Lead, cadmium, and mercury blood levels in schoolchildren in southern Sweden: Time trends over the last decades.

To prevent diseases arising from exposure to toxic metals, more knowledge about their temporal changes is needed, especially in children, the most vulnerable group. This study follows temporal changes in blood lead (BPb), mercury (BHg) and cadmium (BCd) levels in schoolchildren (8-11 years old) from two cities in southern Sweden. One blood sample per 773 children was used for time trend analyses between 2007 and 2022. One further blood sample re-sampled after 2 years, were used to assess intra-individual time trends of BPb (n = 377), BCd (n = 102) and BHg (n = 53) between 1979 and 2019. Geometric mean (range) of BPb, BCd and BHg concentrations during 2007-2022 was 9.9 (2.3-59), 0.09 (0.03-0.34) and 0.73 (0.02-8.2) µg/L, respectively. Living close to a Pb smelter resulted in higher levels of all three metals compared with living in the city or rural area. Annually, the concentrations clearly decreased for BPb (-4.9%, p < 0.001) and weakly for BCd (-0.6%, p = 0.013), while BHg slightly increased (+1.4%, p = 0.029). When stratified by residential area, the decrease of BCd and increase of BHg were significant only in the urban area (-1.8% and +2.8%, respectively; p < 0.01). The BPb decrease rate was the highest in the urban area followed by the rural and Pb smelter areas (-5.8% > -4.5% > -3.9%; p < 0.001). For children re-analysed during 1979-2019, a significant decrease was observed only for BPb (-6.8%; p < 0.001), with a 2% higher decrease rate in the period before than after the Pb-gasoline ban in 1994. The preventive measures against Pb pollution are reflected in the constant decrease of BPb levels over time. However, the area close to a Pb smelter, as indicated by a slower Pb decrease rate, might need further and stricter preventive measures. Exposure to Hg and Cd was low, however, the slight increase in BHg and only a minor decrease in BCd, indicate the need for continuous biomonitoring of children.

Genetic susceptibility to low-level lead exposure in men: Insights from ALAD polymorphisms.

The genetic susceptibility to low-level lead (Pb) exposure in general populations has been poorly investigated and is limited to the single nucleotide polymorphism (SNP) rs1800435 in the delta-aminolevulinic acid dehydratase gene (ALAD). This study explored associations between ten selected ALAD SNPs with Pb concentrations in blood (BPb) and urine (UPb) among 281 men aged 18-49 years from Slovenia, including 20 individuals residing in a Pb-contaminated area. The geometric mean (range) of BPb and UPb were 19.6 (3.86-84.7) µg/L and 0.69 (0.09-3.82) µg/L SG, respectively. The possible genetic influence was assessed by examining SNP haplotypes, individual SNPs, and the combination of two SNPs using multiple linear regression analyses. While no significant associations were found for haplotypes, the presence of variant alleles of rs1800435 and rs1805312 resulted in an 11% and 13% decrease in BPb, respectively, while the presence of variant allele of rs1139488 (homozygous only) exhibited significant 20% increase in BPb, respectively. Additionally, variant allele of rs1800435 resulted in lower UPb. Individual SNPs in the model explained only around 1 additional percentage point of BPb variability. In contrast, combination analyses identified six combinations of two SNPs, which significantly explained 3-22 additional percentage points of BPb variability, with the highest explanatory power observed for the rs1800435-rs1139488 and rs1139488-rs1805313 combinations. Moreover, excluding participants from the Pb-contaminated area indicated that exposure level influenced SNPs-Pb associations. Our results confirm the importance of the ALAD gene in Pb kinetics even at low exposure levels. Additionally, we demonstrated that identifying individuals with specific combinations of ALAD SNPs explained a larger part of Pb variability, suggesting that these combinations, pending confirmation in other populations and further evaluation through mechanistic studies, may serve as superior susceptibility biomarker in Pb exposure compared to individual SNPs.

Exposure to Phthalates in European Children, Adolescents and Adults since 2005: A Harmonized Approach Based on Existing HBM Data in the HBM4EU Initiative.

