3-Methyl-4-nitrophenol Exposure Deteriorates Oocyte Maturation by Inducing Spindle Instability and Mitochondrial Dysfunction.

3-methyl-4-nitrophenol (PNMC), a well-known constituent of diesel exhaust particles and degradation products of insecticide fenitrothion, is a widely distributed environmental contaminant. PNMC is toxic to the female reproductive system; however, how it affects meiosis progression in oocytes is unknown. In this study, in vitro maturation of mouse oocytes was applied to investigate the deleterious effects of PNMC. We found that exposure to PNMC significantly compromised oocyte maturation. PNMC disturbed the spindle stability; specifically, it decreased the spindle density and increased the spindle length. The weakened spindle pole location of microtubule-severing enzyme Fignl1 may result in a defective spindle apparatus in PNMC-exposed oocytes. PNMC exposure induced significant mitochondrial dysfunction, including mitochondria distribution, ATP production, mitochondrial membrane potential, and ROS accumulation. The mRNA levels of the mitochondria-related genes were also significantly impaired. Finally, the above-mentioned alterations triggered early apoptosis in the oocytes. In conclusion, PNMC exposure affected oocyte maturation and quality through the regulation of spindle stability and mitochondrial function.

Hemodiafiltration with endogenous reinfusion for uremic toxin removal in patients undergoing maintenance hemodialysis: a pilot study.

OBJECTIVE: To delineate the efficacy and safety profile of hemodiafiltration with endogenous reinfusion (HFR) for uremic toxin removal in patients undergoing maintenance hemodialysis (MHD). METHODS: Patients who have been on MHD for a period of at least 3 months were enrolled. Each subject underwent one HFR and one hemodiafiltration (HDF) treatment. Blood samples were collected before and after a single HFR or HDF treatment to test uremic toxin levels and to calculate clearance rate. The primary efficacy endpoint was to compare uremic toxin levels of indoxyl sulfate (IS), λ-free light chains (λFLC), and ß2-microglobulin (ß2-MG) before and after HFR treatment. Secondary efficacy endpoints was to compare the levels of urea, interleukin-6 (IL-6), P-cresol, chitinase-3-like protein 1 (YKL-40), leptin (LEP), hippuric acid (HPA), trimethylamine N-oxide (TMAO), asymmetric dimethylarginine (ADMA), tumor necrosis factor-α (TNF-α), fibroblast growth factor 23 (FGF23) before and after HFR treatment. The study also undertook a comparative analysis of uremic toxin clearance between a single HFR and HDF treatment. Meanwhile, the lever of serum albumin and branched-chain amino acids before and after a single HFR or HDF treatment were compared. In terms of safety, the study was meticulous in recording vital signs and the incidence of adverse events throughout its duration. RESULTS: The study enrolled 20 patients. After a single HFR treatment, levels of IS, λFLC, ß2-MG, IL-6, P-cresol, YKL-40, LEP, HPA, TMAO, ADMA, TNF-α, and FGF23 significantly decreased (p < 0.001 for all). The clearance rates of λFLC, ß2-MG, IL-6, LEP, and TNF-α were significantly higher in HFR compared to HDF (p values: 0.036, 0.042, 0.041, 0.019, and 0.036, respectively). Compared with pre-HFR and post-HFR treatment, levels of serum albumin, valine, and isoleucine showed no significant difference (p > 0.05), while post-HDF, levels of serum albumin significantly decreased (p = 0.000). CONCLUSION: HFR treatment effectively eliminates uremic toxins from the bloodstream of patients undergoing MHD, especially protein-bound toxins and large middle-molecule toxins. Additionally, it retains essential physiological compounds like albumin and branched-chain amino acids, underscoring its commendable safety profile.

Impact of dietary digestible aromatic amino acid levels and stachyose on growth, nutrient utilization, and cecal odorous compounds in broiler chickens.

