Comparison of aggregated exposure to di(2-ethylhexyl) phthalate from diet and personal care products with urinary concentrations of metabolites using a PBPK model - Results from the Norwegian biomonitoring study in EuroMix.

Phthalates are widely used as plasticisers in flexible plastics and containers for food and personal care products (PCPs) and contaminates foods and PCPs. A human biomonitoring (BM) study was performed to study exposure of chemicals from foods and PCPs. For two 24-h periods, adult volunteers (n = 144) in Norway kept diaries on food eaten and usage of PCPs, and collected 24-h urine. Aggregated exposure to di(2-ethylhexyl) phthalate (DEHP) from dietary and PCPs was estimated by Monte-Carlo simulation using Oracle Crystal Ball©. Simulated urinary concentrations using physiologically based pharmacokinetic (PBPK) models were compared with measured urinary metabolites of DEHP, mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP) and mono-2-ethyl 5-carboxypentyl phthalate (MECCP). DEHP exposure from food are approximately 10 times higher than exposure than from PCPs. The main contributors to dietary exposure are dairy, grain, fruits and vegetables, meat and fish. Body lotion contribute most to the exposure of DEHP from PCPs. Forward-dosimetry gives good convergence with 24-h urinary concentrations of simulated and measured BM data. The measured concentration of the MECCP metabolite correlated well with simulated high exposure, while the measured concentrations of MEHP, MEHHP and MEOHP partly overlapped with both simulated low, medium and high metabolite exposure.

The Norwegian biomonitoring study from the EU project EuroMix: Levels of phenols and phthalates in 24-hour urine samples and exposure sources from food and personal care products.

BACKGROUND: Exposure to multiple chemicals occurs daily through several routes; diet, inhalation and dermal contact. Real-life exposure assessment is needed to understand the risk. Therefore, a human biomonitoring (BM) study was performed to examine the plausibility of source-to-dose calculations for chemical mixtures in the Horizon 2020 EuroMix project. OBJECTIVES: To provide a detailed description of the design of the EuroMix BM study, and to present the initial results for urinary phenols and phthalates and to describe their exposure determinants from foods and personal care products (PCPs). METHOD: Adults (44 males and 100 females) kept detailed diaries on their food consumption, PCP use and handling of cash receipts. Urine samples were collected over the same 24-hour period. Urinary levels of four parabens, five bisphenols, oxybenzone/benzophenone-3 (OXBE), triclosan (TCS), triclocarban (TCC) and metabolites of eight phthalates and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) were analysed by ultra-high-performance liquid chromatography and tandem mass spectrometry. Multivariable linear regressions were performed between PCPs/food categories and each dependent chemical variable separately, and were only sex-stratified when an interactions between sex and the independent variable was significant. RESULTS: The detection rate for the metabolites of phthalates and DINCH, and bisphenol A (BPA) and TCS in urine was 88-100%, while bisphenol S (BPS) and bisphenol F (BPF) were only found in 29% and 4% of the urine samples, respectively. Bisphenol B (BPB), bisphenol AF (BPAF) and TCC were not detected. Food groups associated with phenol exposure were meat, bread, beverages and butter and oil. Food determinants for phthalate exposure were sweets, butter and oil, fruit and berries and other foods. The only positive association between the use of PCPs and phenols was found between BPA and lip gloss/balm. Phthalate exposure was associated with the use of shower gel, hand cream (females), toothpaste, anti-wrinkle cream (females) and shaving products (males). CONCLUSION: The participants in the EuroMix BM study were exposed to a mixture of phenols and phthalates. A variety of food categories and PCPs were found to be possible sources of these chemicals. This indicates a complex pattern of exposure to numerous chemicals from multiple sources, depending on individual diet and PCP preferences.

Quantitative axial profiles of retinoic acid in the embryonic mouse spinal cord: 9-cis retinoic acid only detected after all-trans-retinoic acid levels are super-elevated experimentally.

