with fish consumption (p < 0.05). CONCLUSION: This analysis demonstrates the existence of morphological brain changes in newborns that are prenatally exposed at mercury concentrations what was diminished in combined analyse including LCPUFA. Our results emphasizes the importance of LCPUFA's and mercury common influence as a predictor of developmental outcome. Fish consumption, not solely LCPUFA contributes to better language development of children at the age of 18 months.

Prenatal selenium status, neonatal cerebellum measures and child neurodevelopment at the age of 18 months.

OBJECTIVES: The aim of this study was to evaluate the association of maternal blood selenium (Se) levels and cord blood Se levels with neonatal cerebellum measures and child neurodevelopment at the age of 18 months. Moreover, to investigate whether the neonatal cerebellum measures could be used as a potential biomarker for selenium homeostasis during pregnancy. STUDY GROUP AND METHODS: The study population consisted of 205 mother-child pairs from Croatian Mother and Child Cohort. Maternal blood and cord blood were obtained at delivery and selenium level was analyzed by Inductively Coupled Plasma Mass Spectrometry. Cranial ultrasonography examination was performed on 49 newborns - cerebellum length and width have been measured. Neurodevelopmental assessment of cognitive, language and motor skills were conducted on 154 children, using The Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), at the age of 18 months. RESULTS: The mean levels of selenium in maternal blood and cord blood were 92.6 ng/g and 97.0 ng/g, respectively. Maternal blood selenium levels were moderately and negatively correlated (r = -0.372; p = 0.008) with cerebellum length, while cord blood selenium levels were positively correlated with cerebellum width (r = 0.613; p = 0.007) among female children group. Maternal blood selenium levels were weakly and positively correlated (r = 0.176; p = 0.029) with child's cognitive abilities. CONCLUSION: To the best of our knowledge, our study is the first one investigating the association between neonatal brain measures and selenium levels in mother-child pairs. Our results indicate that prenatal selenium intake correlated with cerebellum length and width measured by cranial ultrasonography. Hence, cerebellum may be used as a potential biomarker and a target "organ" for early detection of possible adverse effects of prenatal status to various micronutrients.

Mercury speciation in prenatal exposure in Slovenian and Croatian population - PHIME study.

In recent years, several studies have addressed the issue of prenatal exposure to methylmercury (MeHg); however, few have actually analysed MeHg blood concentrations. Our study population included mothers and their new-borns from Slovenia (central region; N = 584) and Croatia (coastal region; N = 234). We have measurements of total Hg (THg) and MeHg in maternal hair, maternal peripheral blood, and cord blood. Cord blood Hg concentrations were low to moderate (median THg = 1.84 ng/g and MeHg = 1.69 ng/g). The proportion of THg as MeHg (%MeHg) in maternal and cord blood varied between 4% and 100% (coefficient of variation, CV = 32%) and between 8% and 100% (CV = 20%), respectively. Our data shows that variability of %MeHg was higher at lower blood THg levels. Concentrations of MeHg in maternal blood and cord blood were highly correlated (Rs = 0.943), in the case of inorganic Hg correlation was significant but weaker (Rs = 0.198). MeHg levels in maternal blood and cord blood were positively associated with seafood intake, maternal age, and negatively associated with pre-pregnancy BMI. Additionally, MeHg in maternal blood was positively associated with plasma selenium levels, and cord blood MeHg was negatively associated with parity. The results of multiple linear regression models showed that speciation analysis provides more defined estimation of prenatal exposure in association modelling. Associations between Hg exposure and cognitive performance of children (assessed using Bayley Scales of Infant and Toddler development) adjusted for maternal or child Apolipoprotein E genotypes showed higher model R2 and lower p-values when adjusted for MeHg compared to THg. This study demonstrates that Hg speciation improves the association between exposure and possible negative health effects.

Biomarkers of exposure in environment-wide association studies - Opportunities to decode the exposome using human biomonitoring data.

BACKGROUND: The European Union's 7th Framework Programme (EU's FP7) project HEALS - Health and Environment-wide Associations based on Large Population Surveys - aims a refinement of the methodology to elucidate the human exposome. Human biomonitoring (HBM) provides a valuable tool for understanding the magnitude of human exposure from all pathways and sources. However, availability of specific biomarkers of exposure (BoE) is limited. OBJECTIVES: The objective was to summarize the availability of BoEs for a broad range of environmental stressors and exposure determinants and corresponding reference and exposure limit values and biomonitoring equivalents useful for unraveling the exposome using the framework of environment-wide association studies (EWAS). METHODS: In a face-to-face group discussion, scope, content, and structure of the HEALS deliverable "Guidelines for appropriate BoE selection for EWAS studies" were determined. An expert-driven, distributed, narrative review process involving around 30 individuals of the HEALS consortium made it possible to include extensive information targeted towards the specific characteristics of various environmental stressors and exposure determinants. From the resulting 265 page report, targeted information about BoE, corresponding reference values (e.g., 95th percentile or measures of central tendency), exposure limit values (e.g., the German HBM I and II values) and biomonitoring equivalents (BEs) were summarized and updated. RESULTS: 64 individual biological, chemical, physical, psychological and social environmental stressors or exposure determinants were included to fulfil the requirements of EWAS. The list of available BoEs is extensive with a number of 135; however, 12 of the stressors and exposure determinants considered do not leave any measurable specific substance in accessible body specimens. Opportunities to estimate the internal exposure stressors not (yet) detectable in human specimens were discussed. CONCLUSIONS: Data about internal exposures are useful to decode the exposome. The paper provides extensive information for EWAS. Information included serves as a guideline - snapshot in time without any claim to comprehensiveness - to interpret HBM data and offers opportunities to collect information about the internal exposure of stressors if no specific BoE is available.