Phthalates are mainly used as plasticizers and are associated inter alia with adverse effects on reproductive functions. While more and more national programs in Europe have started monitoring internal exposure to phthalates and its substitute 1,2-Cyclohexanedicarboxylic acid (DINCH), the comparability of results from such existing human biomonitoring (HBM) studies across Europe is challenging. They differ widely in time periods, study samples, degree of geographical coverage, design, analytical methodology, biomarker selection, and analytical quality assurance level. The HBM4EU initiative has gathered existing HBM data of 29 studies from participating countries, covering all European regions and Israel. The data were prepared and aggregated by a harmonized procedure with the aim to describe-as comparably as possible-the EU-wide general population's internal exposure to phthalates from the years 2005 to 2019. Most data were available from Northern (up to 6 studies and up to 13 time points), Western (11; 19), and Eastern Europe (9; 12), e.g., allowing for the investigation of time patterns. While the bandwidth of exposure was generally similar, we still observed regional differences for Butyl benzyl phthalate (BBzP), Di(2-ethylhexyl) phthalate (DEHP), Di-isononyl phthalate (DiNP), and Di-isobutyl phthalate (DiBP) with pronounced decreases over time in Northern and Western Europe, and to a lesser degree in Eastern Europe. Differences between age groups were visible for Di-n-butyl phthalate (DnBP), where children (3 to 5-year olds and 6 to 11-year olds) had lower urinary concentrations than adolescents (12 to 19-year-olds), who in turn had lower urinary concentrations than adults (20 to 39-year-olds). This study is a step towards making internal exposures to phthalates comparable across countries, although standardized data were not available, targeting European data sets harmonized with respect to data formatting and calculation of aggregated data (such as developed within HBM4EU), and highlights further suggestions for improved harmonization in future studies.

Exposure of children and adolescents from Northeastern Slovenia to per- and polyfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are of high concern for the environment, wildlife, and human health due to their persistence and potential to cause adverse health effects. Despite political measures to restrict the production and distribution of PFAS and to limit the exposure of populations, PFAS can be measured at commonly high detection frequencies in human samples. Thus, this pilot study aimed to determine the serum concentrations of PFPA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, PFHpS, PFOS, PFHxA, PFDoDA, and PFBS in 113 girls and 112 boys (age 7-10 and 12-15) from Northeastern Slovenia - a rural area characterized by agricultural activities - and to identify potential sources of exposure using questionnaire data. PFAS were analysed by liquid chromatography coupled to mass spectrometry after phospholipid removal. 9 out of 12 analytes were detected at detection frequencies above 30%, with the highest geometric means (GM) being observed for PFOS (GM 1.9 ng/mL) > PFOA (GM 1.0 ng/mL) > PFHxS (GM 0.3 ng/mL) = PFNA (GM 0.3 ng/mL). We identified the participants' socio-economic status, age, sex, sampling region, public water supply, and the consumption of fish and seafood, cereals, and locally produced fruits, vegetables, and mushrooms as the predominant determinants of exposure. Furthermore, we compared our results with the serum and plasma concentrations reported for similar age groups in other studies and concluded that PFAS exposure in this highly agricultural area in Slovenia is notably low. This is the first study systematic HBM study of PFAS exposure in Slovenia, although it was conducted on a limited number of participants representative of rural and agricultural areas, it represents a good basis for upgrading the approach to a nationwide HBM study.

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.

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.

Glyphosate and AMPA in Human Urine of HBM4EU Aligned Studies: Part A Children.

Few data are available on the exposure of children to glyphosate (Gly) in Europe. Within HBM4EU, new HBM exposure data were collected from aligned studies at five sampling sites distributed over Europe (studies: SLO CRP (SI); ORGANIKO (CY); GerES V-sub (DE); 3XG (BE); ESTEBAN (FR)). Median Gly concentrations in urine were below or around the detection limit (0.1 µg/L). The 95th percentiles ranged between 0.18 and 1.03 µg Gly/L. The ratio of AMPA (aminomethylphosphonic acid; main metabolite of Gly) to Gly at molar basis was on average 2.2 and the ratio decreased with higher Gly concentrations, suggesting that other sources of AMPA, independent of metabolism of Gly to AMPA in the monitored participants, may concurrently operate. Using reverse dosimetry and HBM exposure data from five European countries (east, west and south Europe) combined with the proposed ADI (acceptable daily intake) of EFSA for Gly of 0.1 mg/kg bw/day (based on histopathological findings in the salivary gland of rats) indicated no human health risks for Gly in the studied populations at the moment. However, the absence of a group ADI for Gly+AMPA and ongoing discussions on e.g., endocrine disrupting effects cast some uncertainty in relation to the current single substance ADI for Gly. The carcinogenic effects of Gly are still debated in the scientific community. These outcomes would influence the risk conclusions presented here. Finally, regression analyses did not find clear associations between urinary exposure biomarkers and analyzed potential exposure determinants. More information from questionnaires targeting exposure-related behavior just before the sampling is needed.

