First metabolic profiling of 4-n-nonylphenol in human liver microsomes by integrated approaches to testing and assessment: Metabolites, pathways, and biological effects.

4-n-nonylphenol (4-n-NP), a typical endocrine disrupting chemical, has been so far frequently detected in various environmental mediums and editable food. However, the specific metabolic pathways in human and potential adverse effects of metabolites have not been elucidated yet. Here, metabolic profiling of 4-n-NP in human liver microsome (HLM) was comprehensively characterized by integrated approaches of testing and assessment. A total of 21 metabolites were identified using nontarget analysis with high-resolution mass spectrum, including three groups of unique phase I metabolites first determined in HLM. Seven various metabolic pathways of 4-n-NP were identified by both in silico and in vitro, and CYP1A2, 2C19, and 2D6 were the mainly participating enzymes. Two secondary metabolites with carbonyl groups on side chains (M4, M7) presented most abundant in HLM, which were also predicted to have high binding affinities towards HPG-axis-related receptors (AR, ER, and PR). ESRs (estrogen receptors) were shared core protein targets for all metabolites revealed by protein-protein interaction networks. Biological functions enrichment analysis indicated that 4-n-NP metabolites might primarily involve in ESR-mediated signaling, GPCR ligand binding, Class A/1 (Rhodopsin-like receptors) and metabolism-related pathways. These findings of 4-n-NP metabolites, pathways, and biological effects provide insightful information for its environmental exposure and risk assessment.

Human risk assessment of 4-n-nonylphenol (4-n-NP) using physiologically based pharmacokinetic (PBPK) modeling: analysis of gender exposure differences and application to exposure analysis related to large exposure variability in population.

As a toxic substance, 4-n-nonylphenol (4-n-NP) or 4-nonylphenol (4-NP) is widely present in the environment. 4-n-NP is a single substance with a linear-alkyl side chain, but 4-NP usually refers to a random mixture containing various branched types. Unfortunately, human risk assessment and/or exposure level analysis for 4-n-NP (or 4-NP) were almost nonexistent, and related research was urgently needed. This study aimed to analyze the various exposures of 4-n-NP (or 4-NP) through development of a physiologically based-pharmacokinetic (PBPK) model considering gender difference in pharmacokinetics of 4-n-NP and its application to human risk assessment studies. A PBPK model was newly developed considering gender differences in 4-n-NP pharmacokinetics and applied to a human risk assessment for each gender. Exposure analysis was performed using a PBPK model that considered gender differences in 4-n-NP (or 4-NP) exposure and high variabilities in several countries. Furthermore, an extended application was attempted as a human risk assessment for random mixture 4-NP, which is difficult to accurately evaluate in reality. External-exposure and margin-of-safety estimated with the same internal exposure amount differed between genders, meaning the need for a differentiated risk assessment considering gender. Exposure analysis based on biomonitoring data confirmed large variability in exposure to 4-n-NP (or 4-NP) by country, group, and period. External-exposures estimated using PBPK model varied widely, ranging from 0.039 to 63.875 mg/kg/day (for 4-n-NP or 4-NP). By country, 4-n-NP (or 4-NP) exposure was higher in females than in males and the margin-of-safety tended to be low. Overall, exposure to 4-n-NP (or 4-NP) in populations was largely not safe, suggesting need for ongoing management and monitoring. Considering low in vivo accumulation confirmed by PBPK model, risk reduction of 4-n-NP is possible by reducing its use.

A Novel Hydrogen Fluoride Assisted-Glass Surface Etching Based Liquid Phase Microextraction for the Determination of 4-n-Nonylphenol in Water by Gas Chromatography-Mass Spectrometry with Matrix Matching Strategy.