Studies using bioassays in normal mice and gene activation in transgenic reporter mice have demonstrated peaks of retinoic acid receptor (RAR) signaling in the brachial and lumbar regions of the spinal cord. Recently, Solomin et al. (Solomin et al. [1998] Nature 395:398-402) detected a retinoid X receptor (RXR) signal in the same region of the developing spinal cord at a slightly later stage than the RAR signal. This finding raises the question of which retinoid ligands underlie RAR and RXR signaling in this part of the embryo. Quantitative measurements of regional differences in retinoid profiles have not been reported previously due to limitation in the sensitivity and specificity of available retinoid detection methods. Here, by using a recently developed ultrasensitive HPLC technique (Sakhi et al. [1998] J. Chromatogr. A 828:451-460), we address this question in an attempt to identify definitively the endogenous retinoids present in different regions of the spinal cord at the stages when regional differences in RAR and RXR signaling have been reported. We find a bimodal distribution of all-trans retinoic acid (at-RA), the ligand for RARs, and relate this to the expression of several retinoid-synthesizing enzymes. However, we do not detect 9-cis-retinoic acid (9-cis-RA), the putative RXR ligand, in any region of the spinal cord unless retinoid levels are massively increased experimentally by gavage feeding pregnant mice with teratogenic doses of at-RA. This study provides for the first time quantitative profiles of endogenous retinoids along the axis of the developing spinal cord, thereby establishing a foundation for more definitive studies of retinoid function in the future. It sets definite limits on how much 9-cis-RA potentially is present and demonstrates that at-RA predominates over 9-cis-RA by at least 30- to 180-fold in different spinal cord regions.

Identification of endogenous retinoids, enzymes, binding proteins, and receptors during early postimplantation development in mouse: important role of retinal dehydrogenase type 2 in synthesis of all-trans-retinoic acid.

Specific combinations of nuclear retinoid receptors acting as ligand-inducible transcription factors mediate the essential role of retinoids in embryonic development. Whereas some data exist on the expression of these receptors during early postimplantation development in mouse, little is known about the enzymes controlling the production of active ligands for the retinoid receptors. Furthermore, at early stages of mouse development virtually no data are available on the presence of endogenous retinoids. In the present study we have used a recently developed high-performance liquid chromatographic (HPLC) technique to identify endogenous retinoids in mouse embryos down to the egg cylinder stage. All-trans-retinoic acid, a ligand for the retinoic acid receptors, was detected in embryos dissected as early as 7.5 dpc (i.e., a combination of midstreak until late allantoic bud stage embryos). At these stages, we detected mRNA coding for all the retinoid receptors, retinoid binding proteins, and two enzymes able to convert retinol to retinal (retinol dehydrogenase 5 (RDH5) and alcohol dehydrogenase 4 (ADH4)). We also detected retinal dehydrogenase type 2 (RALDH2), an enzyme capable of oxidising the final step in the all-trans-retinoic acid synthesis. In egg cylinder stage mouse embryos no all-trans-retinoic acid was detected. However, at this stage its precursor all-trans-retinal was present. In accordance with these HPLC observations, RDH5 and ADH4 were expressed, but no transcripts coding for enzymes that oxidise retinal to retinoic acid. Therefore, our results suggest that RALDH2 is a key regulator in initiating retinoic acid synthesis sometime between the mid-primitive streak stage and the late allantoic bud stage in mouse embryos.

Quantitative determination of endogenous retinoids in mouse embryos by high-performance liquid chromatography with on-line solid-phase extraction, column switching and electrochemical detection.

An isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid and all-trans-retinol in mouse embryos using on-line solid-phase extraction and column switching in combination with electrochemical detection has been developed. The method was validated using retinoids in albumin solutions and 13-cis-acitretin was used as internal standard. About 370 microliters of albumin solution was injected on a 10 x 2.1-mm I.D. pre-column packed with Bondapak C18, 37-53-micron particles. The proteins were washed to waste within 5 min using as mobile phase, a 1:3 dilution of mobile phase 2, which consisted of acetonitrile-methanol-2% ammonium acetate-glacial acetic acid (79:2:16:3, v/v). Components retained on the pre-column were back-flushed to and separated on the 250 x 4.6-mm I.D. Suplex pKb-100 analytical column using mobile phase 2. The retinoids were detected electrochemically at +750 mV using a coulometric electrochemical detector. The total analysis time was about 20 min. Recoveries were in the range of 86-103%. The mass limits of detection were about 10 pg and 25 pg for the retinoic acids and all-trans-retinol, respectively. The intra-assay precision, reported as relative standard deviation, was in general better than 4% (n = 6) for the four retinoids. Inter-assay precision was in the range 3-4% (n = 10). The method was applied for determination of endogenous retinoids in 9.5 day-old mouse embryos. A 340-microliter solution containing 100 microliters of embryo homogenate (1.64 embryos) was analyzed. The concentrations of all-trans-retinol and all-trans-retinoic acid were found to be 279 pg per embryo and 75.8 pg per embryo, respectively. The amount of 13-cis-retinoic acid and 9-cis-retinoic acid was below the detection limit.