Human-Biomonitoring derived exposure and Daily Intakes of Bisphenol A and their associations with neurodevelopmental outcomes among children of the Polish Mother and Child Cohort Study.

BACKGROUND: Bisphenol A (BPA) is an industrial chemical mostly used in the manufacture of plastics, resins and thermal paper. Several studies have reported adverse health effects with BPA exposures, namely metabolic disorders and altered neurodevelopment in children, among others. The aim of this study was to explore BPA exposure, its socio-demographic and life-style related determinants, and its association with neurodevelopmental outcomes in early school age children from Poland. METHODS: A total of 250 urine samples of 7 year-old children from the Polish Mother and Child Cohort Study (REPRO_PL) were analyzed for BPA concentrations using high performance liquid chromatography with online sample clean-up coupled to tandem mass spectrometry (online-SPE-LC-MS/MS). Socio-demographic and lifestyle-related data was collected by questionnaires or additional biomarker measurements. Emotional and behavioral symptoms in children were assessed using mother-reported Strengths and Difficulties Questionnaire (SDQ). Cognitive and psychomotor development was evaluated by Polish adaptation of the Intelligence and Development Scales (IDS) performed by trained psychologists. RESULTS: Urinary BPA concentrations and back-calculated daily intakes (medians of 1.8 µg/l and 46.3 ng/kg bw/day, respectively) were similar to other European studies. Urinary cotinine levels and body mass index, together with maternal educational level and socio-economic status, were the main determinants of BPA levels in Polish children. After adjusting for confounding factors, BPA has been found to be positively associated with emotional symptoms (ß: 0.14, 95% CI: 0.022; 0.27). Cognitive and psychomotor development were not found to be related to BPA levels. CONCLUSIONS: This study represents the first report of BPA levels and their determinants in school age children in Poland. The exposure level was found to be related to child emotional condition, which can have long-term consequences including social functioning and scholastic achievements. Further monitoring of this population in terms of overall chemical exposure is required.

Parabens in urine of children and adolescents in Germany - human biomonitoring results of the german environmental survey 2014-2017 (GerES V).

Parabens are antimicrobial preservatives used in a wide range of consumer products such as personal care products, cosmetics, pharmaceuticals, and food. Consequently, the general population is ubiquitously exposed to these substances via dermal absorption, ingestion, and inhalation. Parabens promote estrogenic activity and are hence under assessment as endocrine disrupting substances. Urine samples from 3- to 17-year-old children and adolescents (N = 516) living in Germany were analysed for concentrations of nine parabens in the population representative German Environmental Survey for Children and Adolescents 2014-2017 (GerES V). Detection rates and urinary concentrations of the parabens decreased with increasing length of the alkyl chain. Methyl paraben was quantified in 97% of the samples with a geometric mean (GM) concentration of 7.724 µg/L (6.714 µg/gcreatinine), ethyl paraben was quantified in 69% (GM: 0.943 µg/L and 0.825 µg/gcrea), and n-propyl paraben in 31% (GM: 0.563 µg/L and 0.493 µg/gcrea). Concentrations of iso-propyl paraben, butyl paraben, iso-butyl paraben, and benzyl paraben were below the limit of quantification in most samples. Pentyl paraben and heptyl paraben were not detected in any of the samples. Paraben concentrations in urine were found to be associated with frequent usage of leave-on personal care products and cosmetics. Cumulative exposure to parabens (back-calculated daily intakes, expressed as hazard index) was found to be on a level raising concern in up to 14% of the population, mainly driven by n-propyl paraben, and depending on the level of conservativeness and point-of departures used for calculation.

Bisphenol A and six other environmental phenols in urine of children and adolescents in Germany - human biomonitoring results of the German Environmental Survey 2014-2017 (GerES V).

