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.

Damage of uremic myocardium by p-cresyl sulfate and the ameliorative effect of Klotho by regulating SIRT6 ubiquitination.

Uremic cardiomyopathy (UCM) is a common complication in patients with chronic kidney disease (CKD) and an important risk factor for death. P-Cresyl sulfate (PCS) is a damaging factor in UCM, and Klotho is a protective factor. However, the molecular mechanisms of Klotho and PCS in UCM and the relationship between PCS and Klotho are unclear. In vitro, Klotho treatment inhibited PCS-induced cardiomyocyte hypertrophy and apoptosis by blocking mTOR phosphorylation and inhibiting DNA double-strand breaks (DSBs), respectively. Moreover, PCS increased SIRT6 protein ubiquitination and downregulated SIRT6 protein expression, while Klotho inhibited SIRT6 protein ubiquitination and upregulated SIRT6 protein expression. In a mouse model of 5/6 nephrectomy (5/6Nx)-induced UCM, the expression of Klotho in the kidney and serum was decreased, and the expression of SIRT6 protein in myocardial tissues was lower. PCS further reduced Klotho and SIRT6 expression, aggravated heart structure and function abnormalities, and increased myocardial cell apoptosis in UCM mice. Administration of Klotho protein inhibited the downregulation of SIRT6 protein expression and improved cardiac structure and function. Furthermore, serum PCS level was associated with the left ventricular mass (LVM) and left ventricular mass index (LVMI) in hemodialysis patients. In conclusion, the uremic toxin PCS injures cardiomyocytes via mTOR phosphorylation and DSBs, and Klotho antagonizes the damaging effects of PCS. Moreover, the SIRT6 protein plays an important role in UCM, and Klotho suppresses SIRT6 ubiquitination induced by PCS, further improves cardiac structure and function in UCM and exerts protective effects.

Passage Number-Induced Replicative Senescence Modulates the Endothelial Cell Response to Protein-Bound Uremic Toxins.

Endothelial aging may be induced early in pathological situations. The uremic toxins indoxyl sulfate (IS) and p-cresol (PC) accumulate in the plasma of chronic kidney disease (CKD) patients, causing accelerated endothelial aging, increased cardiovascular events and mortality. However, the mechanisms by which uremic toxins exert their deleterious effects on endothelial aging are not yet fully known. Thus, the aim of the present study is to determine the effects of IS and PC on endothelial damage and early senescence in cultured human umbilical vein endothelial cells (HUVECs). Hence, we establish an in vitro model of endothelial damage mediated by different passages of HUVECs and stimulated with different concentrations of IS and PC to evaluate functional effects on the vascular endothelium. We observe that cell passage-induced senescence is associated with apoptosis, ROS production and decreased endothelial proliferative capacity. Similarly, we observe that IS and PC cause premature aging in a dose-dependent manner, altering HUVECs' regenerative capacity, and decreasing their cell migration and potential to form vascular structures in vitro. In conclusion, IS and PC cause accelerated aging in HUVECs, thus contributing to endothelial dysfunction associated with CKD progression.

Protein-Bound Uremic Toxins and Immunity.

Protein-bound uremic toxins (PBUTs) are bioactive microbiota metabolites originated exclusively from protein fermentation of the bacterial community resident within the gut microbiota, whose composition and function is profoundly different in the chronic kidney disease (CKD) population. PBUTs accumulate in the later stages of CKD because they cannot be efficiently removed by conventional hemodialysis due to their high binding affinity for albumin, worsening their toxic effects, especially at the cardiovascular level. The accumulation of uremic toxins, along with oxidative stress products and pro-inflammatory cytokines, characterizes the uremic status of CKD patients which is increasingly associated to a state of immune dysfunction including both immune activation and immunodepression. Furthermore, the links between immune activation and cardiovascular disease (CVD), and between immunodepression and infection diseases, which are the two major complications of CKD, are becoming more and more evident. This review summarizes and discusses the current state of knowledge on the role of the main PBUTs, namely indoxyl sulfate and p-cresyl sulfate, as regulators of immune response in CKD, in order to understand whether a microbiota modulation may be useful in the management of its main complications, CVD, and infections. Summarizing the direct effects of PBUT on immune system we may conclude that PCS seemed to be associated to an immune deficiency status of CKD mainly related to the adaptative immune response, while IS seemed to reflect the activation of both innate and adaptative immune systems likely responsible of the CKD-associated inflammation. However, the exact role of IS and PCS on immunity modulation in physiological and pathological state still needs in-depth investigation, particularly in vivo studies.

