Optimizing analytical precision in the identification of synthetic cathinones and isomers: a comparative assessment of diverse GC-MS operating parameters.

Analyzing new psychoactive substances (NPSs) in forensic laboratories present a formidable challenge globally. Within illicit drug analysis, gas chromatography-mass spectrometry (GC-MS) emerges as a robust analytical tool. This study endeavors to assess and compare peak resolution in the analysis of illicit drugs, specifically focusing on 21 synthetic cathinones, encompassing 9 cathinone isomers. Varied GC-MS operating conditions, including distinct GC-MS columns and thermal gradients, were systematically employed for the simultaneous analysis of these synthetic cathinones. The study utilized HP-1 nonpolar and HP-5MS low-bleed columns to achieve optimal analyte resolution through modulation of GC-MS oven conditions. Mass spectra were meticulously recorded within a mass-to-charge (m/z) range spanning from 40 to 500 in full scan mode. The data showed that the cathinone isomers slightly differed in retention times and mass spectra. The GC oven conditions affected the peak resolution for chromatographic separation even with the same column. The peak resolution improved using a slower thermal gradient heat speed with a prolonged analysis time. Conclusively, the interplay of GC columns and thermal gradients emerged as pivotal factors impacting peak resolution in the analysis of illicit drugs. These empirical insights contribute to a nuanced understanding of peak resolution dynamics and facilitate the identification of synthetic cathinones, including their isomers, in seized materials through the judicious application of GC-MS methodologies.

How renal toxins respond to renal function deterioration and oral toxic adsorbent in pH-controlled releasing capsule.

The number of patients with chronic kidney disease (CKD) is increasing. Oral toxin adsorbents may provide some value. Several uremic toxins, including indoxyl sulfate (IS), p-cresol (PCS), acrolein, per- and poly-fluoroalkyl substances (PFAS), and inflammation markers (interleukin 6 [IL-6] and tumor necrosis factor [TNF]-alpha) have been shown to be related to CKD progression. A total of 81 patients taking oral activated charcoal toxin adsorbents (AC-134), which were embedded in capsules that dissolved in the terminal ileum, three times a day for 1 month, were recruited. The renal function, hemoglobulin (Hb), inflammation markers, three PFAS (PFOA, PFOS, and PFNA), and acrolein were quantified. Compared with the baseline, an improved glomerular filtration rate (GFR) and significantly lower acrolein were noted. Furthermore, the CKD stage 4 and 5 group had significantly higher concentrations of IS, PCS, IL-6, and TNF but lower levels of Hb and PFAS compared with the CKD Stage 3 group at baseline and after the intervention. Hb was increased only in the CKD Stage 3 group after the trial (p = .032). Acrolein did not differ between the different CKD stage groups. Patients with improved GFR (responders) (about 77%) and nonresponders had similar baseline GFR. Responders had higher acrolein and PFOA levels throughout the study and a more significant reduction in acrolein, indicating a better digestion function. Responders had higher acrolein and PFOA levels throughout the study and a more significant reduction in acrolein, while PFOA increased in responders. Both the higher PFOA and lower acrolein may be related to improved eGFR (and possibly to improvements in proteinuria, which we did not measure. Proteinuria is associated with PFAS loss in the urine), AC-134 showed the potential to improve the GFR and decrease acrolein, which might better indicate renal function change. Future studies are needed with longer follow-ups.

Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria.

Phenylketonuria (PKU) is an autosomal genetic disorder caused by a deficiency of the phenylalanine hydroxylase (PAH) enzyme. The lack of PAH results in the inability of phenylalanine (PHE) to transform into tyrosine (TYR). Consequently, this leads to the accumulation of PHE in the blood samples of newborns causing metabolic diseases such as irreversible neurological problems. An analysis was required for determining the values of PHE and TYR in blood samples from newborn babies. In this study, therefore, we developed a derivatized method to monitor PHE and TYR in plasma samples using liquid phase chromatography linked with quadrupole mass spectrometry. Accessible formaldehyde isotopes and cyanoborohydride were used to react with PHE and TYR amino groups to generate h2-formaldehyde-modified PHE and TYR (as standards) and d2-formaldehyde-modified PHE and TYR (as internal standards). We used tandem mass spectrometry for multiple reaction monitoring. We demonstrated a derivatized method suitable for the PKU screening of newborns. The recoveries for PHE and TYR were 85% and 90%, respectively. Furthermore, we compared the values of PHE and TYR in different human plasma sample storage methods, including direct plasma and dried blood spots, and the results showed no significant difference.