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

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

Prenatal exposure to low-level methylmercury alters the child's fine motor skills at the age of 18 months.

OBJECTIVES: To compare motor, cognitive and language characteristics in children aged 18 months who were prenatally exposed to low-level methyl-mercury (MeHg), and to analyze the eventual differences in these characteristics in relation to cord blood THg concentration. PATIENTS AND METHODS: The total number of 205 child-mother pairs was included in the study, and total cord blood mercury was measured in 198 of them. Out of the 198 already measured samples, 47 of them have also been tested for methyl-mercury in cord blood. Data regarding the 47 samples of MeHg levels has been used for calculating the correlation between cord blood THg and cord blood MeHg. MeHg and THg showed a significant correlation (r=0.95, p<0.05). One month after the delivery, mothers were asked to complete the questionnaire regarding socioeconomic factors, breastfeeding of their infants, and dietary habits during pregnancy. Neurodevelopmental assessment of motor, cognitive and language skills were conducted on 168 children using The Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III). Regarding the cord blood THg concentration, 135 children were divided in 4 quartile groups. Their neurodevelopmental characteristics have been compared. RESULTS: The cord blood THg concentration median and inter-quartile range was 2.98ng/g (1.41-5.61ng/g). There was a negative correlation between cord blood THg concentration and fine motor skills (rho=-0.22, p=0.01). It is evident that children grouped in 2nd ,3rd and 4th quartile had statistically significant lower fine motor skills assessment related to those grouped in 1st quartile (2nd quartile -1.24, p=0.03; 3rd quartile -1.28, p=0.03; 4th quartile -1.45, p=0.01). The differences in fine motor skills assessments between children in 2nd and 3rd and 3rd and 4th quartile were not statistically significant. CONCLUSION: Intrauterine exposure to low-level THg (MeHg) is associated with alterations in fine motor skills at the age of 18 months.

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

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

Prenatal mercury exposure, neurodevelopment and apolipoprotein E genetic polymorphism.

The aim of the present study was to evaluate the association between prenatal exposure to mercury (Hg) and neurodevelopment of the child, taking into account genetic polymorphism of apolipoprotein E (Apoe) and other relevant confounders. Six hundred and one mother-child pairs were recruited from the central Slovenia region and 243 from Rijeka, on the Croatian coast of the northern Adriatic. The total Hg in cord blood, Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) assessment at 18 months of age and Apoe genotyping was performed on 361 children; 237 of them were from Slovenia and 124 from Croatia. The results showed negative association between low-to-moderate Hg exposure in children with normal neurodevelopmental outcome and cognitive and fine motor scores at 18 months of age as assessed by Bayley III. The Hg-related decrease in cognitive score was observed only in children carrying at least one Apoe ε4 allele, while the decrease in fine motor scores was independent of the Apoe genotype. Adjusting for selenium (Se) and lead (Pb) levels, a positive association between Se and the language score and a negative association between Pb and the motor score was observed, but not in the subgroup of children carrying the ε4 allele.

Urinary BPA measurements in children and mothers from six European member states: Overall results and determinants of exposure.

For the first time in Europe, both European-wide and country-specific levels of urinary Bisphenol A (BPA) were obtained through a harmonized protocol for participant recruitment, sampling and quality controlled biomarker analysis in the frame of the twin projects COPHES and DEMOCOPHES. 674 child-mother pairs were recruited through schools or population registers from six European member states (Belgium, Denmark, Luxembourg, Slovenia, Spain and Sweden). Children (5-12 y) and mothers donated a urine sample. Information on socio-demographic characteristics, life style, dietary habits, and educational level of the parents was provided by mothers. After exclusion of urine samples with creatinine values below 300 mg/L or above 3000 mg/L, 653 children and 639 mothers remained for which BPA was measured. The geometric mean (with 95% confidence intervals) and 90th percentile were calculated for BPA separately in children and in mothers and were named "European reference values". After adjustment for confounders (age and creatinine), average exposure values in each country were compared with the mean of the "European reference values" by means of a weighted analysis of variance. Overall geometric means of all countries (95% CI) adjusted for urinary creatinine, age and gender were 2.04 (1.87-2.24) µg/L and 1.88 (1.71-2.07) µg/L for children (n=653) and mothers (n=639), respectively. Multiple regression analysis was used to identify significant environmental, geographical, personal or life style related determinants. Consumption of canned food and social class (represented by the highest educational level of the family) were the most important predictors for the urinary levels of BPA in mothers and children. The individual BPA levels in children were significantly correlated with the levels in their mothers (r=0.265, p<0.001), which may suggest a possible common environmental/dietary factor that influences the biomarker level in each pair. Exposure of the general European population was well below the current health-based guidance values and no participant had BPA values higher than the health-based guidance values.

Exposure determinants of cadmium in European mothers and their children.

The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother-child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24-52 years of age), and 1689 children (5-12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011-2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1-28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18-86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1-13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9-12 years had 7% (95% CI 1-13%) higher UCd (p=0.04) than children 5-8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.