Uremic endothelial-derived extracellular vesicles: Mechanisms of formation and their role in cell adhesion, cell migration, inflammation, and oxidative stress.

p-Cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) are uremic toxins found in chronic kidney disease (CKD) that are closely related to endothelial extracellular vesicles (EVs) formation. The present study aimed to understand the role of EVs and their role in cell adhesion and migration, inflammation, and oxidative stress. Human endothelial cells were treated with PCS, IS, and Pi in pre-established uremic and kinetic recommendations. EVs were characterized using scanning electron microscopy, flow cytometry, and NanoSight assays. The concentrations of EVs were established using Alamar Blue and MTT assays. Cell adhesion to extracellular matrix proteins was analyzed using an adhesion assay. Inflammation and oxidative stress were assessed by vascular cell adhesion molecule-1 expression/monocyte migration and reactive oxygen species production, respectively. The capacity of EVs to stimulate endothelial cell migration was evaluated using a wound-healing assay. Our data showed that endothelial cells stimulated with uremic toxins can induce the formation of EVs of different sizes, quantities, and concentrations, depending on the uremic toxin used. Cell adhesion was significantly (P < 0.01) stimulated in cells exposed to PCS-induced extracellular vesicles (PCSEVs) and inorganic phosphate-induced extracellular vesicles (PiEVs). Cell migration was significantly (P < 0.05) stimulated by PCSEVs. VCAM-1 expression was evident in cells treated with PCSEVs and IS-induced extracellular vesicles (ISEVs). EVs are not able to stimulate monocyte migration or oxidative stress. In conclusion, EVs may be a biomarker of endothelial injury and the inflammatory process, playing an important role in cell-to-cell communication and pathophysiological processes, although more studies are needed to better understand the mechanisms of EVs in uremia.

Characterization of human sulfotransferases catalyzing the formation of p-cresol sulfate and identification of mefenamic acid as a potent metabolism inhibitor and potential therapeutic agent for detoxification.

p-Cresol sulfate, the primary metabolite of p-cresol, is a uremic toxin that has been associated with toxicities and mortalities. The study objectives were to i) characterize the contributions of human sulfotransferases (SULT) catalyzing p-cresol sulfate formation using multiple recombinant SULT enzymes (including the polymorphic variant SULT1A1*2), pooled human liver cytosols, and pooled human kidney cytosols; and ii) determine the potencies and mechanisms of therapeutic inhibitors capable of attenuating the production of p-cresol sulfate. Human recombinant SULT1A1 was the primary enzyme responsible for the formation of p-cresol sulfate (Km = 0.19 ±â€¯0.02 µM [with atypical kinetic behavior at lower substrate concentrations; see text discussion], Vmax = 789.5 ±â€¯101.7 nmol/mg/min, Ksi = 2458.0 ±â€¯332.8 µM, mean ±â€¯standard deviation, n = 3), while SULT1A3, SULT1B1, SULT1E1, and SULT2A1 contributed negligible or minor roles at toxic p-cresol concentrations. Moreover, human recombinant SULT1A1*2 exhibited reduced enzyme activities (Km = 81.5 ±â€¯31.4 µM, Vmax = 230.6 ±â€¯17.7 nmol/mg/min, Ksi = 986.0 ±â€¯434.4 µM) compared to the wild type. The sulfonation of p-cresol was characterized by Michaelis-Menten kinetics in liver cytosols (Km = 14.8 ±â€¯3.4 µM, Vmax = 1.5 ±â€¯0.2 nmol/mg/min) and substrate inhibition in kidney cytosols (Km = 0.29 ±â€¯0.02 µM, Vmax = 0.19 ±â€¯0.05 nmol/mg/min, Ksi = 911.7 ±â€¯278.4 µM). Of the 14 investigated therapeutic inhibitors, mefenamic acid (Ki = 2.4 ±â€¯0.1 nM [liver], Ki = 1.2 ±â€¯0.3 nM [kidney]) was the most potent in reducing the formation of p-cresol sulfate, exhibiting noncompetitive inhibition in human liver cytosols and recombinant SULT1A1, and mixed inhibition in human kidney cytosols. Our novel findings indicated that SULT1A1 contributed an important role in p-cresol sulfonation (hence it can be considered a probe reaction) in liver and kidneys, and mefenamic acid may be utilized as a potential therapeutic agent to attenuate the generation of p-cresol sulfate as an approach to detoxification.

Demonstration of the first-pass metabolism in the skin of the hair dye, 4-amino-2-hydroxytoluene, using the Chip2 skin-liver microphysiological model.