Assisted Reductive Amination for Quantitation of Tryptophan, 5-Hydroxytryptophan, and Serotonin by Ultraperformance Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Herein, we used isotopic formaldehyde and sodium cyanoborohydride via reductive amination to label two methyl groups on primary amine to arrange the standards (h2-formaldehyde-modified) and internal standards (ISs, d2-formaldehyde-modified) of tryptophan and its metabolites, such as serotonin (5-hydroxytryptamine) and 5-hydroxytryptophan. These derivatized reactions with a high yield are very satisfactory for manufacturing standards and ISs. This strategy will generate one or two methyl groups on amine to create different mass unit shifts with 14 vs. 16 or 28 vs. 32 in individual compounds for biomolecules with amine groups. In other words, multiples of two mass units shift are created using this derivatized method with isotopic formaldehyde. Serotonin, 5-hydroxytryptophan, and tryptophan were used as examples to demonstrate isotopic formaldehyde-generating standards and ISs. h2-formaldehyde-modified serotonin, 5-hydroxytryptophan, and tryptophan are standards to construct calibration curves, and d2-formaldehyde-modified analogs such as ISs spike into samples to normalize the signal of each detection. We utilized multiple reaction monitoring modes and triple quadrupole mass spectrometry to demonstrate the derivatized method suitable for these three nervous biomolecules. The derivatized method demonstrated a linearity range of the coefficient of determinations between 0.9938 to 0.9969. The limits of detection and quantification ranged from 1.39 to 15.36 ng/mL.

Ractopamine at the Center of Decades-Long Scientific and Legal Disputes: A Lesson on Benefits, Safety Issues, and Conflicts.

Ractopamine (RAC) is a synthetic phenethanolamine, ß-adrenergic agonist used as a feed additive to develop leanness and increase feed conversion efficiency in different farm animals. While RAC has been authorized as a feed additive for pigs and cattle in a limited number of countries, a great majority of jurisdictions, including the European Union (EU), China, Russia, and Taiwan, have banned its use on safety grounds. RAC has been under long scientific and political discussion as a controversial antibiotic as a feed additive. Here, we will present significant information on RAC regarding its application, detection methods, conflicts, and legal divisions that play a major role in controversial deadlock and why this issue warrants the attention of scientists, agriculturists, environmentalists, and health advocates. In this review, we highlight the potential toxicities of RAC on aquatic animals to emphasize scientific evidence and reports on the potentially harmful effects of RAC on the aquatic environment and human health.

DEHP mediates drug resistance by directly targeting AhR in human breast cancer.

Resistance to chemotherapy and hormonal therapy is a major clinical problem in breast cancer medicine, especially for cancer metastasis and recurrence. Di(2-ethylhexyl)phthalate (DEHP) affects drug resistance by an unknown mechanism of action. Here we analyzed breast cancer patients (N = 457) and found that Σ4MEHP (the sum of MEHP, MEHHP, MECPP and MEOHP concentrations) in urine was significantly higher (P = 0.018) in the recurrent breast cancer group compared with non-recurrent patients. Σ4MEHP-High was positively and significantly correlated with tumor stage (P = 0.005), lymph node status (P = 0.001), estrogen receptor status (P = 0.010), Her2/Neu status (P = 0.004), recurrence (P = 0.000) and tumor size (P = 0.002), as well as an independent prognostic marker (OR = 1.868; 95% CI = 1.424-2.451; P < 0.000) associated with poor survival rates based on a positive Her2/Neu status (P = 0.035). In addition, we found that DEHP inhibited paclitaxel and doxorubicin effects in breast cancer cell lines MCF-7 and MDA-MB-231 and in zebrafish and mouse tumor initiation models. DEHP induced trefoil factor 3 (TFF3) expression through the vinculin/aryl hydrocarbon receptor (AhR)/ERK signaling pathway and induced CYP2D6, CYP2C8 and CYP3A4 expression through the AhR genomic pathway to increase the epithelial-mesenchymal transition (EMT) and doxorubicin metabolism, respectably. DEHP mediated AhR-related alterations in estrogen receptor expression through the ubiquitination system, which decreased tamoxifen effects in AhR knockout mice. These findings suggest a novel therapeutic avenue by targeting AhR in drug-resistant and recurrent breast cancer.