Exposure to environmental phenols such as bisphenol A, benzophenones, 2-phenylphenol, triclosan, and triclocarban is of concern, because of their endocrine disrupting properties and broad application in consumer products. The current body burden of the 3-17-year-old population in Germany to these substances was assessed in first-morning void urine samples (N = 515-516) collected within the population-representative German Environmental Survey for Children and Adolescents 2014-2017 (GerES V). Bisphenol A was the most prominent phenol analysed here, ubiquitously found in almost all samples with a geometric mean (GM) concentration of 1.905 µg/L (1.669 µg/gcreatinine) and a maximum (MAX) urinary concentration of 399 µg/L. Benzophenone-3 and benzophenone-1 were quantified in 35% and 41% of the samples. GM was below the limit of quantification (LOQ) for benzophenone-3 and 0.559 µg/L (0.489 µg/gcrea) for benzophenone-1, MAX concentrations were 845 µg/L and 202 µg/L, respectively. In 16% of the samples triclosan was found in quantifiable amounts resulting in a GM below LOQ and a MAX concentration of 801 µg/L. Benzophenone-8, 2-phenylphenol and triclocarban were quantified in none or only 1% of the samples. Benzophenone-1 and -3 concentrations were found to be associated with frequent application of personal care products. A comparison with the previous cycle of the survey, GerES IV (2003-2006), showed a decrease of urinary bisphenol A concentrations, mainly in young children. Despite this decrease, the concentration of bisphenol A exceeded the human biomonitoring (HBM) value HBM-I of 0.1 mg/L in 0.11% of the samples. For triclosan, all urinary concentrations were well below the HBM-I value of 2 mg/L. To minimise environmental health risks, it is therefore necessary to maintain a further declining trend for bisphenol A and continue monitoring the exposure to environmental phenols, as well as to monitor substitutes such as bisphenol F and S.

Metabolites of n-Butylparaben and iso-Butylparaben Exhibit Estrogenic Properties in MCF-7 and T47D Human Breast Cancer Cell Lines.

Two oxidized metabolites of n-butylparaben (BuP) and iso-butylparaben (IsoBuP) discovered in human urine samples exhibit structural similarity to endogenous estrogens. We hypothesized that these metabolites bind to the human estrogen receptor (ER) and promote estrogen signaling. We tested this using models of ER-mediated cellular proliferation. The estrogenic properties of 3-hydroxy n-butyl 4-hydroxybenzoate (3OH) and 2-hydroxy iso-butyl 4-hydroxybenzoate (2OH) were determined using the ER-positive, estrogen-dependent human breast cancer cell lines MCF-7, and T47D. The 3OH metabolite induced cellular proliferation with EC50 of 8.2 µM in MCF-7 cells. The EC50 for 3OH in T47D cells could not be reached. The 2OH metabolite induced proliferation with EC50 of 2.2 µM and 43.0 µM in MCF-7 and T47D cells, respectively. The EC50 for the parental IsoBuP and BuP was 0.30 and 1.2 µM in MCF-7 cells, respectively. The expression of a pro-proliferative, estrogen-inducible gene (GREB1) was induced by these compounds and blocked by co-administration of an ER antagonist (ICI 182, 780), confirming the ER-dependence of these effects. The metabolites promoted significant ER-dependent transcriptional activity of an ERE-luciferase reporter construct at 10 and 20 µM for 2OH and 10 µM for 3OH. Computational docking studies showed that the paraben compounds exhibited the potential for favorable ligand-binding domain interactions with human ERα in a manner similar to known x-ray crystal structures of 17ß-estradiol in complex with ERα. We conclude that the hydroxylated metabolites of BuP and IsoBuP are weak estrogens and should be considered as additional components of potential endocrine disrupting effects upon paraben exposure.

Urinary parabens and triclosan concentrations and associated exposure characteristics in a Korean population-A comparison between night-time and first-morning urine.

