Short-term test for the toxicogenomic assessment of ecotoxic modes of action in Myriophyllum spicatum.

In environmental risk assessment of substances, the 14-day growth inhibition test following OECD test guideline 239 is employed to assess toxicity in the macrophyte Myriophyllum spicatum. Currently, this test evaluates physiological parameters and does not allow the identification of the mode of action (MoA) by which adverse effects are induced. However, for an improved ecotoxicity assessment of substances, knowledge about their ecotoxic MoA in non-target organisms is required. It has previously been suggested that the identification of gene expression changes can contribute to MoA identification. Therefore, we developed a shortened three-day assay for M. spicatum including the transcriptomic assessment of global gene expression changes and applied this assay to two model substances, the herbicide and photosynthesis inhibitor bentazone and the pharmaceutical and HMG-CoA reductase inhibitor atorvastatin. Due to the lack of a reference genome for M. spicatum we performed a de novo transcriptome assembly followed by a functional annotation to use the toxicogenomic results for MoA discrimination. The gene expression changes induced by low effect concentrations of these substances were used to identify differentially expressed genes (DEGs) and impaired biological functions for the respective MoA. We observed both concentration-dependent numbers and differentiated patterns of DEGs for both substances. While bentazone impaired genes involved in the response to reactive oxygen species as well as light response, and also genes involved in developmental processes, atorvastatin exposure led to a differential regulation of genes related to brassinosteroid response as well as potential metabolic shifts between the mevalonate and methyl erythritol 4-phosphate pathway. Based on these responses, we identified biomarker candidates for the assessment of MoA in M. spicatum. Utilizing the shortened assay developed in this study, the investigation of the identified biomarker candidates may contribute to the development of future MoA-specific screening approaches in the ecotoxicological hazard prediction using aquatic non-standard model organisms.

Determination of orally administered 1,8-Cineol in nasal polyp tissues from chronic rhinosinusitis patients using gas chromatography: mass spectrometry.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common inflammatory disease causing considerable disease burden. The anti-inflammatory monoterpene 1,8-Cineol is a natural plant-based therapeutic agent that is well established to treat chronic and acute airway diseases. Aim of this study was to investigate whether the herbal drug 1,8-Cineol reaches the nasal tissue via the gut and the blood stream upon its oral administration. A highly sensitive gas chromatography mass spectrometry-based method with stir bar sorptive extraction (SBSE) for sample preparation has been developed and validated for the extraction, detection and quantification of 1,8-Cineol in tissue samples of nasal polyps from 30 CRSwNP patients. Data revealed a highly sensitive detection of 1,8-Cineol in nasal tissue samples after 14 days of oral administration of 1,8-Cineol prior to surgical treatment. There was no significant correlation between the measured 1,8-Cineol concentrations and bodyweight or BMI values of the analyzed patients, respectively. Our data indicate a systemic distribution of 1,8-Cineol in the human body after its oral administration. Individual differences in terms of metabolic characteristics and have to be further investigated. The study increases our understanding of the systemic effects of 1,8-Cineol upon its therapeutic application and benefit in patients with CRSwNP.

Nonylphenol (NP) exposure in Germany between 1991 and 2021: Urinary biomarker analyses in the German Environmental Specimen Bank (ESB).