Polycyclic aromatic hydrocarbons (PAHs) in men and lactating women in Slovenia: Results of the first national human biomonitoring.

In the first national human biomonitoring study in the Slovenian population of adults (18-49 years), including men (n = 297) and lactating primiparous women (n = 304), exposure to polycyclic aromatic hydrocarbons (PAHs) was evaluated. Nine urinary metabolites of four parent PAHs were determined. These included 1-hydroxypyrene (1-OHPYR), 2-hydroxynaphthalene (2-OHNAP), 2-hydroxyphenanthrene (2-OHPHE), 3-hydroxyphenanthrene (3-OHPHE), 4-hydroxyphenanthrene (4-OHPHE), a combination of 2-hydroxyfluorene and 3-hydroxyfluorene (2/3-OHFLU) and a combination of 1-hydroxyphenanthrene and 9-hydroxyphenanthrene (1/9-OHPHE). For comparison, the analysed phenanthrene metabolites were reported as a sum (ΣOHPHE = 1/9-OHPHE + 2-OHPHE + 3-OHPHE + 4-OHPHE) and all the analysed PAH metabolites were reported as a sum (ΣOHPAH = 1-OHPYR + 2/3-OHFLU + 2-OHNAP + ΣOHPHE). All metabolites or their combinations were determined in more than 91% of the samples, except 4-OHPHE, which was determined in only 5% of the samples. The highest concentration was found for 2-OHNAP. This was followed by 2/3-OHFLU and the phenanthrene metabolites, while the lowest concentration was determined for 1-OHPYR. Among the phenanthrene metabolites, the highest concentration was determined for 2-OHPHE, followed by 1/9-OHPHE and then by 3-OHPHE. Values in units of volume and values adjusted for specific gravity were significantly higher in men than in lactating primiparous women for all metabolites, whereas values in units adjusted for creatinine were generally higher in lactating primiparous women than in men. The difference between the two study groups, men and lactating primiparous women, was no longer significant in statistical models adjusted for specific gravity, suggesting that smoking, wood-burning exposure, and/or education largely explained the difference in PAH exposure in both study groups. For most metabolites, predictors of exposure were less significant in lactating primiparous women than in men. Also, site-specific patterns of exposure were observed, with additional predictors identified in certain areas, namely, proximity to roads and release of particulate matter (PM10) from industry. The time of year in which sampling took place appeared to be an important determinant in urban areas and in the case of participants who used wood for heating. Specific dietary factors could not be identified, as the study questionnaire did not include information on PAH-related diet. Despite the low number of paired partners (women and men living in the same household, n = 84), significant positive correlations for all metabolites were observed. This indicated that 31%-56% of variability in exposure could be explained by shared exposure to sources within the households (such as diet and wood-burning-related determinants).

Assessment of susceptibility to phthalate and DINCH exposure through CYP and UGT single nucleotide polymorphisms.