We used TissUse's HUMIMIC Chip2 microfluidic model, incorporating reconstructed skin models and liver spheroids, to investigate the impact of consumer-relevant application scenarios on the metabolic fate of the hair dye, 4-amino-2-hydroxytoluene (AHT). After a single topical or systemic application of AHT to Chip2 models, medium was analysed for parent and metabolites over 5 days. The metabolic profile of a high dose (resulting in a circuit concentration of 100 µM based on 100% bioavailability) of AHT was the same after systemic and topical application to 96-well EpiDerm™ models. Additional experiments indicated that metabolic capacity of EpiDerm™ models were saturated at this dose. At 2.5 µM, concentrations of AHT and several of its metabolites differed between application routes. Topical application resulted in a higher Cmax and a 327% higher area under the curve (AUC) of N-acetyl-AHT, indicating a first-pass effect in the EpiDerm™ models. In accordance with in vivo observations, there was a concomitant decrease in the Cmax and AUC of AHT-O-sulphate after topical, compared with systemic application. A similar alteration in metabolite ratios was observed using a 24-well full-thickness skin model, EpiDermFT™, indicating that a first-pass effect was also possible to detect in a more complex model. In addition, washing the EpiDermFT™ after 30 min, thus reflecting consumer use, decreased the systemic exposure to AHT and its metabolites. In conclusion, the skin-liver Chip2 model can be used to (a) recapitulate the first-pass effect of the skin and alterations in the metabolite profile of AHT observed in vivo and (b) provide consumer-relevant data regarding leave-on/rinse-off products.

Knockdown of CK2α reduces P-cresol-induced fibrosis in human renal proximal tubule epithelial cells via the downregulation of profilin-1.

Renal fibrosis is one of the main causes of chronic kidney disease. Many studies have focused on fibroblasts and myofibroblasts involved in renal fibrogenesis. Recently, several studies have reported that renal proximal tubule epithelial cells are possible initiators of renal fibrosis. However, the mechanism through which cells induce renal fibrosis is poorly understood. In this study, we found that CK2α induces fibrosis in renal proximal tubule epithelial cells (TH1) by regulating the expression of profilin-1 (Pfn1). CKD mouse model and TH1 cells treated with P-cresol also showed an increased level of Pfn1. The knockdown of CK2α suppressed fibrosis in TH1 cells via the downregulation of Pfn1. In particular, CK2α knockdown inhibited the expression of stress fibers and fibrosis-related proteins in P-cresol-treated TH1 cells. Furthermore, the knockdown of CK2α inhibited mitochondrial dysfunction and restored cellular senescence and cell cycle in P-cresol-treated TH1 cells. These results indicate that CK2α induces renal fibrosis through Pfn1, which makes CK2α a key target molecule in the treatment of fibrosis related to chronic kidney disease.

Effects of p-Cresol on Senescence, Survival, Inflammation, and Odontoblast Differentiation in Canine Dental Pulp Stem Cells.

Aging, defined by a decrease in the physical and functional integrity of the tissues, leads to age-associated degenerative diseases. There is a relation between aged dental pulp and the senescence of dental pulp stem cells (DPSCs). Therefore, it is important to investigate the molecular processes underlying the senescence of DPSCs to elucidate the dental pulp aging mechanisms. p-Cresol (PC), a uremic toxin, is strongly related to cellular senescence. Here, age-related phenotypic changes including senescence, apoptosis, inflammation, and declining odontoblast differentiation in PC-treated canine DPSCs were investigated. Under the PC condition, cellular senescence was induced by decreased proliferation capacity and increased cell size, senescence-associated ß-galactosidase (SA-ß-gal) activity, and senescence markers p21, IL-1ß, IL-8, and p53. Exposure to PC could stimulate inflammation by the increased expression of IL-6 and cause the distraction of the cell cycle by the increased level of Bax protein and decreased Bcl-2. The levels of odontoblast differentiation markers, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, and osterix, were decreased. Consistent with those findings, the alizarin red staining, alkaline phosphatase, and DSPP protein level were decreased during the odontoblast differentiation process. Taken together, these findings indicate that PC could induce cellular senescence in DPSCs, which may demonstrate the changes in aging dental pulp.

Role of endothelial microvesicles released by p-cresol on endothelial dysfunction.