This study evaluated the impact of dietary digestible aromatic amino acid (DAAA) levels and stachyose on growth, nutrient utilization and cecal odorous compounds in broiler chickens. A 3×2 two-factor factorial design: Three dietary DAAA levels (1.40, 1.54, 1.68%) supplemented with either 5 g/kg of stachyose or without any stachyose were used to create 6 experimental diets. Each diet was fed to 6 replicates of 10 birds from d 22 to 42. Findings revealed that broilers receiving a diet with 1.54% DAAA levels supplemented with 5 g/kg stachyose exhibited a significant boost in average daily gain and improved utilization of crude protein, ether extract, tryptophan, and methionine compared to other diet treatments (P < 0.05). As the dietary DAAA levels increased, there was a significant rise in the concentrations of indole, skatole, p-methylphenol, and butyric acid in the cecum of broilers (P < 0.05). The addition of stachyose to diets reduced concentrations of indole, skatole, phenol, p-methylphenol, acetic acid and propionic acid in the cecum (P < 0.05). The lowest concentrations of indole, phenol, p-methylphenol, volatile fatty acids and pH in cecum of broilers were observed in the treatment which diet DAAA level was 1.40% with stachyose (P < 0.05). In conclusion, dietary DAAA levels and stachyose had significant interactions on the growth, main nutrient utilization and cecal odorous compounds in broilers. The dietary DAAA level was 1.54% with 5 g/kg of stachyose can improve the growth performance, nutrient utilization. However, the dietary DAAA level was 1.40% with stachyose was more beneficial to decrease the cecal odor compound composition in broilers.

Can our microbiome break our hearts? Collaborative production of p-cresol sulfate and indoxyl sulfate by commensal microbes increases susceptibility to thrombosis.

A recent study published in mBio by Nemet et al. revealed the critical role played by two gut microbiota members in producing the metabolites indoxyl sulfate (IS) and p-cresol sulfate (pCS) (I. Nemet, M. Funabashi,X. S. Li, M. Dwidar, et al., 2023, mBio 14:e01331-23, https://doi.org/10.1128/mbio.01331-23). Understanding microbial pathways leading to IS and pCS production is crucial because they are connected to a pre-thrombotic profile, and having high levels of these metabolites increases the risk of cardiovascular diseases (CVD). Hence, this study can offer vital insights into assessing the risk for CVD and identifying potential treatment targets for this disease.

Comparative analysis of Zn(II)-complexes as model metalloenzymes for mimicking Jack bean urease.

The inhibitory action of Schiff base complexes of 3d metals against the urease enzyme is well explored in the scientific community. However, the ability of such complexes in mimicking active metallobiosites of urease enzymes, possessing ureolytic behavior, still remains unexplored. With this aim firstly, two Zn(II)-complexes (PPR-HMB-Zn and PZ-HMB-Zn) have been developed from two different Schiff base ligands (HL1 = 2-((E)-(2-(piperidin-1-yl)ethylimino)methyl)-5-methylphenol and HL2 = 2-((E)-(2-(piperizin-1-yl)ethylimino)methyl)-5-methylphenol) and structurally characterized using single crystal XRD. The hydrolytic enzymatic activity of both complexes was demonstrated by the gradual increase in the absorption maxima at 425 nm for the formation of the p-nitrophenolate ion from catalytic hydrolysis mediated by the Zn(II) complexes with a disodium salt of p-nitrophenyl phosphate as a model substrate. Associated kinetic parameters, pH dependency and a relevant hydrolysis mechanism have also been explored. After confirming the hydrolytic ability, the complexes were exploited to mimic the hydrolytic activity of Jack bean urease that catalytically hydrolyses urea into ammonia and CO2. The change in the pH of the solution owing to the formation of ammonia under the complex catalysed hydrolytic action of urea has been monitored spectrophotometrically using the pH dependent structural change of phenol red. The amount of ammonia has been quantified using the Nessler's reagent spectrophotometric method. The ureolytic reaction mechanism has been investigated using density functional theory (DFT) calculations using the B3LYP and TPSSH methods for the systematic calculation of the interaction energy. In contrast to PZ-HMB-Zn, PPR-HMB-Zn functions more effectively as a catalyst due to the existence of a lattice-occluded water molecule in its crystal structure and the protonation of the non-terminal N to attract urea by H-bonding, which was further confirmed by AIM analysis.

Embryonic Exposure to the Benzotriazole Ultraviolet Stabilizer 2-(2H-benzotriazol-2-yl)-4-methylphenol Decreases Fertility of Adult Zebrafish (Danio rerio).