A novel extraction method named hydrogen fluoride assisted-glass surface etching based liquid phase microextraction (HF-GSE-LPME) was proposed to determine 4-n-nonylphenol at trace levels by gas chromatography-mass spectrometry (GC-MS). After the evaluation of system analytical performance for the HF-GSE-LPME-GC-MS system, limit of detection (LOD) and limit of quantification (LOQ) values were calculated as 7.1 and 23.8 ng/g, respectively. Enhancement in detection power of the method was determined to be 22 fold when LOD values of the GC-MS and HF-GSE-LPME-GC-MS systems were compared with each other. Applicability and accuracy of the established method were checked by performing spiking experiments. A matrix matching calibration strategy was applied to boost the accuracy of quantification in both matrices, and the percent recovery results obtained for bottled drinking water and dam lake water samples were in the range of 98 - 107 and 90 - 117%, respectively.

The comparison of transcriptomic response of green microalga Chlorella sorokiniana exposure to environmentally relevant concentration of cadmium(II) and 4-n-nonylphenol.

The transcriptomic response of green microalga Chlorella sorokiniana exposure to environmentally relevant concentration of cadmium(II) (Cd) and 4-n-nonylphenol (4-n-NP) was compared in the present study. Cd and 4-n-NP exposure showed a similar pattern of dys-regulated pathways. The photosystem was affected due to suppression of chlorophyll biosynthesis via down-regulation of Mg-protoporphyrin IX chelatase subunit ChlD (CHLD) and divinyl chlorophyllide a 8-vinyl-reductase (DVR) in Cd group and via down-regulation of DVR in 4-n-NP group. Furthermore, the reactive oxygen species (ROS) could be induced through down-regulation of solanesyl diphosphate synthase 1 (SPS1) and homogentisate phytyltransferase (HPT) in Cd group and via down-regulation of HPT in 4-n-NP group. Additionally, Cd and 4-n-NP would both cause the dys-regulation of carbohydrate metabolism and protein synthesis. On the other hand, there are some different responses or detoxification mechanism of C. sorokiniana to 4-n-NP stress compared to Cd exposure. The increased ROS would cause the DNA damage and protein destruction in Cd exposure group. Simultaneously, the RNA transcription was dys-regulated and a series of changes in gene expressions were observed. This included lipid metabolism, protein modification, and DNA repair, which involved in response of C. sorokiniana to Cd stress or detoxification of Cd. For 4-n-NP exposure, no effect on lipid metabolism and DNA repair was observed. The nucleotide metabolism including pyrimidine metabolism and purine metabolism was significantly up-regulated in the 4-n-NP exposure group, but not in the Cd exposure group. In addition, 4-n-NP would induce the ubiquitin-mediated proteolysis and proteasomal degradation to diminish the misfolded protein caused by ROS and down-regulation of heat shocking protein 40. In sum, the Cd and 4-n-NP could cause the same toxicological effects via the common pathways and possess similar detoxification mechanism. They also showed different responses in nucleotide metabolism, lipid metabolism, and DNA repair.

The steroid receptor coactivator 1 (SRC1) and 3 (SRC3) recruitment as a novel molecular initiating event of 4-n-nonylphenol in estrogen receptor α-mediated pathways.

Recently, the action of steroid receptor coactivators (SRCs) has been recognized to be an important molecular initiating event (MIE) in estrogenic adverse outcome pathways (AOPs). However, the role of SRCs in the molecular mechanisms of many highly concerned environmental estrogens remains poorly understood. In this study, the widely studied environmental estrogen, 4-n-nonylphenol (4-n-NP), was used as a typical pollutant to study SRCs recruitment in its estrogenic effects. In MCF7 cell proliferation (E-SCREEN) assay and MVLN cell assay, 4-n-NP showed significant estrogenic potency that involved an increase in estrogen receptor α (ERα), SRC1 and SRC3 transcript levels. Moreover, 4-n-NP was found to induce estrogen response element (ERE)-mediated activity via ERα in MVLN cells. To investigate the mechanism by which SRCs recruitment is induced by 4-n-NP-ERα, a coactivators recruitment assay was performed, and the results showed that 4-n-NP-ERα recruited both SRC1 and SRC3, whereas it failed to recruit SRC2. Similarly, it had no interaction with SRC2 in the ERα-SRC2 two-hybrid yeast assay. This is the first report to investigate the novel MIE of SRCs recruitment in 4-n-NP-ERα-induced estrogenicity. Overall, our results suggest that the action of 4-n-NP on estrogenic effects involves the following MIEs: the activation of ERα, the recruitment of SRC1 and SRC3, and the induction of ERE-mediated activity. The findings also provide valuable insights into the MIE associated with the different SRCs that are recruited in the adverse outcome pathways of environmental estrogens.