Nonylphenol (NP) is a high production volume chemical with a wide range of uses, e.g. in NP ethoxylates (NPEO). NP and NPEO have become ubiquitous in the environment and are considered of concern due to their general ecotoxicity and endocrine disrupting properties. However, knowledge on human exposure is scarce. In this study, we analyzed novel NP metabolites (OH-NP and oxo-NP) as robust biomarkers of exposure in 24h-urine samples from the German Environmental Specimen Bank (ESB). This enables us to reliably determine the individual NP body burden and to retrospectively evaluate NP exposure over the past 30 years. We analyzed 660 urine samples from eleven sampling years between 1991 and 2021. All samples were from young German adults between 20 and 29 years of age. OH-NP was quantifiable in all samples until 2017. In 2019 and 2021, the frequency of samples above the LOQ dropped to 90% and 77%, respectively. Median OH-NP concentrations significantly decreased from 4.32 µg/L in 1991 to 0.70 µg/L in 2021. OH-NP and oxo-NP levels correlated strongly, but oxo-NP concentrations and detections were considerably lower, in line with its known lower metabolic conversion. Reverse dosimetry back-calculated daily intakes (DI) of NP, based on OH-NP, decreased by almost a factor of four from medians of 0.16 µg/(kg bw*d) in 1991 to 0.04 µg/(kg bw*d) in 2021, respectively. The major drop took place only after 2012. This came as a surprise, because strict restrictions had been enacted much earlier in the EU, in 2003. All NP DIs were below the provisional tolerable daily intake of 5 µg/(kg bw*d) from the Danish Environmental Agency. DIs back-calculated from the ESB biomonitoring data agree well with calculations from food. This indicates to contaminated foodstuff as a major source of exposure. The time lag of regulatory restrictions to decreasing human exposure levels, the general lack of knowledge on exposure levels in susceptible populations such as children, and the ongoing worldwide use of NP underline the urgent need to continue monitoring NP exposures in Germany and worldwide. With these novel NP biomarkers, we provide a robust and sensitive tool for exposure and risk assessments, complementing environmental monitoring.

Biomarker-Determined Nonylphenol Exposure and Associated Risks in Children of Thailand, Indonesia, and Saudi Arabia.

Nonylphenol (NP) is an endocrine disruptor and environmental contaminant. Yet, data on individual body burdens and potential health risks in humans, especially among children, are scarce. We analyzed two specific urinary NP metabolites, hydroxy-NP (OH-NP) and oxo-NP. In contrast to parent NP, OH-NP has a much higher urinary excretion fraction (Fue), and both are insusceptible to external contamination. We investigated spot urine samples from school children of Thailand (n = 104), Indonesia (n = 89), and Saudi Arabia (n = 108) and could quantify OH-NP in 100% of Indonesian and Saudi children (median concentrations: 8.12 and 8.57 µg/L) and in 76% of Thai children (1.07 µg/L). Median oxo-NP concentrations were 0.95, 1.10, and <0.25 µg/L, respectively, in line with its lower Fue. Median daily NP intakes (DIs), back-calculated from urinary OH-NP concentrations, were significantly higher in Indonesia and Saudi Arabia [0.47 and 0.36 µg/(kg bw·d), respectively] than in Thailand [0.06 µg/(kg bw·d)]. Maximum DIs were close to the preliminary tolerable DI of 5 µg/(kg bw·d) from the Danish Environmental Protection Agency. Dominant sources of exposure or relevant exposure pathways could not be readily identified by questionnaire analyses and also potentially varied by region. The novel biomarkers provide long-needed support to the quantitative exposure and risk assessment of NP.

Nonylphenol exposure in 7-year-old Japanese children between 2012 and 2017- Estimation of daily intakes based on novel urinary metabolites.

Nonylphenol (NP) has been under scrutiny for decades due to its endocrine-disrupting properties and its ubiquity in the environment. Despite its widespread occurrence, robust and reliable exposure data are rare. In this study, we used human biomonitoring (HBM) measuring the novel urinary alkyl-chain-oxidized biomarkers OH-NP and oxo-NP to determine NP exposure in 7-year-old Japanese children. The new biomarkers are advantageous over measuring unchanged NP because they are not prone to external contamination. We analyzed 180 first morning void urine samples collected between 2012 and 2017. OH-NP and oxo-NP were detected in 100% and 66% of samples at median concentrations of 2.69 and 0.36 µg/L, respectively. 10-fold concentration differences between OH-NP and oxo-NP are in line with recent findings on human NP metabolism. Based on OH-NP we back-calculated median and maximum NP daily intakes (DI) of 0.14 and 0.95 µg/(kg bw*d). These DIs are rather close to but still below the current provisional tolerable daily intake of 5 µg/(kg bw*d) by the Danish Environmental Protection Agency. Between 2012 and 2017 the DIs decreased by an average of 4.7% per year. We observed no seasonal changes or gender differences and questionnaire data on food consumption, housing characteristics or pesticide use showed no clear associations with NP exposure. Urinary OH-NP was weakly associated with the oxidative stress (lipid peroxidation) biomarkers N-ε-hexanoyl-lysine (HEL) and trans-4-hydroxy-2-nonenal (HNE) (Spearman ρ = 0.30 and 0.22, respectively), but not with 8-hydroxy-2'-deoxyguanosine (8-OHdG). Further research is needed to identify and understand the major sources of NP exposure and to investigate a potential role in oxidative stress. This study is the first to investigate NP exposure in Japanese children based on robust and sensitive HBM data. It is a first step to fill the long-standing gap in quantitative human NP exposure monitoring and risk assessment.

