Infant Exposure to PCBs and PBDEs Revealed by Hair and Human Milk Analysis: Evaluation of Hair as an Alternative Biomatrix.

Human hair, as an emerging biological monitoring matrix, has begun to be used in various human exposure studies, but little research has been done on persistent organic pollutants (POPs), especially for the body burden of POPs in infants. In this study, 36 breast-fed infants in Shanghai were recruited for a study to determine their exposure to POPs, including 12 dioxin-like polychlorinated biphenyls (dl-PCBs), 6 indicator PCBs, and 8 polybrominated diphenyl ethers (PBDEs) in the inner layer (internal) and outer layer (external) of infant hair and human milk. The similarity or difference of the POP distribution pattern or concentration among these matrices was investigated, and only weak correlations (r < 0.4) were observed between the POP concentration in human milk and infant hair (internal or external). POPs in human milk have a different profile than those in infant hair, while they have stable concentration ratios (0.58-2.72), similar distribution patterns, fine Spearman's rank correlations, and tangled principal component analysis (PCA) plots in each POP family between external and internal hair samples. The result suggested that POPs in internal hair can be easily affected by those in external hair, but POPs in human milk seem to have little contribution to the POP profile in internal hair. Although infant hair cannot reflect the POPs from diet or from body burden, it can be an ideal biomatrix that estimates infant exposure to POPs from exogenous sources like house dust when considering the similar pattern of POPs and their proper accumulation period in hair.

Flurochloridone induces responses of free radical reactions and energy metabolism disorders to BRL-3A cell.

Flurochloridone (FLC), a wildly used herbicide, could induce hepatotoxicity after long-term exposure to male rat, in addition to its reactive oxygen species (ROS)-dependent reproductive toxicity. The hepatotoxicity effect and mechanism was investigeted using 1, 10 and 100 µmol L-1 FLC treated BRL-3A liver cell in this study. The function of mitochondrial respiration, glycolysis rate and real time ATP production rate are determined by seahorse XF analyzer, and the bio-transformers of FLC, intermediates of TCA cycle and glycolysis, and related amino acids are determined and identified by [U-13C] Glucose metabolic flux technology based on UPLC-HRMS. The mRNA expression of cytochrome P450s and the key regulatory enzymes of glucose metabolism and γ- glutamyl cycle pathway. The protein expressions of protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK-3ß) were determined. The results show dechlorination and glutathione (GSH) conjugate products of FLC are predominant bio-transformmers after 24 h treatment in BRL-3A cell. FLC could enhance glycolysis function and inhibit mitochondrial aerobic respiratory, which is accompanied by the decreased total ATP level and ATP produced rate. Increased glucose-6-phosphate, fructose-6-phosphate, pyruvate and lactate levels, and elevated level of GSH and its precursor 5-glutamate-cysteine (γ-Glu-Cys) are observed in FLC treated cells, which indicates that energy metabolism dysfunction and GSH accumulation could be potentially mediated by activating γ- Glutamyl cycle pathway. Conclusively, FLC induced hepatotoxicity could be potentially related to some free radical reactions, including inhibiting mitochondrial function, glucose metabolism via glycolysis, regulating γ- glutamyl cycle pathway to promote reactive oxygen species (ROS) level, and then induced cell apoptosis by inhibiting AKT/GSK-3ß signal.

Profiling of pesticides and pesticide transformation products in Chinese herbal teas.

Herbal teas have potential health benefits, but they also contain a variety of pesticides and pesticide transformation products (PTPs) that might brings health risks. Our study maps the pesticides and PTPs in two herbal teas (chrysanthemum and Lusterleaf Holly) from two main producing areas in China. Almost all 122 samples contain pesticides, with concentration ranging from 0.0005 to 10.305 mg/kg. Nearly 40% carbendazim and imidacloprid in chrysanthemum teas and λ-cyhalothrin in Lusterleaf Holly have higher concentration levels than the values permitted in EC Regulation No. 396/2005. Distinct distributions of pesticides were found in different teas and production areas. Eight PTPs were identified along with their parents, and were confirmed using a biosynthetic strategy. Acute, chronic and cumulative health risk assessments of pesticides revealed acceptable results. Our study uncovers the profile of pesticides in herbal teas, and provides new insight into discovering the potential environmental pollution and food contaminants.

Novel Strategy for Mining and Identification of Acylcarnitines Using Data-Independent-Acquisition-Based Retention Time Prediction Modeling and Pseudo-Characteristic Fragmentation Ion Matching.