Single nucleotide polymorphisms (SNPs) of cytochrome P450 (CYPs) and UDP-glucuronosyltransferase (UGTs) genes have been proposed to influence phthalates and 1,2-cyclo-hexanedicarboxylic acid diisononyl ester (DINCH) biotransformation but have not been investigated on a populational level. We investigated the role of SNPs in CYP2C9, CYP2C19, CYP2D6, UGT2B15, and UGT1A7 genes in the biotransformation of phthalates (DEHP, DEP, DiBP, DnBP, BBzP, DiNP, DidP) and DINCH by determining their urine metabolites. From the Slovenian study population of 274 men and 289 lactating primiparous women we obtained data on phthalate and DINCH urine metabolite levels (MEHP, 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP, MEP, MiBP, MnBP, MBzP, cx-MINP, OH-MiDP, MCHP, MnPeP, MnOP, 5OH-MINCH, 5oxo-MINCH), SNP genotypes (rs1057910 = CYP2C9*3, rs1799853 = CYP2C9*2, rs4244285 = CYP2C19*2, rs12248560 = CYP2C19*17, rs3892097 = CYP2D6*4, rs1902023 = UGT2B15*2, and rs11692021 = UGT1A7*3) and questionnaires. Associations of SNPs with levels of metabolites and their ratios were assessed by multiple linear regression and ordinary logistic regression analyses. Significant associations were observed for CYP2C9*2, CYP2C9*3, CYP2C19*17, and UGT1A7*3 SNPs. The most pronounced was the influence of CYP2C9*2 and *3 on the reduced DEHP biotransformation, with lower levels of metabolites and their ratios in men and women. In contrast, carriers of CYP2C19*17 showed higher urine levels of DEHP metabolites in both genders, and in women also in higher DiNP, DiDP, and DINCH metabolite levels. The presence of UGT1A7*3 was associated with increased metabolite levels of DINCH in men and of DiBP and DBzP in women. Statistical models explained up to 27% of variability in metabolite levels or their ratios. Our observations confirm the effect of CYP2C9*2 and *3 SNPs towards reduced DEHP biotransformation. We show that CYP2C9*2, CYP2C9*3, CYP2C19*17, and UGT1A7*3 SNPs might represent biomarkers of susceptibility or resilience in phthalates and DINCH exposure that have been so far unrecognised.

Exposure of Slovenian children and adolescents to bisphenols, parabens and triclosan: Urinary levels, exposure patterns, determinants of exposure and susceptibility.

Chemicals such as bisphenols, parabens and triclosan are endocrine disrupting chemicals. They are used in a wide variety of consumer products, making human exposure to those chemicals widespread. In the present study, levels of three bisphenols (bisphenol A, F and S), 7 parabens (methyl-, ethyl-, isopropyl-, propyl-, isobutyl-, butyl-, benzyl paraben) and triclosan were measured in first morning void from 246 Slovenian children and adolescents, aged 6-9 and 11-15 years and living in a rural region of Slovenia. Median levels of specific-gravity corrected levels for bisphenol A, bisphenol F, methyl paraben and ethyl paraben were 1.9, 0.085, 5.4 and 2.5 µg/L for children and 1.6, 0.11, 7.2 and 6.0 µg/L for adolescents, respectively. Median levels for all other endocrine disrupting chemicals were < LOQ. The levels are comparable with the levels reported in studies across the world. Exposure was age, sex, and location specific. Higher levels of bisphenol F and ethyl paraben were found in the samples of adolescents, while higher levels of methyl paraben were found in samples from girls. Furthermore, individuals living in one of the sampling locations, Goricko, were exposed to higher levels of bisphenol F and ethyl paraben than those in the remaining two sampling locations. Information about participants' dietary habits, use of food packaging and personal care products was obtained through questionnaires, and used to investigate associations between urinary levels of the biomarkers and potential exposure sources. High fat foods were associated with bisphenol A exposure, and cosmetics items such as lipstick and perfume with methyl paraben exposure. Significant correlation between methyl- and propyl paraben was observed in children's samples, suggesting similar exposure sources, while other compounds were not largely correlated, indicating independent sources. Furthermore, association between a single nucleotide polymorphism (SNP) in UGT2B15 gene and urinary levels of methyl and ethyl paraben was observed, showing the role of UGT2B15 isoform in methyl and ethyl paraben metabolism as well as indicating the SNP rs1902023 as a potential biomarker of susceptibility to adverse effects caused by the exposure. The present study reports exposure of children and adolescents in Slovenia to a wide range of different endocrine disrupting chemicals for the first time, connecting it to exposure patterns and exposure sources. The study is to the authors' knowledge the first that investigates direct connection between levels of urinary endocrine disrupting chemical biomarkers and genetic polymorphism in UGT2B15.

Trace elements and APOE polymorphisms in pregnant women and their new-borns.