The Long-Term DEHP Exposure Confers Multidrug Resistance of Triple-Negative Breast Cancer Cells through ABC Transporters and Intracellular ROS.

The characteristics of phthalates had been thought to be similar to endocrine disruptors, which increases cancer risk. The role of phthalates in acquired drug resistance remains unclear. In this study, we investigated the effect of di-(2-ethylhexyl) phthalate (DEHP) on acquired drug resistance in breast cancer. MCF7 and MDA-MB-231 breast cancer cells were exposed to long-term physiological concentration of DEHP for more than three months. Long-exposure DEHP permanently attenuated the anti-proliferative effect of doxorubicin with estrogen receptor-independent activity even after withdrawal of DEHP. Long term DEHP exposure significantly reduced ROS (O2-) level in MDA-MB-231 cells while increased in MCF7 cells. ATP-binding cassette (ABC) transporters possess a widely recognized mechanism of drug resistance and are considered a target for drug therapy. Upregulation of ABC family proteins, ABCB-1 and ABCC-1 observed in DEHP-exposed clones compared to doxorubicin-resistant (DoxR) and parental MDA-MB-231 cells. A viability assay showed enhanced multidrug resistance in DEHP-exposed clones against Dox, topotecan, and irinotecan. Inhibition of ABC transporters with tariquidar, enhanced drug cytotoxicity through increased drug accumulation reversing acquired multidrug resistance in MDA-MB-231 breast cancer cells. Tariquidar enhanced Dox cytotoxicity by increasing intracellular ROS production leading to caspase-3 mediated apoptosis. Activation of PI3K/Akt signaling enhanced proliferation and growth of DEHP-exposed MDA-MB-231 cells. Overall, long-term DEHP exposure resulted in acquired multidrug resistance by upregulating ABCB-1 and ABCC1; apart from proliferation PI3K/Akt may be responsible for acquired drug resistance through ABC transporter upregulation. Targeting ABCB1 and ABCC1 with tariquidar may be a promising strategy for reversing the acquired multidrug resistance of triple-negative breast cancer cells.

Anti-Inflammatory Effects Induced by Near-Infrared Light Irradiation through M2 Macrophage Polarization.

Near-infrared (NIR) can penetrate the dermis. NIR is able to regulate cutaneous component cells and immune cells and shows significant anti-inflammatory therapeutic effects. However, the mechanisms of these effects are largely unknown. The purpose of this study is to elucidate NIR-induced molecular mechanisms on macrophages because macrophages play initial roles in directing immune responses by their M1 or M2 polarizations. Proteomic analysis revealed that NIR radiation enhanced the expression of mitochondrial respiratory gene citrate synthase. This increased citrate synthase expression was triggered by NIR-induced H3K4 hypermethylation on the citrate synthase gene promoter but not by heat, which led to macrophage M2 polarization and finally resulted in TGFß1 release from CD4+ cells. These cellular effects were validated in human primary macrophages and abdominal NIR-irradiated mouse experiments. In a phorbol 12-myristate 13-acetate‒induced inflammatory model on mouse ear, we confirmed that NIR irradiation induced significant anti-inflammatory effects through decreased M1 counts, reduced TNF-α, and increased CCL22 and/or TGFß1 levels.