Gender differences in pharmacokinetics and tissue distribution of 4-n-nonylphenol in rats.

The aim of this study was to newly identify and investigate the gender differences in pharmacokinetics (PKs) and tissue distribution of 4-n-nonylphenol (4-n-NP) in both male and female Sprague-Dawley rats. For this study, a UPLC-ESI-MS/MS system for 4-n-NP was developed as a sensitive and rapid analysis method and validated according to the accepted criteria of the international guidelines. The method was finally applied to the analysis of plasma, urine, feces, and nine different tissue samples of rats. PK parameters were calculated after single oral or intravenous administration of 4-n-NP at a dose of 10 or 50 mg/kg. Mean half-life of 4-n-NP in female rats was shorter and its clearance was larger for all doses than those in male rats. There were statistically significant differences in excretion patterns of urine and feces between male and female rats. Distribution of nine different tissues for 4-n-NP was greater in male than in female, and 4-n-NP was highly distributed in the liver or kidney. It was also specific that the distribution of 4-n-NP into brain was considerable. These results suggest that there are gender differences in the PKs of 4-n-NP in rats. Although, 4-n-NP is known to be a reproductive toxicant, reports on its PKs, excretion pattern, tissue distribution, and gender difference are limited. Therefore, our results will be useful data for gender differences as well as toxicokinetic information for 4-n-NP. In addition, it is expected to be very important for future risk assessment and PBPK model establishment of 4-n-NP.

A comparison of endocrine disruption potential of nonylphenol ethoxylate, vanillin ethoxylate, 4-n-nonylphenol and vanillin in vitro.

The widely used surfactant nonylphenol ethoxylate (NPEO) and its raw material 4-n-nonylphenol (4-n-NP), as well as its degradation products, are recognized as endocrine disrupting chemicals. The USA Environmental Protection Agency (EPA) released an assessment that looked for safe alternatives to NPEO. Vanillin ethoxylate (VAEO) is a novel substitute for NPEO and is quite similar to NPEO in structure; there is a risk that it has similar endocrine disrupting effects to NPEO. However, their effects on various nuclear hormone receptors have not been thoroughly examined. In this study, the effects of NPEO, VAEO, 4-n-NP and Vanillin on the estrogen receptor α (ERα), androgen receptor (AR), thyroid hormone receptor (TR), retinoic X receptor ß (RXRß) and estrogen-related receptor γ (ERRγ) were determined and compared using a battery of recombined yeast strains expressing ß-galactosidase. The results showed that NPEO and 4-n-NP acted as significant antagonists of ER, AR, TR and ERRγ. In addition, 4-n-NP also had antagonistic activity toward RXRß. Moreover, VAEO was shown to be a very weak antagonist of TR and ERRγ, and Vanillin had no interaction with any nuclear receptors. For the first time, it was found that NPEO had AR, TR and ERRγ antagonistic effects and that 4-n-NP was an antagonist of RXRß. The in vitro data indicated that NPEO, 4-n-NP and VAEO have the potential to act as endocrine disruptors involving more than one nuclear hormone receptor, but VAEO has much lower endocrine disrupting potential than NPEO. Thus, it is critical to find safe substitutes for NPEO and a substitute of NPEO with structural analogues should be carried out with caution. Furthermore, to look for preferable alternatives for NPEO, more in vivo and in vitro studies of the alternatives concerning endocrine disruption are needed, especially in vitro studies need to involve various target points, not only focus on their effects on ER but also take other nuclear hormone receptor pathways into consideration.