We investigated the relationship between lipid binding glycoprotein apolipoprotein E (apoE; gene APOE) polymorphisms (ε4 allele carriers versus no carriers = Îµ4+/ε4-) and trace elements (TEs) (e.g., (methyl)mercury, arsenic, lead, cadmium, selenium, manganese, copper, and zinc) in mothers (N = 223) and their new-borns (N = 213) exposed to potentially toxic metal(loid)s from seafood consumption. The apoE isoform encoded by the ε4 allele is believed to have beneficial effects in early life but represents a risk factor for age-associated diseases. Under certain conditions ε4 carriers are more susceptible to oxidative stress and metal(loid) toxicity. DNA from Croatian pregnant women (N = 223, third trimester) and their new-borns (N = 176), was genotyped for APOE by TaqMan® SNP assay - rs429358 and rs7412. Seafood intake data and TE levels in maternal urine, milk, hair, peripheral venous blood, mixed cord blood, and new-borns' urine were available from previous studies. We compared TEs between ε4+ and ε4- carriers using Mann-Whitney U tests and applied multiple linear regression models to analyse the TE's dependence on the presence of allele ε4 (genotypes ε3/ε4, ε4/ε4) in combination with other explanatory variables. We identified 17% (n = 37) and 20% (n = 35) ε4 allele carriers in mothers and new-borns, respectively. The Mann-Whitney U test showed that mothers with the ε4 allele had significantly higher mean levels of (methyl)mercury in peripheral venous blood, cord blood, and hair; arsenic in urine and cord blood; and selenium in peripheral venous blood and plasma. However, taking confounders into account, only the maternal plasma selenium remained statistically significant in the linear regression models (ε4 carriers vs non-carriers: 62.6 vs 54.9 ng/mL, p < 0.001). Literature suggestions of possible ε4 allele impact on Hg levels were not observed, while superior selenium status observed in healthy pregnant women carrying allele ε4 could be linked to the proposed APOE ε4 beneficial effects early in life.

Seasonal glyphosate and AMPA levels in urine of children and adolescents living in rural regions of Northeastern Slovenia.

There are extensive data on the toxicity of glyphosate (GLY) based herbicides (GBH), however the interpretation of some data (e.g. carcinogenic effect) are subject to controversy. For the appropriate health risk assessment more data on exposure levels in the general population, especially in susceptible groups such as pregnant women, the elderly and children are needed. The aims of the present study were to estimate the exposure to GLY and its major metabolite aminomethylphosphonic acid (AMPA) in children and adolescents living in agricultural areas, to identify possible determinants of the exposure, and to assess co-exposure with elements. In total, 149 children (aged 7-10 years, 55% girls) and 97 adolescents (aged 12-15 years; 44% girls) were recruited in 2018 from rural areas of Northeastern Slovenia. The effect of seasonal GLY application on the exposure was estimated using GLY and AMPA levels determined by GC-MS/MS in first morning urine in winter (n = 246) and in late-spring/early-summer seasons (n = 225). Levels of elements were determined by ICP-MS in urine in both samplings and in blood or plasma in the first sampling. Questionnaire data on basic characteristics, dietary habits, living environments and use of pesticides were obtained for all participants. GLY and AMPA were detected in 27% and 50% of urine samples from the first sampling period, respectively; and in 22% and 56% from the second sampling period, respectively. Geometric means and medians of both AMPA and GLY were below or at the limit of quantification (≤LOQ; 0.1 µg/L). Children rather than adolescents tended to have higher exposure, as did, boys rather than girls among adolescents. The exposure did not significantly differ between both sampling periods. Except for one individual, exposure was not higher among participants who reported use of GLY or herbicides in the vicinity of child's home or live in close vicinity of agriculture, orchards, vineyards, gardens, sport courts or cemeteries. The extensive food consumption frequency data revealed higher exposure to GLY and AMPA only among individuals with higher consumption of nuts and wholegrain rice. Levels of AMPA and GLY were significantly positively correlated, with considerably stronger correlation in urine of the second than the first sampling (Spearman's rank coefficient: 0.49 vs 0.22, respectively). Urine levels of As, Pb, Co, Zn and Cu were significantly higher in participants with GLY and/or AMPA levels ≥LOQ than with levels

Trace elements and ALAD gene polymorphisms in general population from three uranium legacy sites - A case study in Kyrgyzstan.