The relationship of indoxyl sulfate and p-cresyl sulfate with target cardiovascular proteins in hemodialysis patients.

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C-C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Synbiotics Alleviate the Gut Indole Load and Dysbiosis in Chronic Kidney Disease.

Chronic kidney disease (CKD) has long been known to cause significant digestive tract pathology. Of note, indoxyl sulfate is a gut microbe-derived uremic toxin that accumulates in CKD patients. Nevertheless, the relationship between gut microbiota, fecal indole content, and blood indoxyl sulfate level remains unknown. In our study, we established an adenine-induced CKD rat model, which recapitulates human CKD-related gut dysbiosis. Synbiotic treatment in CKD rats showed a significant reduction in both the indole-producing bacterium Clostridium and fecal indole amount. Furthermore, gut microbiota diversity was reduced in CKD rats but was restored after synbiotic treatment. Intriguingly, in our end-stage kidney disease (ESKD) patients, the abundance of indole-producing bacteria, Bacteroides, Prevotella, and Clostridium, is similar to that of healthy controls. Consistently, the fecal indole tends to be higher in the ESKD patients, but the difference did not achieve statistical significance. However, the blood level of indoxyl sulfate was significantly higher than that of healthy controls, implicating that under an equivalent indole production rate, the impaired renal excretion contributes to the accumulation of this notorious uremic toxin. On the other hand, we did identify two short-chain fatty acid-producing bacteria, Faecalibacterium and Roseburia, were reduced in ESKD patients as compared to the healthy controls. This may contribute to gut dysbiosis. We also identified that three genera Fusobacterium, Shewanella, and Erwinia, in the ESKD patients but not in the healthy controls. Building up gut symbiosis to treat CKD is a novel concept, but once proved effective, it will provide an additional treatment strategy for CKD patients.

Direct Binding of Cisplatin to p22phox, an Endoplasmic Reticulum (ER) Membrane Protein, Contributes to Cisplatin Resistance in Oral Squamous Cell Carcinoma (OSCC) Cells.

Prolonged treatment with cisplatin (CDDP) frequently develops chemoresistance. We have previously shown that p22phox, an endoplasmic reticulum (ER) membrane protein, confers CDDP resistance by blocking CDDP nuclear entry in oral squamous cell carcinoma (OSCC) cells; however, the underlying mechanism remains unresolved. Using a fluorescent dye-labeled CDDP, here we show that CDDP can bind to p22phox in both cell-based and cell-free contexts. Subsequent detection of CDDP-peptide interaction by the Tris-Tricine-based electrophoresis revealed that GA-30, a synthetic peptide matching a region of the cytosolic domain of p22phox, could interact with CDDP. These results were further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis, from which MA-11, an 11-amino acid subdomain of the GA-30 domain, could largely account for the interaction. Amino acid substitutions at Cys50, Met65 and Met73, but not His72, significantly impaired the binding between CDDP and the GA-30 domain, thereby suggesting the potential CDDP-binding residues in p22phox protein. Consistently, the p22phox point mutations at Cys50, Met65 and Met73, but not His72, resensitized OSCC cells to CDDP-induced cytotoxicity and apoptosis. Finally, p22phox might have binding specificity for the platinum drugs, including CDDP, carboplatin and oxaliplatin. Together, we have not only identified p22phox as a novel CDDP-binding protein, but further highlighted the importance of such a drug-protein interaction in drug resistance.

Visceral obesity and cell cycle pathways serve as links in the association between bisphenol A exposure and breast cancer.