Benzotriazole ultraviolet stabilizers (BUVSs) are emerging contaminants of concern. They are added to a variety of products, including building materials, personal care products, paints, and plastics, to prevent degradation caused by ultraviolet (UV) light. Despite widespread occurrence in aquatic environments, little is known regarding the effects of BUVSs on aquatic organisms. The aim of the present study was to characterize the effects of exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol (UV-P) on the reproductive success of zebrafish (Danio rerio) following embryonic exposure. Embryos were exposed, by use of microinjection, to UV-P at <1.5 (control), 2.77, and 24.25 ng/g egg, and reared until sexual maturity, when reproductive performance was assessed, following which molecular and biochemical endpoints were analyzed. Exposure to UV-P did not have a significant effect on fecundity. However, there was a significant effect on fertilization success. Using UV-P-exposed males and females, fertility was decreased by 8.75% in the low treatment group and by 15.02% in the high treatment group relative to control. In a reproduction assay with UV-P-exposed males and control females, fertility was decreased by 11.47% in the high treatment group relative to the control. Embryonic exposure to UV-P might have perturbed male sex steroid synthesis as indicated by small changes in blood plasma concentrations of 17ß-estradiol and 11-ketotestosterone, and small statistically nonsignificant decreases in mRNA abundances of cyp19a1a, cyp11c1, and hsd17b3. In addition, decreased transcript abundances of genes involved in spermatogenesis, such as nanos2 and dazl, were observed. Decreases in later stages of sperm development were observed, suggesting that embryonic exposure to UV-P impaired spematogenesis, resulting in decreased sperm quantity. The present study is the first to demonstrate latent effects of BUVSs, specifically on fish reproduction. Environ Toxicol Chem 2024;43:385-397. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Constructing 3D flower-like S-scheme N-Bi2O2CO3/g-C3N4 heterojunction with enhanced photocatalytic performance.

Mineral processing wastewater contains a lot of organic matter and heavy metal ions, and poor self-degradation ability makes it a key treatment object in environmental treatment. Photocatalysis is a promising technology to efficiently mineralize refractory contaminants from wastewater. In this work, 3D flower-like S-scheme N-Bi2O2CO3/g-C3N4 heterostructures were successfully constructed by hydrothermal method with the auxiliary of ionic liquids. The photocatalytic experiments show that the catalytic activity of heterojunction photocatalysts was significantly higher than that of bare g-C3N4 and N-Bi2O2CO3 for the degradation of two pollutants. NBOC/CN-2 shows the highest photocatalytic performance, and the degradation efficiency of sodium isobutyl xanthate (SIBX) on NBOC/CN-2 is 1.85 and 3 times that of bare g-C3N4 and Bi2O2CO3, respectively. The degradation efficiency of m-Cresol on NBOC/CN-2 is 8.34 and 6.93 times that of bare g-C3N4 and N-Bi2O2CO3, respectively. This significantly enhanced photocatalytic activity is attributed to the formation of flower-like heterojunctions, which can greatly increase the specific surface area and facilitate the separation and migration of photogenerated carriers. Total organic carbon (TOC) experiment proves that the two pollutants are effectively mineralized under the action of the prepared photocatalyst. The degradation path of m-Cresol degradation products was inferred based on the ion fragments. The capture experiment and Nitro-blue tetrazolium (NBT)-•O2- measurement show that superoxide radical plays a major role in photocatalytic degradation. The outstanding stability of the prepared flower-like heterojunction samples was examined by cyclic experiments. The S-scheme charge transfer mechanism has been proposed to explain the boosted activity of the flower-like heterojunction photocatalyst. This work provides a new idea for the design of efficient and stable g-C3N4-based photocatalyst for the photocatalytic degradation of refractory wastewater.

Suitability of different reconstructed human skin models in the skin and liver Chip2 microphysiological model to investigate the kinetics and first-pass skin metabolism of the hair dye, 4-amino-2-hydroxytoluene.

The HUMIMIC skin-liver Chip2 microphysiological systems model using the epidermal model, EpiDerm™, was reported previously to mimic application route-dependent metabolism of the hair dye, 4-amino-2-hydroxytoluene (AHT). Therefore, we evaluated the use of alternative skin models-SkinEthic™, EpiDermFT™ and PhenionFT™-for the same purpose. In static incubations, AHT permeation was similar using SkinEthic™ and EpiDerm™ models. Older Day 21 (D21) SkinEthic™ models with a thicker stratum corneum did not exhibit a greater barrier to AHT (overall permeation was the same in D17 and D21 models). All epidermal models metabolised AHT, with the EpiDerm™ exhibiting higher N-acetylation than SkinEthic™ models. AHT metabolism by D21 SkinEthic™ models was lower than that by D17 SkinEthic™ and EpiDerm™ models, thus a thicker stratum corneum was associated with fewer viable cells and a lower metabolic activity. AHT permeation was much slower using PhenionFT™ compared to epidermal models and better reflected permeation of AHT through native human skin. This model also extensively metabolised AHT to N-acetyl-AHT. After a single topical or systemic application of AHT to Chip2 model with PhenionFT™, medium was analysed for parent and metabolites over 5 days. The first-pass metabolism of AHT was demonstrated, and the introduction of a wash step after 30 min decreased the exposure to AHT and its metabolites by 33% and 40%-43%, respectively. In conclusion, epidermal and FT skin models used in the Chip2 can mimic the first-pass skin metabolism of AHT. This highlights the flexibility of the Chip2 to incorporate different skin models according to the purpose.