It is a challenging work to screen, identify, and quantify acylcarnitines in complex biological samples. A method, based on the retention time (RT) prediction and data-independent acquisition strategies, was proposed for the large-scale identification of acylcarnitines using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Relative cumulative eluotropic strength was introduced as a novel descriptor in building a linear prediction model, which not only solves the problem that acylcarnitines with long carbon chains cannot be well predicted in traditional models but also proves its robustness and transferability across instruments in two data sets that were acquired in distinct chromatography conditions. The accessibility of both predictive RT and MS2 spectra of suspect features effectively reduced about 30% false-positive results, and consequently, 150 and 186 acylcarnitines were identified in the rat liver and human plasma (NIST SRM 1950), respectively. This method provides a new approach in large-scale analysis of acylcarnitine in lipidomic studies and can also be extended to the analysis of other lipids.

Evaluation and application of machine learning-based retention time prediction for suspect screening of pesticides and pesticide transformation products in LC-HRMS.

Computational QSAR models have gradually been preferred for retention time prediction in data mining of emerging environmental contaminants using liquid chromatography coupled with mass spectrometry. Generally, the model performance relies on the components such as machine learning algorithms, chemical features, and example data. In this study, we evaluated the performances of four algorithms on three feature sets, using 321 and 77 pesticides as the training and validation sets, respectively. The results were varied with different combinations of algorithms on distinct feature sets. Two strategies including enhancing the complexity of chemical features and enlarging the size of the training set were proved to improve the results. XGBoost, Random Forest, and lightGBM algorithms exhibited the best results when built on a large-scale chemical descriptors, while the Keras algorithm preferred fingerprints. These four models have comparable prediction accuracies that at least 90% of pesticides in validation set can be successfully predicted with ΔRT <1.0 min. Meanwhile, a blended prediction strategy using average results from four models presented a better result than any single model. This strategy was used for assisting identification of pesticides and pesticide transformation products in 120 strawberry samples from a national survey of food contamination. Twenty pesticides and twelve pesticide transformation products were tentatively identified, where all pesticides and two pesticide transformation products (bifenazate diazene and spirotetramat-enol) were confirmed by standard materials. The outcome of this study suggested that retention time prediction is a valuable approach in compound identification when integrated with in silico MS2 spectra and other MS identification strategies.

Exposure and health risk assessment of secondary contaminants closely related to brominated flame retardants (BFRs): Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in human milk in shanghai.

Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs), as the secondary environmental pollutants of the widely used brominated flame retardants (BFRs), possess the similar physicochemical and toxic properties as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). However, studies on human body exposure to them are extremely limited. In this study, forty human milk samples collected in Shanghai were measured for 13 PBDD/F congeners using gas chromatography-high resolution mass spectrometry (GC-HRMS), to investigate their exposure level and characteristics, potential source and corresponding health risks to breastfed infants. The results showed no PBDDs but three PBDF congeners including 2,3,7,8-TBDF, 1,2,3,4,6,7,8-HpBDF and OBDF (mean concentration (detection rates) are 3.2 pg/g (72.5%), 9.5 pg/g (100%) and 28 pg/g (67.5%), respectively) were detected. The average toxic equivalent quantity (TEQ, 0.42 pg/g lw) presented the highest concentration level compared to other regions reported. The contribution of PBDFs to the total TEQ of PBDD/Fs and PCDD/Fs is 6.8%. The correlation between PBDD/Fs and age or dietary habits was not observed, which normally existed in their chlorinated analogues-PCDD/Fs. Significant correlations were observed between PBDFs and highly brominated polybrominated diphenyl ethers (PBDEs) (especially for BDE 183 and BDE 209). The correlation between PCDD/Fs and PBDFs was not observed except 2,3,7,8-TBDF. The high PBDFs exposure in Shanghai may originate from the emission of PBDEs and/or non-PBDE BFRs in environment, according to the consistency of the environmental data previously reported. The average estimated dietary intakes (EDI) for breastfed infants is 2.0 pg TEQ/kg·bw/day (0.13-13 pg TEQ/kg·bw/day), within the range of the tolerable daily intake (TDI) for TCDD (1-4 pg TEQ/kg·bw/day) suggested by the World Health Organization (WHO). However, given the high toxicity of PBDD/Fs, the potential health risks of these pollutants for breastfed infants should be of concern.

Comprehensive strategy for analysis of pesticide multi-residues in food by GC-MS/MS and UPLC-Q-Orbitrap.