At three uranium (U) legacy sites in Kyrgyzstan, namely, Kadji Sai, Mailuu-Suu and Sumsar, an initial human bio-monitoring programme was introduced as a complementary activity to environmental impact studies in these areas. The aim was to assess trace element (TE) contents in blood and genetic susceptibility for Pb as one of the contaminants. The programme included the determination of 9 TE in blood samples from 123 residents living permanently in this environment. The analyses included U and the potentially toxic TE, lead (Pb), cadmium, mercury (Hg), and arsenic (As), together with essential elements iron (Fe), copper, selenium (Se) and manganese (Mn). TE were analysed by inductively coupled plasma mass spectrometry (ICPMS) and genetic background effect by three single nucleotide polymorphisms (SNPs) of delta-aminolevulinic acid dehydratase (ALAD; rs1805313, rs818708, rs1800435) genotyped by quantitative polymerase chain reaction (qPCR). The obtained results were generally similar to literature reference values obtained from the U non-exposed environments. However, some significant findings indicated elevated levels of certain contaminants typical of the studied environment (U, Pb). Several essential (Se, Mn) and toxic TE (Pb, Hg, As, U) in blood showed statistically significant differences among the studied areas. All areas showed diminished Fe blood levels. Altogether, this indicated specific and different environmental conditions at three industrial legacy sites for U milling and processing along with the accompanying chemical (pollutant) elements. Blood U concentrations were slightly higher at Mailuu-Suu, known for elevated technogenic and naturally occurring U. At Sumsar, the distribution of elevated blood Pb concentrations indicated an airborne source of pollution that was different from the anticipated aqueous exposure pathway. Pb blood variability was found associated with ALAD polymorphisms (SNPs rs1805313, rs1800435). Results are confirming that human data will be a useful and scientifically important additional tool for environmental impact assessment studies at industrial legacy sites in Kyrgyzstan.

Results of the first national human biomonitoring in Slovenia: Trace elements in men and lactating women, predictors of exposure and reference values.

The first national human biomonitoring in Slovenia surveyed cadmium (Cd), lead (Pb), mercury (Hg), arsenic (As), manganese (Mn), selenium (Se), copper (Cu) and zinc (Zn) in a childbearing population (18-49 years) selected from lactating primiparous women and men (N = 1084). The overall aim was to estimate trace elements' levels and geographical variations in order to identify sources of possible exposures and set the national reference values. The study population was selected evenly from 12 study areas across Slovenia, including rural, urban and known or potentially contaminated environments. Within 6-8 weeks after delivery, venous blood, spot urine, scalp hair and breast milk samples were collected to determine the selected elements. The data analysis included descriptive statistics and multiple linear regression using elemental concentrations in biological matrices, questionnaire data and environmental datasets. Essential elements showed no significant deficiencies or excessive levels in the study population and were largely determined by sex and/or the participating women's physiological status (postpartum, lactation), as well as by certain dietary sources. Toxic elements' levels were mainly below the levels considered to present increased health risk. Lifestyle and nutritional habits appeared as significant determinants of exposure to Cd (smoking and game meat consumption), Hg (seafood and amalgam fillings), As (seafood) and Pb (alcohol consumption, smoking, game meat consumption and type of water supply). A distinctive geographical pattern was confirmed, due to past mining activities combined with naturally elevated background levels in the cases of Pb (Mezica Valley), Hg (Idrija and Posocje) and As exposure (Zasavje). Increased seafood consumption in the coastal study area contributed to higher Hg and As (arsenobetaine) levels. Extensive sample size database accompanied with life-style and environmental data improved the prediction of exposure patterns, set the reference values for the childbearing population living in Slovenia, and provided a strong basis for evaluating spatial and temporal trends in exposure. To our best knowledge, this is the first study to establish reference values for lactating primiparous women.

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.

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.

Bioaccumulation of arsenic species in rays from the northern Adriatic Sea.

The difference in arsenic concentration and speciation between benthic (Pteromylaeus bovinus, Myliobatis aquila) and pelagic rays (Pteroplatytrygon violacea) from the northern Adriatic Sea (Gulf of Trieste) in relation to their size (age) was investigated. High arsenic concentrations were found in both groups with tendency of more efficient arsenic accumulation in benthic species, particularly in muscle (32.4 to 362 µg·g-1 of total arsenic). This was attributed to species differences in arsenic access, uptake and retention. In liver most arsenic was present in a form of arsenobetaine, dimethylarsinic acid and arsenoipids, whereas in muscle mainly arsenobetaine was found. The good correlations between total arsenic/arsenobetaine and size reflect the importance of accumulation of arsenobetaine with age. Arsenobetaine is an analogue of glycine betaine, a known osmoregulator in marine animals and both are very abundant in mussels, representing an important source of food for benthic species P. bovinus and M. aquila.