Metabolic Communication by SGLT2 Inhibition.

BACKGROUND: SGLT2 (sodium-glucose cotransporter 2) inhibitors (SGLT2i) can protect the kidneys and heart, but the underlying mechanism remains poorly understood. METHODS: To gain insights on primary effects of SGLT2i that are not confounded by pathophysiologic processes or are secondary to improvement by SGLT2i, we performed an in-depth proteomics, phosphoproteomics, and metabolomics analysis by integrating signatures from multiple metabolic organs and body fluids after 1 week of SGLT2i treatment of nondiabetic as well as diabetic mice with early and uncomplicated hyperglycemia. RESULTS: Kidneys of nondiabetic mice reacted most strongly to SGLT2i in terms of proteomic reconfiguration, including evidence for less early proximal tubule glucotoxicity and a broad downregulation of the apical uptake transport machinery (including sodium, glucose, urate, purine bases, and amino acids), supported by mouse and human SGLT2 interactome studies. SGLT2i affected heart and liver signaling, but more reactive organs included the white adipose tissue, showing more lipolysis, and, particularly, the gut microbiome, with a lower relative abundance of bacteria taxa capable of fermenting phenylalanine and tryptophan to cardiovascular uremic toxins, resulting in lower plasma levels of these compounds (including p-cresol sulfate). SGLT2i was detectable in murine stool samples and its addition to human stool microbiota fermentation recapitulated some murine microbiome findings, suggesting direct inhibition of fermentation of aromatic amino acids and tryptophan. In mice lacking SGLT2 and in patients with decompensated heart failure or diabetes, the SGLT2i likewise reduced circulating p-cresol sulfate, and p-cresol impaired contractility and rhythm in human induced pluripotent stem cell-derived engineered heart tissue. CONCLUSIONS: SGLT2i reduced microbiome formation of uremic toxins such as p-cresol sulfate and thereby their body exposure and need for renal detoxification, which, combined with direct kidney effects of SGLT2i, including less proximal tubule glucotoxicity and a broad downregulation of apical transporters (including sodium, amino acid, and urate uptake), provides a metabolic foundation for kidney and cardiovascular protection.

Severe 25E-NBOH intoxication associated with MDPHP intake: a case report, metabolism study, and literature review.

N-Benzylphenethylamine derivatives are 5-HT2A receptor agonists with hallucinogenic properties, including NBOMe (N-(2-methoxybenzyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-amine) and NBOH (2-(((2,5-dimethoxyphenethyl)amino)methyl)phenol). We reported here the case of a 23-year-old man who presented a serotoninergic syndrome and a loss of consciousness following the consumption of a powder labelled as 25I-NBOH. Toxicological analyses of biological samples were carried out using a liquid chromatography high-resolution mass spectrometry. Two new psychoactive substances were identified and confirmed with certified reference materials: 25E-NBOH (2-(((4-ethyl-2,5-dimethoxyphenethyl)amino)methyl)phenol) and MDPHP (1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)hexan-1-one). Pharmaceuticals administered to the patient during his medical care were found in plasma and urine. 25E-NBOH and MDPHP concentrations were respectively at 2.3 ng/mL and 3.4 ng/mL in plasma, and 25.7 ng/mL and 30.5 ng/mL in urine. 25I-NBOH (2-(((4-iodo-2,5-dimethoxyphenethyl)amino)methyl)phenol) was specifically searched in both samples and was not detected. These results are discussed along with a literature review on human cases of exposure to N-benzylphenethylamine derivatives. Using molecular networking approach, we propose the first 25E-NBOH metabolism study using authentic biological samples (plasma and urine). We described seven metabolites (M1 to M7), including two phase I (m/z 330.172; m/z 288.160) and five phase II metabolites (m/z 464.191, m/z 478.207, m/z 492.223, m/z 508.218; m/z 396.156). The M6 (m/z 492.223) was the most intense ion detected in plasma and urine and could be proposed as a relevant 25E-NBOH consumption marker. Overall, we described an original case of 25E-NBOH poisoning and identified metabolites that could potentially be used as consumption markers to detect 25E-NBOH intoxications with a higher confidence level and probably a longer detection window.