A rapid and high-throughput method using both GC-MS/MS and UPLC-Q-Orbitrap systems was applied for pesticide multi-residues analysis in food samples. Strategies based on QuEChERs extraction, intelligent data mining tools with in-house/online database, and in-silico fragment prediction system were introduced to screen and identify target/untargeted features. Full-scan combined with data-independent-acquisition modes was evaluated in real sample in an attempt to improve and facilitate the pesticide screening process, of which the results showed that FS-vDIA provided equal detection rate (100%) and far less false positive results than FS-AIF did. The proposed methodology was evaluated in analysis of pesticide multi-residues in several proficiency test samples provided by EURL, and exhibited a high detection rate (>90%) of various pesticide residues with satisfactory recoveries (70-130%) without reporting false positive results. The method was also applied in China's national surveys from 2016 to 2019, and results showed its high performance in pesticide analysis in different food matrices.

A systemic workflow for profiling metabolome and lipidome in tissue.

Simple metabolome and lipidome sample preparation procedures involving two successive extractions using small pieces of tissue, and a subsequent metabolite identification (MetID) strategy were developed. The sample preparation can significantly circumvent incomplete analysis due to insufficient amounts of tissue as a result of splitting into several aliquots for multiple measurements, with advantages over the similar previously reported methods in metabolite coverage, extraction efficiency, method robustness and friendly experimental operation. A MetID strategy, based on the integration of MS information mining (including adduct ions, in-source CID, MS information from both ESI (+) and ESI (-), characteristic fragmentation ions (CFIs), constant neutral losses (CNLs) and multimers) and in silico MS simulation, was demonstrated. A large number of adduct ions (83 features), in-source CID (123 features), ESI (+/-) ionization (20 features), CFIs& CNLs (more than 120 features) and multimers (17 features) were mined by manually or in silico recognition/filtering, which provide the most suspicious structures for subsequent in silico MS simulation. The unknown features presented the same score distribution as the known (83 features) features with scores ≥25% (geomean score: 52%) and with satisfactory match for the main ions of interest. The MS/MS noise and fragment ions of coeluted quasi-molecular ions of interest are the main reason for the low score in the simulation. Manual check/evaluation is always suggested for the simulation with a score less than 50%. This strategy presents satisfactory performance with 2.5 times more metabolites structurally characterized compared with that of the traditional method based on accurate-mass-based MS and MS/MS library matching. This strategy would be useful for potentially identifying metabolites without available MS/MS information in the library.

Multi-analyte method development for analysis of brominated flame retardants (BFRs) and PBDE metabolites in human serum.

Commonly, analytical methods measuring brominated flame retardants (BFRs) of different chemical polarities in human serum are labor consuming and tedious. Our study used acidified diatomaceous earth as solid-phase extraction (SPE) adsorbent and defatting material to simultaneously determine the most abundant BFRs and their metabolites with different polarities in human serum samples. The analytes include three types of commercial BFRs, tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) isomers, and polybrominated biphenyl ethers (PBDEs), and dominant hydroxylated BDE (OH-PBDE) and methoxylated BDE (MeO-PBDE) metabolites of PBDEs. The sample eluents were sequentially analyzed for PBDEs and MeO-BDEs on online gel permeation chromatography/gas chromatography-electron capture-negative ionization mass spectrometry (online GPC GC-ECNI-MS) and for TBBPA, HBCD, and OH-BDEs on liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method recoveries were 67-134% with a relative standard deviation (RSD) of less than 20%. Method detection limits (MDLs) were 0.30-4.20 pg/mL fresh weight (f.w.) for all analytes, except for BDE-209 of 16 pg/mL f.w. The methodology was also applied in a pilot study, which analyzed ten real samples from healthy donors in China, and the majority of target analytes were detected with a detection rate of more than 80%. To our knowledge, it is the first time for us in effectively determining BFRs of most types in one aliquot of human serum samples. This new analytical method is more specific, sensitive, accurate, and time saving for routine biomonitoring of these BFRs and for integrated assessment of health risk of BFR exposure.

A validated method for rapid determination of dibenzo-p-dioxins/furans (PCDD/Fs), polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in human milk: focus on utility of tandem solid phase extraction (SPE) cleanup.