Parabens and triclosan have been widely used in many personal care products and cosmetics. The endocrine disrupting potential of these compounds is of increasing public health concern. The aim of this study is to understand the current exposure profile of these chemicals in last void before bedtime (night-time) and first-morning void (first-morning) urines among a Korean population and to characterize their exposure sources and pathways. A total of 261 people, including infants (0-2 years), toddlers (3-6 years), children (7-12 years), adolescents (13-18 years), and adults (≥19 years), were recruited, and sampled for night-time urine and first-morning urine of the following day. Methyl (MeP), ethyl (EtP), propyl (PrP) and butyl paraben (BuP), and triclosan were measured in urine. The demographic characteristics, use of personal care products, and food consumption were obtained through a questionnaire. Among the target compounds, EtP and MeP were most frequently detected at the highest concentrations. The median concentration of EtP in night-time urine was 32.4 µg/L (interquartile range: 8.37-82.8 µg/L), which is higher than previously reported worldwide. Unlike other test compounds, compared to those measured from first-morning urine, the EtP concentrations were significantly higher in night-time urine, suggesting the presence of different exposure sources. Among adults, the MeP and PrP concentrations in night-time urine were associated with frequent use of skin care products, colored cosmetics, bath products, toothpaste, vinyl food packaging, or consumption of canned food. The MeP and PrP concentrations were higher in females than in males, especially in night-time urine. The results of this study also show that multiple urine samples are necessary to capture the diurnal variation of non-occupational exposure to environmental chemicals, such as parabens.

Daily intake and hazard index of parabens based upon 24 h urine samples of the German Environmental Specimen Bank from 1995 to 2012.

In recent years, exposure to parabens has become more of a concern because of evidence of ubiquitous exposure in the general population, combined with evidence of their potency as endocrine disruptors. New human metabolism data from oral exposure experiments enable us to back calculate daily paraben intakes from urinary paraben levels. We report daily intakes (DIs) for six parabens based on 660 24 h urine samples from the German Environmental Specimen Bank collected between 1995 and 2012. Median DI values ranged between 1.1 µg/kg bw/day for iso-butyl paraben and 47.5 µg/kg bw/day for methyl paraben. The calculated DIs were compared with acceptable levels of exposure to evaluate the hazard quotients (HQs) that indicate that acceptable exposure is exceeded for values of >1. Approximately 5% of our study population exceeded this threshold for individual paraben exposure. The hazard index (HI) that takes into account the cumulative risk of adverse estrogenic effects was 1.3 at the 95th percentile and 4.4 at maximum intakes, mainly driven by n-propyl paraben exposure. HI values of >1 indicate some level of concern. However, we have to point out that we applied most conservative assumptions in the HQ/HI calculations. Also, major exposure reduction measures were enacted in the European Union after 2012.

Metabolism and elimination of methyl, iso- and n-butyl paraben in human urine after single oral dosage.

Parabens are used as preservatives in personal care and consumer products, food and pharmaceuticals. Their use is controversial because of possible endocrine disrupting properties. In this study, we investigated metabolism and urinary excretion of methyl paraben (MeP), iso-butyl paraben (iso-BuP) and n-butyl paraben (n-BuP) after oral dosage of deuterium-labeled analogs (10 mg). Each volunteer received one dosage per investigated paraben separately and at least 2 weeks apart. Consecutive urine samples were collected over 48 h. In addition to the parent parabens (free and conjugated) which are already used as biomarkers of internal exposure and the known but non-specific metabolites, p-hydroxybenzoic acid (PHBA) and p-hydroxyhippuric acid (PHHA), we identified new, oxidized metabolites with hydroxy groups on the alkyl side chain (3OH-n-BuP and 2OH-iso-BuP) and species with oxidative modifications on the aromatic ring. MeP represented 17.4 % of the dose excreted in urine, while iso-BuP represented only 6.8 % and n-BuP 5.6 %. Additionally, for iso-BuP, about 16 % was excreted as 2OH-iso-BuP and for n-BuP about 6 % as 3OH-n-BuP. Less than 1 % was excreted as ring-hydroxylated metabolites. In all cases, PHHA was identified as the major but non-specific metabolite (57.2-63.8 %). PHBA represented 3.0-7.2 %. For all parabens, the majority of the oral dose captured by the above metabolites was excreted in the first 24 h (80.5-85.3 %). Complementary to the parent parabens excreted in urine, alkyl-chain-oxidized metabolites of the butyl parabens are introduced as valuable and contamination-free biomarkers of exposure.