Impact of plastic-related compounds on the gene expression signature of HepG2 cells transfected with CYP3A4.

The presence of plastic and microplastic within the oceans as well as in marine flora and fauna have caused a multitude of problems that have been the topic of numerous investigations for many years. However, their impact on human health remains largely unknown. Such plastic and microplastic particles have been detected in blood and placenta, underlining their ability to enter the human body. Plastics also contain other compounds, such as plasticizers, antioxidants, or dyes, whose impact on human health is currently being studied. Critical enzymes within the metabolism of endogenous molecules, especially of xenobiotics, are the cytochrome P450 monooxygenases (CYPs). Although their importance in maintaining cellular balance has been confirmed, their interactions with plastics and related products are poorly understood. In this study, the possible relationship between different plastic-related compounds and CYP3A4 as one of the most important CYPs was analyzed using hepatic cells overexpressing this enzyme. Beginning with virtual compound screening and molecular docking of more than 1000 plastic-related compounds, several candidates were identified to interact with CYP3A4. In a second step, RNA-sequencing was used to study in detail the transcriptome-wide gene expression levels affected by the selected compounds. Three candidate molecules ((2,2'-methylenebis(6-tert-butyl-4-methylphenol), 1,1-bis(3,5-di-tert-butyl-2-hydroxyphenyl)ethane, and 2,2'-methylenebis(6-cyclohexyl-4-methylphenol)) had an excellent binding affinity to CYP3A4 in-silico as well as cytotoxic effects and interactions with several metabolic pathways in-vitro. We identified common pathways influenced by all three selected plastic-related compounds. In particular, the suppression of pathways related to mitosis and 'DNA-templated DNA replication' which were confirmed by cell cycle analysis and single-cell gel electrophoresis. Furthermore, several mis-regulated metabolic and inflammation-related pathways were identified, suggesting the induction of hepatotoxicity at different levels. These findings imply that these compounds may cause liver problems subsequently affecting the entire organism.

Interaction between preservatives and a monoclonal antibody in support of multidose formulation development.

Multidose formulations have patient-centric advantages over single-dose formats. A major challenge in developing multidose formulations is the prevention of microbial growth that can potentially be introduced during multiple drawings. The incorporation of antimicrobial preservatives (APs) is a common approach to inhibit this microbial growth. Selection of the right preservative while maintaining drug product stability is often challenging. We explored the effects of three APs, 1.1 % (w/v) benzyl alcohol, 0.62 % (w/v) phenol, and 0.42 % (w/v) m-cresol, on a model immunoglobulin G1 monoclonal antibody, termed the "NIST mAb." As measured by hydrogen exchange-mass spectrometry (HX-MS) and differential scanning calorimetry, conformational stability was decreased in the presence of APs. Specifically, flexibility (faster HX) was significantly increased in the CH2 domain (HC 238-255) across all APs. The addition of phenol caused the greatest conformational destabilization, followed by m-cresol and benzyl alcohol. Storage stability studies conducted by subvisible particle (SVP) analysis at 40 °C over 4 weeks further revealed an increase in SVPs in the presence of phenol and m-cresol but not in the presence of benzyl alcohol. However, as monitored by size exclusion chromatography, there was neither a significant change in the monomeric content nor an accumulation of soluble aggregate in the presence of APs.

SLC22A11 Inserts the Uremic Toxins Indoxyl Sulfate and P-Cresol Sulfate into the Plasma Membrane.

Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.

Phenolic compounds in the freshwater environment in South Korea: Occurrence and tissue-specific distribution.