An improved method based on tandem solid phase extraction (SPE) cleanup and gas chromatography-high resolution mass spectrometry (GC-HRMS) has been validated for a rapid determination of dibenzo-p-dioxins/furans (PCDD/Fs), dioxin-like polychlorinated biphenyls (PCBs), marker polychlorinated biphenyls (M-PCBs), and polybrominated diphenyl ethers (PBDEs) using a large volume (50 mL) of human milk. This method was well validated for the measurement of these analytes in human milk from the general population with low limits of detection (LODs, 0.004-0.12 ng/g lipid), satisfactory accuracy (75-120 % of recoveries), and precision [less than 10 % of relative standard deviations (RSDs)]. To comprehensively evaluate the performance of this method, a good, presently validated and routinely used method based on an automated sample clean-up system (ASCS, based on the commercial acid multilayer silica, basic alumina, and carbon columns) was used in parallel for comparison. Compared with the ASCS method, this method presented comparable specificity. Additionally, this method, in contrast to ASCS method, highly reduced consumption of solvents (40 mL versus 500 mL), which results in much lower background in the procedural blank, reduced time, and enhanced sample pretreatment throughput. This method was also applied in a pilot study to measure a batch of human milk samples with satisfactory results. Graphical Abstract Characteristics of the application of tandem SPE cleanup for determination of PCDD/Fs, DL-PCBs,M-PCBs and PBDEs in human milk.

Identification of flurochloridone metabolites in rat urine using liquid chromatography/high resolution mass spectrometry.

It is of great interest to develop strategic methods to enable chemicals' metabolites to be accurately and rapidly screened and identified. To screen and identify a category of metabolites with distinct isotopic distribution, this study proposed a generic strategy using in silico metabolite prediction plus accurate-mass-based isotopic pattern recognition (AMBIPR) and library identification on the data acquired via the data dependent MS/MS scan of LC-Q Exactive Orbitrap mass spectrometry. The proposed method was evaluated by the analysis of flurochloridone (FLC) metabolites in rat urine sample collected from toxicity tests. Different from the traditional isotopic pattern recognition (IPR) approach, AMBIPR here was performed based on the potential metabolites predicted via in silico metabolite prediction tools. Thus, the AMBIPR treated FLC data was only associated with FLC metabolites, consequently not only avoiding great efforts made to remove FLC-unrelated information and reveal FLC metabolites, but also increasing the percent of positive hits. Among the FLC metabolite peaks screened using AMBIPR, 87% of them (corresponding 97 metabolites and 49 biotransformation) were successfully identified via multiple MS identification techniques packaged in an established FLC's metabolites library based on Mass Frontier. Noteworthy, 34 metabolites (89%) were identified without distinct naturally isotopic distribution. The universal strategic approach based on background subtraction (BS) and mass defect filtering (MDF) was used to evaluate the AMBIPR and no more false positive and negative metabolites were detected. Furthermore, our results revealed that AMBIPR is very effective, inherently sensitive and accurate, and is easily automated for the rapidly screening and profiling chemicals related metabolites.

Determination of urinary bromophenols (BrPs) as potential biomarkers for human exposure to polybrominated diphenyl ethers (PBDEs) using gas chromatography-tandem mass spectrometry (GC-MS/MS).

Bromophenols (BrPs), as the metabolites of PBDEs, would be the potential exposure markers for human biomonitoring (HB) of PBDEs in urine. An analytical method using solid-phrase extraction (SPE) and gas chromatography coupled to tandem mass spectrometer (GC-MS/MS) was developed and validated for simultaneous determination of nineteen BrPs in human urine. The method detection limits (MDLs) were below 23pgmL(-1), with recovery ranged from 63% to 133% and inter-day repeatability ranged from 3% to 11% for the majority of target analytes. This method was applied in a pilot study and 2-Bromophenol (2-BrP), 4-Bromophenol (4-BrP), 2,4-Dibromophenol (2,4-DBP) and 2,4,6-Tribromophenol (2,4,6-TBP) as the predominant analytes were detected in human urine samples collected from the general population. Among the four detected analytes, 2-BrP and 4-BrP as the mono-brominated BrP congeners were firstly reported. To our knowledge, it is the first study covering all BrP congeners (from mono-brominated to penta-brominated, totally 19 congeners) in human urine. Therefore, this study is very useful for profiling urinary BrPs and discovering potential relationship between urinary BrPs and human internal exposure to PBDEs. The mechanism of fragmentation pathway of silanized BrPs was firstly illustrated in this study.

Determination of polybrominated diphenyl ethers and polychlorinated biphenyls in fishery and aquaculture products using sequential solid phase extraction and large volume injection gas chromatography/tandem mass spectrometry.