It has been identified that bisphenol A (BPA) exposure causes developmental toxicity in breast cells. However, the exact molecular mechanisms underlying the association between exposure to BPA and breast cancer remain unclear. The aim of the present study was to investigate the BPA-regulated signaling pathways associated with the aggressiveness and the development of breast cancer. Microarray technology and functional gene set analyses were used to evaluate BPA and breast cancer-associated biomarkers and pathways in a discovery-driven manner. Using individual dataset analyses, it was indicated that two BPA-associated gene sets, the visceral obesity pathway, involved in visceral fat deposits and the metabolic syndrome, and the cell cycle pathway, involved in cyclins and cell cycle regulation, were significantly associated with a high grade of aggressiveness and the development of estrogen receptor (ER)-positive breast cancer (between P<0.05 and 0.0001). The pooled analysis indicated that the most significant pathway was G1/S checkpoint regulation, and the cyclin and cell cycle regulation pathway for BPA-associated ER-positive cancer. Cancer cell signaling pathways were associated with healthy breast cells developing into breast cancer. The visceral obesity and the cell cycle pathways were indicated to link BPA exposure to breast cancer. The results of the present study demonstrate a significant association between breast cancer and BPA-regulated gene pathways.

Therapeutic Targeting of Aristolochic Acid Induced Uremic Toxin Retention, SMAD 2/3 and JNK/ERK Pathways in Tubulointerstitial Fibrosis: Nephroprotective Role of Propolis in Chronic Kidney Disease.

The nephrotoxicity of aristolochic acids (AAs), p-cresyl sulfate (PCS) and indoxyl sulfate (IS) were well-documented, culminating in tubulointerstitial fibrosis (TIF), advanced chronic kidney disease (CKD) and fatal urothelial cancer. Nonetheless, information regarding the attenuation of AAs-induced nephropathy (AAN) and uremic toxin retention is scarce. Propolis is a versatile natural product, exerting anti-oxidant, anti-cancer and anti-fibrotic properties. We aimed to evaluate nephroprotective effects of propolis extract (PE) in a murine model. AAN was developed to retain circulating PCS and IS using C57BL/6 mice, mimicking human CKD. The kidney sizes/masses, renal function indicators, plasma concentrations of PCS/IS, tissue expressions of TIF, α-SMA, collagen IaI, collagen IV and signaling pathways in transforming growth factor-ß (TGF-ß) family were analyzed among the control, PE, AAN, and AAN-PE groups. PE ameliorated AAN-induced renal atrophy, renal function deterioration, TIF, plasma retention of PCS and IS. PE also suppressed α-SMA expression and deposition of collagen IaI and IV in the fibrotic epithelial-mesenchymal transition. Notably, PE treatment in AAN model inhibited not only SMAD 2/3-dependent pathways but also SMAD-independent JNK/ERK activation in the signaling cascades of TGF-ß family. Through disrupting fibrotic epithelial-mesenchymal transition and TGF-ß signaling transduction pathways, PE improves TIF and thereby facilitates renal excretion of PCS and IS in AAN. In light of multi-faced toxicity of AAs, PE may be capable of developing a new potential drug to treat CKD patients exposed to AAs.

Fabrication of Biodegradable Poly(caprolactone) Spherical-Microcarriers for Arterial Embolization.

This study aimed to develop emulsification assisted with ultrasonic atomization (EUA) to make embolic biodegradable poly(caprolactone) (PCL) spherical-microcarriers with uniform particle size for mass production which was used to cure hepatocellular carcinoma, because this kind of embolic drugs is expensive at the current market due to their complex manufacturing process. The embolic spherical-microcarriers with sustained-releasing therapeutic agents can shrink an unresectable tumor into a respectable size. Through high frequency vibrating surface on the ultrasonic atomizer nozzle, the thin liquid film for PCL oil-phase solution was broken into the uniform PCL microdroplets (particle sizes are from 20 to 55 µm) with less medicine loss. To determine the optimal parameters to make PCL microcarriers, the ultrasonic module parameters including the concentration of PCL solution, vibrating amplitude of atomizer, feeding rate of PCL oil-phase solution and collection distance on the particle size of microdroplets were analyzed. Besides, a vertical circulation flow field of aqueous-phase poly(vinyl alcohol) (PVA) solution was created to enhance the separation of the microdroplets and increase the production of the PCL microcarriers, and about 8~11 wt% of PVA solution with high stable dispersion property was used to effectively improve the yield rate of PCL spherical-microcarriers (89.8~98.2 wt%). The final particle size of PCL microcarriers was ca. 5-18 µm, indicating an about 25-50% volume shrinkage from microdroplets to solid spherical-microcarriers.