In this study, we investigated the occurrence and distribution of phenolic compounds, including phenol, cresols, chlorophenols, nitrophenol, and bromophenols, in freshwater environments. We also focused on phenolic compounds in crucian carp (Carassius auratus) tissues, specifically the muscle, gills, brain, blood, liver, and gonads, to assess their potential bioaccumulation in fish and human health risks. Phenolic compounds were found to be widespread in various freshwater environments throughout South Korea. Phenol was predominant in all matrices, with median concentrations of 57.0 ng/L in freshwater, 54.3 ng/g dry weight (dw) in sediment, and ranging from 71 ng/g wet weight (ww) to 621 ng/g ww in crucian carp tissues. Cresols were the second most dominant compound, with m-cresol exhibiting the highest prevalence. Most of the compounds detected in crucian carp samples were also detected in freshwater and sediment, whereas pentachlorophenol and 2,4,6-tribromophenol were exclusively detected in crucian carp tissues. A high bioaccumulation potential in the liver was observed for most phenolic compounds [median log bioconcentration factor (BCF): 3.2-3.7]. Interestingly, only m-cresol showed high bioaccumulation potential in the gills (median log BCF: 3.1). The estimated daily intake of phenolic compounds suggested that it does not pose an immediate concern for human exposure owing to crucian carp consumption. These findings enhance our understanding of the exposure status, distribution, and bioaccumulation potency of phenolic compounds in aquatic ecosystems and emphasize the importance of ongoing monitoring and risk assessment efforts.

Total analysis system for the determination of uremic toxins in human plasma based on bead injection solid phase extraction hyphenated to mass spectrometry.

Indoxyl sulfate (INDS) and p-cresol sulfate (pCS) are two of the most relevant uremic toxins that are recognized to have an essential role in chronic kidney disease (CKD) progression and associated cardiovascular risk. Thus, it is crucial to accurately assess their circulating levels in the body. Aiming at establishing an analytical strategy for quantification of INDS and pCS in human plasma, an automatic on-line micro-solid-phase extraction (µSPE) procedure hyphenated to tandem mass spectrometry (MS/MS) detection without previous chromatographic separation was herein developed. The bead injection (BI) concept was used to implement the µSPE procedure in the lab-on-valve (LOV) format. After studying the extraction conditions, the anion-exchange OASIS WAX sorbent beads (10 mg) and 99% ACN-H2O (15:85, v/v)-1% (v/v) NH4OH were chosen as sorbent and eluent, respectively, as they provided the highest analyte recoveries. Subsequently, the µSPE-BI-LOV system was hyphenated on-line to a MS/MS detector and the full analytical cycle, comprising sample preparation and analytes detection, was completed in <20 min. The developed µSPE-BI-LOV-MS methodology presented good linearity (r2 > 0.999) for quantification of the target analytes at concentrations ranging from 18 to 360 µg mL-1 in plasma. LOQ values were 2 µg mL-1 for INDS and 7 µg mL-1 for pCS in plasma. Human plasma samples from healthy subjects and individuals with CKD were successfully analyzed using the developed approach. The proposed automatic methodology can be described as an eco-friendly strategy, with a favorable score of 0.64 after greenness evaluation using the AGREE metric.

Microbial metabolite p-cresol inhibits gut hormone expression and regulates small intestinal transit in mice.

p-cresol is a metabolite produced by microbial metabolism of aromatic amino acid tyrosine. p-cresol and its conjugated forms, p-cresyl sulfate and p-cresyl glucuronide, are uremic toxins that correlate positively with chronic kidney disease and diabetes pathogenesis. However, how p-cresol affects gut hormones is unclear. Here, we expose immortalized GLUTag cells to increasing concentrations of p-cresol and found that p-cresol inhibited Gcg expression and reduced glucagon-like peptide-1 (GLP-1) secretion in vitro. In mice, administration of p-cresol in the drinking water for 2 weeks reduced the transcript levels of Gcg and other gut hormones in the colon; however, it did not affect either fasting or glucose-induced plasma GLP-1 levels. Furthermore, it did not affect glucose tolerance but promoted faster small intestinal transit in mice. Overall, our data suggest that microbial metabolite p-cresol suppresses transcript levels of gut hormones and regulates small intestinal transit in mice.

Refrigerated multi-dose insulin vials remain sterile through 6 months of use.