Parabens in 24 h urine samples of the German Environmental Specimen Bank from 1995 to 2012.

Parabens are widely used as antimicrobial preservatives in personal care and consumer products, food and pharmaceuticals. Due to their ubiquity, humans are constantly exposed to these chemicals. We assessed exposure to nine parabens (methyl-, ethyl-, n- and iso-propyl-, n- and iso-butyl-, benzyl-, pentyl- and heptyl paraben) in the German population from 1995 to 2012 based on 660 24h urine samples from the German Environmental Specimen Bank (ESB) using on-line HPLC coupled to isotope dilution tandem mass spectrometry. The limit of quantification (LOQ) was 0.5 µg/L for all parabens. We detected methyl-, ethyl- and n-propyl paraben in 79-99% of samples, followed by n-butyl paraben in 40% of samples. We infrequently detected iso-butyl-, iso-propyl- and benzyl paraben in 24%, 4% and 1.4% of samples, respectively. Urinary concentrations were highest for methyl paraben (median 39.8 µg/L; 95th percentile 319 µg/L) followed by n-propyl paraben (4.8 µg/L; 95th percentile 74.0 µg/L) and ethyl paraben (2.1 µg/L; 95th percentile 39.1 µg/L). Women had significantly higher urinary levels for all parabens than men, except for benzyl paraben. Samples from the ESB revealed that over the investigation period of nearly 20 years urinary paraben levels remained surprisingly constant; only methyl paraben had a significant increase, for both men and women. We found strong correlations between methyl- and n-propyl paraben and between n- and iso-butyl paraben. These results indicate that parabens are used in combination and arise from common sources of exposure. Urinary excretion factors are needed to extrapolate from individual urinary concentrations to actual doses.

Inter- and intra-individual variation in urinary biomarker concentrations over a 6-day sampling period. Part 1: metals.

The aim of the current HBM-study is to further the understanding of the impact of inter- and intra-individual variability in HBM surveys as it may have implications for the design and interpretation of the study outcomes. As spot samples only provide a snapshot in time of the concentrations of chemicals in an individual, it remains unclear to what extent intra-individual variability plays a role in the overall variability of population-wide HBM surveys. The current paper describes the results of an intensive biomonitoring study, in which all individual urine samples of 8 individuals were collected over a 6-day sampling period (a total of 352 unique samples). By analyzing different metals (As, Cd, Mn, Ni) in each individual sample, inter- and intra-individual variability for these four metals could be determined, and the relationships between exposure, internal dose, and sampling protocol assessed. Although the range of biomarker values for different metals was well within the normal range reported in large-scale population surveys, large intra-individual differences over a 6-day period could also be observed. Typically, measured biomarker values span at least an order of magnitude within an individual, and more if specific exposure episodes could be identified. Fish consumption for example caused a twenty- to thirty-fold increase in urinary As-levels over a period of 2-6h. Intra-class correlation coefficients (ICC) were typically low for uncorrected biomarker values (between 0.104 and 0.460 for the 4 metals), but improved when corrected for creatinine or specific gravity (SG). The results show that even though urine is a preferred matrix for HBM studies, there are certain methodological issues that need to be taken into account in the interpretation of urinary biomarker data, related to the intrinsic variability of the urination process itself, the relationship between exposure events and biomarker quantification, and the timing of sampling. When setting up HBM-projects, this expected relationship between individual exposure episode and urinary biomarker concentration needs to be taken into account.

Rapid determination of nine parabens and seven other environmental phenols in urine samples of German children and adults.