A core-shell structured magnetic covalent organic framework (type Fe3O4@COF) as a sorbent for solid-phase extraction of endocrine-disrupting phenols prior to their quantitation by HPLC.

A magnetic covalent organic framework (Fe3O4@COF) with core-shell structure was fabricated at room temperature and used as an adsorbent for magnetic solid-phase extraction of polar endocrine-disrupting phenols (4-n-nonylphenol, 4-n-octylphenol, bisphenol A and bisphenol AF). The sorbent was characterized by transmission electron microscopy, FTIR, powder X-ray diffraction and other techniques. The main parameters governing the extraction efficiency were optimized. The phenols were quantified by HPLC with fluorometric detection. The method has attractive features such as low limits of detection (0.08-0.21 ng.mL-1), wide linear ranges (0.5-1000 ng.mL-1), and good repeatability (intra-day: 0.39%-4.99%; inter-day: 1.57%-5.21%). Satisfactory results were obtained when the developed method was applied to determine the four target pollutants in real world drink samples with spiked recoveries over the range of 81.3~118.0%. This indicates that the method is a powerful tool for the enrichment and determination of endocrine-disrupting phenols in drink samples. Graphical abstract A magnetite based covalent organic framework (Fe3O4@COFs) was synthesized with TPAB, TPA and Fe3O4. It was used for magnetic solid-phase extraction of endocrine-disrupting phenols from plastic-packaged tea drink samples coupled with liquid chromatography (LC) for determination.

Synergistic effect of co-exposure to cadmium (II) and 4-n-nonylphenol on growth inhibition and oxidative stress of Chlorella sorokiniana.

Toxicological effect of freshwater algae co-exposure to Cd and 4-n-nonylphenol (4-n-NP) was seldom reported. In the present study, Chlorella sorokiniana was selected for testing the single and combined effect of Cd and 4-n-NP by detecting the growth inhibition and oxidative stress after exposure for 48 h, 72 h, and 96 h. The combined effects were evaluated by using toxic units (TU) method and concentration addition（CA）model. The synergistic effect of mixture on algal growth inhibition was both observed at 48 h and 72 h, and the additive effect was observed at 96 h. In addition, the significant alterations of superoxide, thiobarbituric acid reactive substances and antioxidant defenses (superoxide dismutase, catalase, glutathione) have been detected. It could be observed that the mixture predominantly lead to synergistic effects in superoxide induction, and the antagonistic effects in the GSH induction. A similar trend between the superoxide induction and growth inhibition were observed, which may indicate that the oxidative effects of Chlorella sorokiniana contributed to the growth inhibition after exposure to Cd and 4-n-NP. These findings may have important implications in the risk assessments of heavy metals and endocrine disruptors in the aquatic environment.

Urine sample collection methods for measuring urinary concentrations of phenolic endocrine disrupting chemicals and their comparison in pregnant women / 上海交通大学学报（医学版）

Objective · To compare urine sample collection methods for measuring urinary concentrations of phenolic endocrine disrupting chemicals including bisphenol A (BPA),triclosan (TCS),and 4-n-nonylphenol (4-n-NP) in pregnant women.Methods· Urine samples were collected from women at late pregnancy by two methods:urine catheter and collection bag (n=176),urine collecting containers made of polypropylene (PP) (n=642).Urinary concentrations of BPA,TCS and 4-n-NP were measured with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)method.Confirmation experiment used PP containers,urine catheter and collection bags,as well as low-density polyethylene (LDPE) tubes and glass containers as both negative controls to collect urine samples from each of the 5 women.Results · Geometric mean (GM) of urinary BPA concentration collected by urine catheter and collection bags was 82.5 ng/mL (95％ CI 71.4-95.4 ng/mL),which was 63 times higher than that from PP containers (GM 1.3 ng/mL;95％ CI 1.3-1.5 ng/mL).Concentrations of urinary 4-n-NP and creatinine were similar between two collection methods.Confirmation experiment showed that urinary BPA concentration collected by urine catheter and collection bags was much higher than those collected by other three methods.Conclusion· In collection of urine samples for measuring phenolic chemicals,PP urine collection container as well as LDPE containers are adequate for use in epidemiologic studies,but urine catheter and collection bag is not.

Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat.

Thyroperoxidase (TPO), the enzyme that catalyzes the synthesis of thyroid hormone, is a known target for thyroid-disrupting chemicals. In vivo toxicological evidence supporting TPO-inhibition as one molecular-initiating event that leads to thyroid disruption is derived largely from rat models; however, a significant fraction of research on the inhibition of TPO by xenobiotics has been conducted using porcine TPO. The current work tested the hypothesis that porcine and rat thyroid microsomes exposed to TPO-inhibiting chemicals would demonstrate different responses in a guaiacol oxidation assay. A primary objective of this work is to establish the degree of concordance between rat and porcine TPO inhibition data. Microsomes were isolated from both rat and pig thyroid glands, and the guaiacol oxidation assay was performed for a training set of 12 chemicals, including previously reported TPO inhibitors, thyroid-disrupting chemicals thought to perturb other targets, and several previously untested chemicals, to determine the relative TPO inhibition responses across species. Concentration-response curves were derived for methimazole (MMI), dibutylphthalate (DBP), diethylhexylphthalate (DEHP), diethylphthalate (DEP), 3,5-dimethylpyrazole-1-methanol (DPM), iopanoic acid (IOA), 2-mercaptobenzothiazole (MBT), sodium perchlorate (PERC), p-nonylphenol (PNP), 4-propoxyphenol (4POP), 6-propylthiouracil (PTU), and triclosan (TCS). MMI, PTU, MBT, DPM, 4POP, and at extremely high concentrations, PERC, inhibited TPO activity. Results demonstrated a strong qualitative concordance of response between the two species. All chemicals that inhibited TPO in porcine microsomes also inhibited TPO in rat microsomes. Hill model-derived IC50 values revealed approximate 1.5- to 50-fold differences in relative potency to MMI between species for positive chemicals. DPM, MBT, 4POP, and PTU exhibited greater relative potency to MMI using rat TPO versus porcine TPO, but rank order potency for inhibition was similar for the other test chemicals, with: PTU>MBT>DPM>4POP>PERC for rat TPO and MBT>PTU>DPM>4POP>PERC for porcine TPO. These data support the extrapolation of porcine TPO data to potential thyroid-disrupting activity in rodent models to evaluate TPO-inhibiting chemicals.

Parental phenols exposure and spontaneous abortion in Chinese population residing in the middle and lower reaches of the Yangtze River.

Widespread use of phenols has led to ubiquitous exposure to phenols. In experimental animals, phenols increased resorptions, reduced live litter size and fetal body weights. However, there are limited epidemiological evidences of the relationships between exposure to phenols and pregnancy outcomes. We evaluated the associations between parental urinary levels of various phenols and spontaneous abortion in a Chinese population residing in the middle and lower reaches of the Yangtze River. A case-control study was conducted that included 70 case couples with medically unexplained spontaneous abortion and 180 control couples who did not have a history of spontaneous abortion and had at least one living child. Both parental urinary phenols were measured by ultra-high performance liquid chromatography-tandem mass spectrometry including bisphenol A (BPA), benzophenone-3 (BP-3), 2,3,4-trichlorophenol (2,3,4-TCP), pentachlorophenol (PCP), 4-n-octylphenol (4-n-OP) and 4-n-nonylphenol (4-n-NP). Compared with the low exposure group, there was an increased risk of spontaneous abortion with high paternal urinary PCP concentration [odds ratio (OR)=2.09, 95% Confidence Interval (CI), 1.05-4.14], and maternal exposure to 4-n-OP and alkylphenol(s) also significantly increased the risk of spontaneous abortion (OR=2.21, 95% CI, 1.02-4.80; OR=2.81, 95% CI, 1.39-5.65, respectively). Our study firstly provides the evidence that paternal PCP exposure, maternal 4-n-OP and alkylphenol(s) exposure are associated with spontaneous abortion in humans.