Canned food intake and urinary bisphenol a concentrations: a randomized crossover intervention study.

Bisphenol A (BPA) is an endocrine disruptor. To evaluate the effect of canned food consumption on internal BPA dose, urinary BPA concentrations were measured before and after intake of canned foods. This study applied a randomized crossover design, recruited 20 healthy volunteers, and divided them into two groups. One group consumed canned food; the other group consumed fresh food. After a 1-day washout, the dietary interventions were reversed. In each period, urine samples were collected immediately before meals and then 2 h, 4 h, and 6 h after meals. A mixed-effects model was used to assess BPA changes over time. Our results showed urinary BPA concentrations increased after consumption of canned food. Specifically, urinary BPA concentrations significantly differed between consumption of canned food and fresh food at 2 h, 4 h, and 6 h after intake (p values of 0.001, < 0.001, and < 0.001, respectively). Mean BPA concentrations at 2 h, 4 h, and 6 h after meals were 152%, 206%, and 79% higher, respectively, than mean BPA concentrations before meals. Urine concentration profiles of canned food intake showed that peaks were at 4 h, the increase diminished at 6 h, and returned to baseline levels at 24 h after intake. Therefore, dietary intervention and a 1-day washout period are effective for limiting internal BPA burden. This study provides convincing evidence of a human exposure route to BPA and a basis for designing interventions to mitigate exposure.

A standard addition method to quantify histamine by reductive amination and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

Histamine is an organic nitrogenous compound that acts as a neurotransmitter in the uterus, spinal cord, and brain and is involved in local immune responses. In this study, we developed a fast and simple derivatization method based on reductive amination that can be used to quantify histamine by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Histamine isotope analogs were synthesized via reductive amination. Histamine was modified with H2-formaldehyde to form N-dimethylated histamine to act as a standard or with D2-formaldehyde to form N-dimethylated histamine-d4 to act as an internal standard. Using this method, we achieved a limit of detection of 3.6 ng/mL, a limit of quantification of 7.9 ng/mL, and a linear calibration curve with a coefficient of determination (R2) of 0.9987. Furthermore, the intra-day relative standard deviations ranged from 0.9% to 3.7% and the inter-day relative standard deviations ranged from 2.0% to 17.6%. After derivatization, N-dimethylated histamine showed 382.5% signal enhancement compared to unmodified histamine in mass spectrometry detection. To demonstrate the applicability of this method for biological samples, we utilized standard addition method to quantify histamine in fetal bovine serum and achieved a recovery of 86.7%.

Quantification of the ß2-Adrenergic Feed Additives Ractopamine and Salbutamol by Reductive Amination-Assisted Modification.

HIGHLIGHTS: A reductive amination-assisted method was used to synthesize standards and internal standards of ractopamine and salbutamol. Standard and internal standard analogs were fabricated by isotopic formaldehydes and sodium cyanoborohydride. A quantitative method of modified ractopamine and salbutamol was successfully validated. The reductive amination-assisted method enhances the signal for MS detection.

Reduced camptothecin sensitivity of estrogen receptor-positive human breast cancer cells following exposure to di(2-ethylhexyl)phthalate (DEHP) is associated with DNA methylation changes.