We developed a fast, selective and sensitive on-line LC/LC-MS/MS method for the simultaneous determination of nine parabens and seven environmental phenols in urine. Parabens are widely used as antimicrobial preservatives. Bisphenol A, triclosan, triclocarban, 2-phenylphenol, and benzophenones are used inter alia in disinfectants, sunscreens and in polymers. Some of these substances are suspected endocrine disruptors. Limits of quantification and analytical quality criteria fully met the needs for determining exposure levels occurring in the general population. We analyzed 157 spot urine samples from the general German population (59 females, 39 males and 59 children). For the parabens, we found methyl, ethyl and n-propyl paraben with high detection rates (77-98%), followed by n-butyl (36%), iso-butyl (17%), iso-propyl (3%) and benzyl paraben (3%). We detected no pentyl and heptyl paraben. Urinary concentrations were highest for methyl paraben (median 24.5 µg/L; 95th percentile 379 µg/L) followed by ethyl (1.4 µg/L; 35.2 µg/L) and n-propyl paraben (1.2 µg/L; 68.1 µg/L). Other environmental phenols with high detection rates were BPA (95%), triclosan (45%) and benzophenone 1 and 3 (26%). For most of the parabens/environmental phenols we found higher urinary levels in females than in males or children, probably due to differences in (personal care) product use. However, high levels (in the mg/L range) were also observed in children. Exposure to the above substances is occurring worldwide. Differences between countries do seem to exist and might be caused by different product compositions or different use habits. Human metabolism data is urgently needed to extrapolate from urinary biomarker levels to doses actually taken up.

Inter- and intra-individual variation in urinary biomarker concentrations over a 6-day sampling period. Part 2: personal care product ingredients.

An intensive study was conducted to provide data on intra- and inter-individual variation in urinary excretion of a series of ingredients in personal care products (parabens, triclosan, benzophenones) and bisphenol A (BPA, not expected to be an ingredient) in 8 volunteers over 6 days. Exposure diaries recorded use of personal care products with identified target analytes as ingredients. Participants' usual products were replaced with products without the target analytes for 2 of the 6 days. Urine void volumes and times were recorded. Methyl, ethyl, and n-propylparabens, triclosan, benzophenone-3, and BPA were frequently detected (≥70% of samples). Urinary concentrations of the parabens and triclosan were lower on product replacement days. First morning void concentrations correlated moderately to highly with 24-h composite concentrations for all analytes. Intraclass correlation coefficients (ICCs) for spot samples collected on days with usual product use were low for BPA (0.15), moderate for n-propylparaben and methylparaben (0.39 and 0.56, respectively), and high for ethylparaben, benzophenone-3, and triclosan (0.76, 0.81, and 0.934, respectively); ICCs were consistently higher on the basis of cr-adjusted concentrations. Hydration status adjustment methods were assessed by comparing unadjusted and adjusted concentrations to urinary excretion rates (ER, ng/kg-h) for all analytes and samples. Specific gravity-adjusted concentrations correlated slightly better with ER than creatinine-adjusted concentrations. Within-individual variation in biomarker concentrations was highest for methyl and ethylparabens (2 orders of magnitude variation in spot sample concentrations) and lower for the other analytes (1-1.5 orders of magnitude). This dataset provides insight into the design and interpretation of urinary biomonitoring studies for non-persistent chemicals.

Quantitative matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis of synthetic polymers and peptides.

Matrix-assisted laser desorption ionization (MALDI) is a very powerful and widely used mass spectrometric technique to ionize high molecular weight compounds. The most commonly used dried droplet (DD) technique can lead to a concentration distribution of the analyte on the target and is therefore often not suitable for reproducible analyses. We developed a new solvent-free deposition technique, called compressed sample (CS), to prevent the distribution of the analytes caused by the crystallization of the compounds. The CS technique presented in this work allows the quantitative analysis of synthetic polymers such as derivatized maltosides with correlation coefficients of 0.999 and peptides up to 3500 Da with correlation coefficients of at least 0.982 without the use of stable-isotope-labeled standards.