A new method was developed to determine polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in fishery and aquaculture products. Samples were extracted by an accelerated solvent extraction system and cleaned up by sequential solid phase extraction (SPE) including dispersive SPE (D-SPE) and tandem SPE. PBDEs and PCBs were analyzed by a large-volume injection gas chromatography triple quadrupole mass spectrometry (LVI-GC-QqQ-MS/MS). Good linearity (R(2)≥0.9958) was achieved. Method detection limits (MDLs) were 0.16-3.3pgg(-1) (wet weight, ww) for PBDEs and 0.13-0.97pgg(-1)ww for PCBs. Mean recoveries were 60-140% with relative standard deviations (RSDs) of less than 20% in weever fish, scallop and shrimp samples spiked at a lower level of 13-31pgg(-1)ww and a higher level of 50-125pgg(-1)ww. Certified reference materials were analyzed with acceptable results. The method reduced solvent consumption, analytical time and labor, and is suitable for the routine analysis of PBDEs and PCBs in fishery and aquaculture products.

Urinary concentrations of metabolites of pyrethroid insecticides in textile workers, Eastern China.

Pyrethroid insecticides have been applied in the production of cotton, wool and textile. In order to examine whether textile workers are exposed to pyrethroid insecticides, we recruited 50 textile workers in two textile plants in Eastern China. Their urine samples were collected for the measurement of pyrethroid metabolites: cis- and trans-isomers of 2,2-dichlorovinyl-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl2CA and trans-Cl2CA) and 3-phenoxybenzoic acid (3-PBA). Our results showed that textile workers were exposed to high levels of pyrethroid insecticides. cis-Cl2CA and 3-PBA were dominant metabolites with concentrations of 0.17-261µg/L, while concentrations of trans-Cl2CA were in the range of 0.26-11µg/L. Levels of three metabolites were in a descending order: cis-Cl2CA, 3-PBA, and trans-Cl2CA. Levels of the metabolites were associated with ages and job responsibilities of textile workers. Sewing workers, cutting workers, machine operators, reorganizers, and older workers were more likely in contact with pyrethroid insecticides in the textile production. trans- to cis-Cl2CA ratios might indicate that exposure of textile workers was via dermal absorption and inhalation.

Simultaneous determination of 45 pesticides in fruit and vegetable using an improved QuEChERS method and on-line gel permeation chromatography-gas chromatography/mass spectrometer.

In this study, a method was developed to determine 45 selected pesticides (of different chemical families) in fruit and vegetable (including apple, spinach and cucumber). Samples were extracted using an improved QuEChERS method with salting out and phase separation in two steps. The target pesticides in concentrated extracts were analyzed by an on-line gel permeation chromatography-gas chromatography/mass spectrometer (online-GPC-GC/MS). Online GPC effectively removed matrix interferences and greatly improved the method sensitivity, recoveries and automation. Method limits of quantification were 10 ng/g for uniconazole and metalaxyl, and 5 ng/g for other 43 target analytes. In three fruit and vegetable matrices each spiked with 45 pesticides (0.01 µg/g), mean recoveries ranged from 80 to 118% for most of the tested pesticides except for profenofos (77% in apple) and chlorpyrifos (68% in apple and 75% in cucumber), with relative standard deviations (RSDs) of less than 14%. The results of the proficiency testing showed that the method is very successful in measuring the certified pesticides with less than 1.3 of the absolute value of Z-score. This method has been applied for routinely monitoring pesticides in fresh fruit and vegetable.

Determination of palmatine in canine plasma by liquid chromatography-tandem mass spectrometry with solid-phase extraction.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C(18) reversed-phase column at 30 degrees C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile -0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor-->product ion transitions at m/z 352-->336 and m/z 338-->322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 microL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.

Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver.

Palladin was originally found up-regulated with NB4 cell differentiation induced by all-trans retinoic acid. Disruption of palladin results in neural tube closure defects, liver herniation, and embryonic lethality. Here we further report that Palld(-/-) embryos exhibit a significant defect in erythropoiesis characterized by a dramatic reduction in definitive erythrocytes derived from fetal liver but not primitive erythrocytes from yolk sac. The reduction of erythrocytes is accompanied by increased apoptosis of erythroblasts and partial blockage of erythroid differentiation. However, colony-forming assay shows no differences between wild-type (wt) and mutant fetal liver or yolk sac in the number and size of colonies tested. In addition, Palld(-/-) fetal liver cells can reconstitute hematopoiesis in lethally irradiated mice. These data strongly suggest that deficient erythropoiesis in Palld(-/-) fetal liver is mainly due to a compromised erythropoietic microenvironment. As expected, erythroblastic island in Palld(-/-) fetal liver was found disorganized. Palld(-/-) fetal liver cells fail to form erythroblastic island in vitro. Interestingly, wt macrophages can form such units with either wt or mutant erythroblasts, while mutant macrophages lose their ability to bind wt or mutant erythroblasts. These data demonstrate that palladin is crucial for definitive erythropoiesis and erythroblastic island formation and, especially, required for normal function of macrophages in fetal liver.