Di(2-ethylhexyl)phthalate (DEHP) has been considered as an estrogen receptor alpha (ERα) agonist due to its ability to interact with ERα and promote the cell proliferation of ERα-positive breast cancer cells. The impact of DEHP on the chemical therapy in breast cancer is little known. Two breast cancer cell lines, MCF-7 (ERα-dependent) and MDA-MB-231 (ERα-independent) were examined. We found that DEHP impaired the effectiveness of camptothecin (CPT) and alleviated the CPT-induced formation of reactive oxygen species in ERα-positive MCF-7 cells, but not in ERα-negative MDA-MB-231 cells. DEHP also significantly protected MCF-7 cells against the genotoxicity of CPT. Genome-wide DNA methylation profiling revealed that after 48 hours of exposure to 100 µM DEHP, MCF-7 cells exhibited a significant change in their DNA methylation pattern, including hypermethylation of 700 genes and hypomethylation of 221 genes. The impaired therapeutic response to CPT in DEHP-exposed MCF-7 cells is probably mediated by epigenetic changes, especially through Wnt/ß-catenin signaling. A zebrafish xenograft model confirmed the disruptive effect of DEHP on CPT-induced anti-growth of MCF-7 cells. In summary, DEHP exposure induces acquired CPT-resistance in breast cancer cells and epigenetic changes associated with Wnt/ß-catenin signaling activation are probably depending on an ER-positive status.

Inhibition of the formation of autophagosome but not autolysosome augments ABT-751-induced apoptosis in TP53-deficient Hep-3B cells.

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel antimicrotubule drug, ABT-751, in a tumor protein p53 ( TP53)-deficient hepatocellular carcinoma-derived Hep-3B cells. A series of in vitro assays indicated that ABT-751 caused the disruption of the mitotic spindle structure, collapse of mitochondrial membrane potential, generation of reactive oxygen species, DNA damage, G 2 /M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Hep-3B cells accompanied by alteration of the expression levels of several DNA damage checkpoint proteins and cell cycle regulators. Subsequently, ABT-751 triggered apoptosis along with markedly upregulated several proapoptotic proteins involving in extrinsic, intrinsic, and caspase-mediated apoptotic pathways. A pan-caspase inhibitor suppressed ABT-751-induced apoptosis. ABT-751 also induced autophagy soon after the occurrence of apoptosis through the suppression of AKT serine/threonine kinase/mechanistic target of rapamycin signaling pathway. Exogenous expression of the TP53 gene significantly incurred both apoptosis and autophagy in Hep-3B cells. Pharmacological inhibition of autophagosome (early autophagy) but not autolysosome (late autophagy) enhanced ABT-751-induced apoptosis in TP53-deficient Hep-3B cells. Our study provided a new strategy to augment ABT-751-induced apoptosis in TP53-deficient cells.

Protein-bound uremic toxins are associated with cognitive function among patients undergoing maintenance hemodialysis.

Patients with chronic kidney disease have a greater risk of cognitive impairment. Cerebral uremic solute accumulation causes uremic encephalopathy; however, the association of protein-bound uremic toxins on cognitive function remains unclear. The present study aimed to investigate the association of two protein-bound uremic toxins, namely indoxyl sulfate (IS) and p-cresyl sulfate (PCS), on cognitive function in patients receiving hemodialysis (HD) for at least 90 days. Circulating free form IS and PCS were quantified by liquid chromatography/mass spectrometry. Mini-Mental State Examination (MMSE) and Cognitive Abilities Screening Instrument (CASI) were used to evaluate cognitive function. In total, 260 HD patients were recruited with a mean age of 58.1 ± 11.3 years, of which, 53.8% were men, 40% had diabetes, and 75.4% had hypertension. The analysis revealed that both free IS and free PCS were negatively associated with the CASI score and MMSE. After controlling for confounders, circulating free IS levels persisted to be negatively associated with MMSE scores [ß = -0.62, 95% confidence interval (CI): -1.16 to -0.08] and CASI scores (ß = -1.97, 95% CI: -3.78 to -0.16), mainly in the CASI domains of long-term memory, mental manipulation, language ability, and spatial construction. However, there was no correlation between free PCS and total MMSE or total CASI scores after controlling for confounders. In conclusion, circulating free form IS, but not PCS is associated with lower cognitive function test scores in HD patients. Thus, a further study is needed to evaluate whether a decrease in free IS levels can slow down cognitive decline in HD patients.