Human Metabolism and Urinary Excretion Kinetics of Nonylphenol in Three Volunteers after a Single Oral Dose.

Nonylphenol (NP) is an endocrine-disrupting anthropogenic chemical that is ubiquitous in the environment. Human biomonitoring data and knowledge on internal NP exposure are still sparse, and its human metabolism is largely unknown. Therefore, in this study, we investigated human metabolism and urinary excretion of NP. Three male volunteers received a single oral dose of 1 mg 13C6-labeled NP (10.6-11.7 µg/kg body weight). Consecutive full urine voids were collected for 48 h. A metabolite screening identified nine ring- and/or side chain-oxidized metabolites. We chose the most promising hits, the alkyl chain-oxidized metabolites hydroxy-NP (OH-NP) and oxo-NP, for quantitative investigation next to the parent NP. For this purpose, we newly synthesized specific n - 1-oxidized monoisomeric analytical standards. Quantification of the polyisomeric metabolites was performed via online-solid phase extraction-LC-MS/MS with stable isotope dilution using a previously published consensus method. Alkyl chain hydroxylation (OH-NP) constituted the major metabolism pathway representing 43.7 or 62.2% (depending on the mass transition used for quantification) of the NP dose excreted in urine. The urinary excretion fraction (FUE) for oxo-NP was 6.0 or 9.3%. The parent NP, quantified via an analogous isomeric 13C6-NP standard, represented 6.6%. All target analytes were excreted predominately as glucuronic acid conjugates. Excretion was rather quick, with concentration maxima in urine 2.3-3.4 h after dosing and biphasic elimination kinetics (elimination half-times first phase: 1.0-1.5 h and second phase: 5.2-6.8 h). Due to its high FUE and insusceptibility to external contamination (contrary to parent NP), OH-NP represents a robust and sensitive novel exposure biomarker for NP. The novel FUEs enable to robustly back-calculate the overall NP intakes from urinary metabolite levels in population samples for a well-informed cumulative exposure and risk assessment.

Urinary Concentrations of Major Phthalate and Alternative Plasticizer Metabolites in Children of Thailand, Indonesia, and Saudi Arabia, and Associated Risks.

Phthalates are widely used in consumer products and are well-known for adverse endocrine outcomes. Di-(2-ethylhexyl) phthalate (DEHP), one of the most extensively used phthalates, has been rapidly substituted with alternative plasticizers in many consumer products. The aim of this study was to assess urinary phthalate and alternative plasticizer exposure and associated risks in children of three Asian countries with different geographical, climate, and cultural characteristics. Children were recruited from elementary schools of Saudi Arabia (n = 109), Thailand (n = 104), and Indonesia (n = 89) in 2017-2018, and their urine samples were collected. Metabolites of major phthalates and alternative plasticizers were measured in the urine samples by HPLC-MS/MS. Urinary metabolite levels differed substantially between the three countries. Metabolite levels of diisononyl phthalate (DiNP), diisodecyl phthalate (DiDP), di(2-ethylhexyl) terephthalate (DEHTP), and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) were the highest in Saudi children: Median urinary concentrations of oxo-MiNP, OH-MiDP, 5cx-MEPTP, and OH-MINCH were 8.3, 8.4, 128.0, and 2.9 ng/mL, respectively. Urinary DEHP metabolite concentrations were the highest in the Indonesian children. The hazard index (HI) derived for the plasticizers with antiandrogenicity based reference doses (RfDAA) was >1 in 86%, 80%, and 49% of the Saudi, Indonesian, and Thai children, respectively. DEHP was identified as a common major risk driver for the children of all three countries, followed by DnBP and DiBP depending on the country. Among alternative plasticizers, urinary DEHTP metabolites were detected at levels comparable to those of DEHP metabolites or higher among the Saudi children, and about 4% of the Saudi children exceeded the health based human biomonitoring (HBM)-I value. Priority plasticizers that were identified among the children of three countries warrant refined exposure assessment for source identification and relevant exposure reduction measures.