OBJECTIVES: To evaluate sterility in refrigerated multi-dose insulin vials through 6 months of routine aspiration. MATERIALS AND METHODS: Twelve vials of insulin, six of insulin glargine U100 (Lantus®, 10 mL multi-dose vial, Sanofi, Bridgewater, NJ) containing the preservative metacresol, and six of protamine zinc insulin U40 (ProZinc®, 10 mL multi-dose vial, Boehringer Ingelheim, Duluth, GA) containing the preservative phenol, were refrigerated and aspirated twice daily for 6 months, using a new insulin syringe each time. Three vials of each insulin type were wiped with a single-use alcohol swab before sampling. Three times weekly, aspirated samples were inoculated in Tryptic Soy Broth enrichment media and incubated for evidence of microbial growth. Positive broth was cultured and speciated. Endpoints were microbial vial contamination (defined as three consecutive positive cultures of the same organism) and completion of the six-month study period. RESULTS: Microbial contamination was not identified in any vial throughout the study period. A total of 454 aspirated samples were cultured, one of which exhibited non-repeatable growth of Staphylococcus epidermidis. This vial was prematurely lost to breakage after 59 culture samples (29 after the positive growth). CLINICAL SIGNIFICANCE: Refrigerated phenol- and metacresol-containing multi-dose insulin products carry minimal risk for iatrogenic infection through 6 months of use, regardless of alcohol swab preparation.

Development of methods based on low-temperature partitioning (LTP) for monitoring cresols and chlorophenols in sewage sludge, soil, and water in column leaching.

Cresols and chlorophenols are chemical contaminants that are potentially toxic to humans and can be found in sewage sludge. These chemical contaminants can migrate into the sludge-soil-water system when sludge is used as a conditioner for agricultural soils. Thus, the objective of this study was to develop methodologies based on extraction with low-temperature partitioning (LTP) to determine cresols and chlorophenols in sewage sludge, soil, and water. The analysis was performed by gas chromatography coupled with mass spectrometry (GC-MS). The validated methods were applied to monitor cresols and chlorophenols in a column-leaching study of a sludge-soil-water system. Satisfactory results were achieved for selectivity, limit of quantification (LOQ), linearity, accuracy, and precision. In the column leaching study, only 2,4,6-trichlorophenol was quantified in sludge samples after 20 days of the experiment. None of the studied compounds were quantified in soil and leached water samples, due to the degradation promoted by the microorganisms present in the sewage sludge. Finally, validated methods were suitable for monitoring cresols and chlorophenols in the sludge-soil-water system.

Effect of cresol isomers on reproduction and gene expression in Daphnia magna.

Cresols are frequently detected in freshwater systems due to their wide use as disinfectants. However, there is limited knowledge regarding their adverse long-term toxicity effects on reproduction and gene expression patterns of aquatic species. Therefore, this study aimed to investigate the chronic toxic effects on reproduction and gene expression using D. magna. In addition, the bioconcentration behaviour of cresol isomers was also investigated. Based on the 48 h EC50 value, p-cresol's toxicity unit (TU) (13.77 TU, very toxic) was higher than o-cresol (8.05 TU, toxic) and m-cresol (5.52 TU, toxic). Regarding population-level effects, cresols resulted in a decrease in offspring and a delay in reproduction. However, cresols did not significantly affect daphnia's body weight throughout the course of the exposure period of 21 day, while third brood neonates' average body length (m-cresol and p-cresol) was significantly affected in sub-lethal concentrations. In addition, the gene transcription did not vary significantly across treatments. For bioconcentration exposure experiments, D. magna quickly eliminated all cresols from their body, suggesting that cresol isomers are unlikely to bioaccumulate in aquatic species.

4,4'-Thiobis(2-tert-butyl-5-methylphenol), an antioxidant in medical devices that may cause allergic contact dermatitis.

BACKGROUND: Antioxidants in medical devices, added to protect polymers or adhesives, may also cause contact dermatitis in some individuals. OBJECTIVES: To present data on sensitization to 4,4'-thiobis(2-tert-butyl-5-methylphenol), an antioxidant detected in some types of medical devices, for six patients that experienced eczematous reactions to different medical devices. METHODS: Patch testing with 4,4'-thiobis(2-tert-butyl-5-methylphenol), 1% pet was performed. Gas chromatography-mass spectrometry (GC-MS) was used for identification of 4,4'-thiobis(2-tert-butyl-5-methylphenol) in different medical device products. RESULTS: Six patients with contact allergy to 4,4'-thiobis(2-tert-butyl-5-methylphenol) also had relevant contact allergic reactions to medical devices containing the antioxidant. The presence of the antioxidant in products was detected using GC-MS analysis. CONCLUSIONS: The antioxidant 4,4'-thiobis(2-tert-butyl-5-methylphenol) may cause allergic contact dermatitis after exposure to different medical devices.