Determination of specific urinary nonylphenol metabolites by online-SPE-LC-MS/MS as novel human exposure biomarkers.

Nonylphenol (NP) is an endocrine disrupting and ecotoxic substance that has been detected in a variety of environmental matrices. It is utilized for the production of non-ionic nonylphenol ethoxylate (NPEO) detergents and other high production volume chemicals. Human biomonitoring data are scarce and mostly limited to the non-oxidized NP, which is ubiquitous in the (laboratory) environment and susceptible to external contamination. Here, we describe a sensitive, precise, accurate and rugged analytical method for the determination of OH-NP and oxo-NP, two potential alkyl-chain-oxidized metabolites of NP in human urine. We used single isomer standards, obtained by custom synthesis, for the quantification of the sum of the respective isomers. After enzymatic hydrolysis of potential urinary phase II conjugates, urine samples were analyzed by online turbulent flow chromatography for analyte enrichment and matrix depletion coupled to reversed phase liquid chromatography with negative electrospray-ionization triple quadrupole tandem mass spectrometry detection (online-SPE-LC-MS/MS). Quantification was performed by stable isotope dilution analysis. Limits of quantification in urinary matrix were 0.5 µg/L for OH-NP and 0.25 µg/L for oxo-NP. Mean relative recoveries were 101-105% (OH-NP) and 112-117% (oxo-NP) and the method imprecision (CV) in matrix was below 5%. In spite of extensive use restrictions in the EU since 2003, we could quantify OH-NP and oxo-NP in 94% and 47% of spot urine samples from the general German population (n = 32) collected in 2014. Thus, both metabolites seem suitable as sensitive and specific urinary biomarkers of NP exposure for future human biomonitoring population studies. Currently this method is used to quantitatively investigate human NP metabolism and to derive urinary metabolite excretion fractions that can be used to calculate external doses based on urinary biomarker concentrations.

Human metabolism and urinary excretion kinetics of di-n-butyl adipate (DnBA) after oral and dermal administration in three volunteers.

Di-n-butyl adipate (DnBA) is used as a plasticizer and in various consumer products (e.g. personal care products) replacing, in part, the endocrine disruptor di-n-butyl phthalate (DnBP). We provide quantitative in vivo data on human DnBA metabolism and excretion after oral dose (105-185 µg/kg bw) and dermal application to three volunteers each as a tool for exposure and risk assessment. Complete and consecutive urine samples were collected for two (oral) and four days (dermal), respectively, and analyzed for the metabolites mono-n-butyl adipate (MnBA), 3- and tentative 4-hydroxy-mono-n-butyl adipate (3OH-MnBA, 4OH-MnBA), and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA), as well as the hydrolysis product adipic acid (AA) using stable isotope dilution quantification. Metabolites were excreted within 24 h after oral dose with one or two concentration maxima at 0.8-3.0 h (n = 3) and 4.8-6.3 h (n = 2). AA was the major but unspecific metabolite with urinary excretion fractions (FUEs) of 14-26 %. Mean FUEs (range) of 3cx-MnPrA, MnBA, 3OH-MnBA, and tentative 4OH-MnBA were low, but consistent between volunteers (0.47 % (0.35-0.63 %), 0.079 % (0.065-0.091 %), 0.012 % (0.006-0.016 %), and 0.005 % (0.002-0.009 %), respectively). MnBA and 3OH-MnBA seem to be suitable, specific exposure biomarkers for DnBA, whereas 3cx-MnPrA and 4OH-MnBA seem to originate also from other, unknown sources not related to DnBA. Compared to the oral study, metabolite excretion in the dermal study was delayed and MnBA excretion was somewhat higher compared to the oxidized metabolites. Based on urinary concentrations and the above excretion fractions, calculated uptakes in the dermal study did not exceed the adipate ester ADI of 5 mg/(kg bw*day).

Determination of di-n-butyl adipate (DnBA) metabolites as possible biomarkers of exposure in human urine by online-SPE-LC-MS/MS.

Di-n-butyl adipate (DnBA) is an alternative to the anti-androgenic and strictly regulated di-n-butyl phthalate (DnBP) used as a cosmetic ingredient, plasticizer, and in various articles of everyday life. Hence, exposures of the general population have to be expected. Currently, biomarkers of DnBA exposure and methods for their determination are not available. Here, we describe a sensitive, rugged and precise analytical method for the determination of the DnBA monoester metabolite mono-n-butyl adipate (MnBA), as well as its potential downstream metabolites 3-hydroxy-mono-n-butyl adipate (3OH-MnBA) and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA) in human urine. Glucuronic acid conjugates present in urine were deconjugated using a pure ß-glucuronidase. The metabolites were then analyzed by liquid chromatography on a C18 column with superficially porous particles coupled to electrospray ionization-triple quadrupole-tandem mass spectrometry, applying online turbulent flow chromatography for analyte enrichment and matrix depletion (online-SPE-LC-MS/MS). The metabolites were quantified using stable isotope dilution analysis with limits of quantification of 0.05 µg/L (MnBA), 0.1 µg/L (3OH-MnBA), and 0.5 µg/L (3cx-MnPrA). Method imprecision in urinary matrix was below 7% (coefficient of variation) for all analytes. Mean relative recoveries were between 93% and 107%. The suitability of the DnBA metabolites as biomarkers of exposure was demonstrated after dermal application of a commercially available sunscreen containing DnBA. Maximum concentrations were reached 6.5 h after dose (219 µg/L 3cx-MnPrA, 91 µg/L MnBA, and 3.9 µg/L 3OH-MnBA). Elimination kinetics were similar for all three metabolites. We were able to quantify 3cx-MnPrA and MnBA until 4 d after sunscreen application. In a sample set of 35 urine samples from the general German population, 3cx-MnPrA was quantified in 94% (median 2.54 µg/L, maximum 78.3 µg/L) and MnBA in 3% (median < LOQ, maximum 0.18 µg/L) of the samples. The method will be applied in future human metabolism and human biomonitoring population studies.

Metabolism and urinary excretion kinetics of di(2-ethylhexyl) adipate (DEHA) in four human volunteers after a single oral dose.

Di(2-ethylhexyl) adipate (DEHA) is used as a substitute for the reprotoxic phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP). This study reports the first quantitative data on human in vivo DEHA metabolism and urinary metabolite excretion with the aim of providing tools for DEHA exposure and risk assessments. After DEHA was administered to four healthy volunteers (107-164 µg/kg body weight (bw)), urine samples were continuously and completely collected for 48 h and analyzed for the specific oxidized monoester metabolites mono-2-ethyl-5-hydroxyhexyl adipate (5OH-MEHA), mono-2-ethyl-5-oxohexyl adipate (5oxo-MEHA), and mono-5-carboxy-2-ethylpentyl adipate (5cx-MEPA), as well as for the non-specific hydrolysis product adipic acid (AA) using stable isotope dilution analysis. AA was confirmed as a major (urinary excretion fraction (FUE): 10-40%), yet non-specific DEHA metabolite. 5cx-MEPA was the major specific DEHA metabolite with an FUE of 0.20% (range: 0.17-0.24%). FUEs for 5OH-MEHA and 5oxo-MEHA were 0.07% (0.03-0.10%) and 0.05% (0.01-0.06%), respectively. The three specific metabolites were excreted with two concentration maxima (tmax1 = 1.5-2.3 h, tmax2 = 3.8-6.4 h). Elimination half-lives (t1/2, calculated after the second tmax) for 5cx-MEPA were calculated between 2.1-3.8 h. The majority (98-100%) of metabolites was excreted within 24 h. The FUE of 5cx-MEPA was applied to demonstrate its applicability for calculating daily intakes based on urinary metabolite levels from three pilot populations. Daily intakes were generally far below the tolerable daily intake (TDI) for DEHA (300 µg/kg bw/day). The highest daily intake (114 µg/kg bw/day) was calculated in individuals after consuming food that had been wrapped in DEHA containing cling film.