Protein bound uremic toxins, such as p-cresol, cannot be effectively removed by conventional dialysis techniques and are accumulated in plasma, thus contributing to progression of both chronic kidney disease (CKD) and cardiovascular disease (CVD). Pathological effects of uremic toxins include activation of inflammatory response, endothelial dysfunction and release of endothelial microvesicles. To date, the role of p-cresol in endothelial microvesicles formation has not been analyzed. The aim of the present study was evaluate the effects of endothelial microvesicles released by p-cresol (PcEMV) on endothelial dysfunction. An in vitro model of endothelial damage mediated by p-cresol was proposed to evaluate the functional effect of PcEMV on the endothelial repair process carried out by endothelial cells and microRNA (miRNA) that could be involved in this process. We observed that p-cresol induced a greater release of microvesicles in endothelial cells. These microvesicles altered regenerative capacity of endothelial cells, decreasing their capacity for cell migration and their potential to form vascular structures in vitro. Moreover, we observed increased cellular senescence and a deregulation of miRNA-146b-5p and miRNA-223-3p expression in endothelial cells treated with endothelial microvesicles released by p-cresol. In summary our data show that microvesicles generated in endothelial cells treated with p-cresol (PcEMV) interfere with the endothelial repair process by decreasing the migratory capacity, the ability to form new vessels and increasing the senescence of mature endothelial cells. These alterations could be mediated by the upregulation of miRNA-146b-5p and miRNA-223-3p.

P-Cresylsulfate, the Protein-Bound Uremic Toxin, Increased Endothelial Permeability Partly Mediated by Src-Induced Phosphorylation of VE-Cadherin.

The goal of our study was to investigate the impact of p-cresylsulfate (PCS) on the barrier integrity in human umbilical vein endothelial cell (HUVEC) monolayers and the renal artery of chronic kidney disease (CKD) patients. We measured changes in the transendothelial electrical resistance (TEER) of HUVEC monolayers treated with PCS (0.1-0.2 mM) similar to serum levels of CKD patients. A PCS dose (0.2 mM) significantly decreased TEER over a 48-h period. Both PCS doses (0.1 and 0.2 mM) significantly decreased TEER over a 72-h period. Inter-endothelial gaps were observed in HUVECs following 48 h of PCS treatment by immunofluorescence microscopy. We also determined whether PCS induced the phosphorylation of VE-cadherin at tyrosine 658 (Y658) mediated by the phosphorylation of Src. Phosphorylated VE-cadherin (Y658) and phosphorylated Src levels were significantly higher when the cells were treated with 0.1 and 0.2 mM PCS, respectively, compared to the controls. The endothelial barrier dysfunction in the arterial intima in CKD patients was evaluated by endothelial leakage of immunoglobulin G (IgG). Increased endothelial leakage of IgG was related to the declining kidney function in CKD patients. Increased endothelial permeability induced by uremic toxins, including PCS, suggests that uremic toxins induce endothelial barrier dysfunction in CKD patients and Src-mediated phosphorylation of VE-cadherin is involved in increased endothelial permeability induced by PCS exposure.

RIFM fragrance ingredient safety assessment, p-tolyl acetate, CAS Registry Number 140-39-6.

The existing information supports the use of this material as described in this safety assessment. p-Tolyl acetate was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog ethyl p-tolyl carbonate (CAS # 22719-81-9) show that p-tolyl acetate is not expected to be genotoxic. Data on read-across materials p-cresol (CAS # 106-44-5) and acetic acid (CAS # 64-19-7) provide a calculated MOE >100 for the repeated dose and reproductive toxicity endpoints. The skin sensitization endpoint was completed using DST for reactive materials (64 µg/cm2); exposure is below the DST. The phototoxicity/photoallergenicity endpoints were evaluated based on UV spectra; p-tolyl acetate is not expected to be phototoxic/photoallergenic. The local respiratory toxicity endpoint was evaluated using the TTC for a Cramer Class I material, and the exposure to p-tolyl acetate is below the TTC (1.4 mg/day).The environmental endpoints were evaluated; p-tolyl acetate was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

A novel water-soluble fluorescent probe with ultra-sensitivity over a wider pH range and its application for differentiating cancer cells from normal cells.

A novel pH-sensitive fluorescent probe has been designed and synthesized for sensing intracellular pH. This probe showed excellent water solubility, it was sensitive toward the pH range from 4 to 12, and it was especially sensitive in alkaline environments. During the pH changes from acidic to alkaline environments, the color of the solution turned from yellow to purple, thus the difference in color can be used to distinguish between acidic and alkaline systems. The other major features of probe pH-DCN including high selectivity, low toxicity, good reversibility and stability allowed pH-DCN to visualize fluctuations of the pH in live cells. Moreover, probe pH-DCN has successfully discriminated cancer cells from normal cells by monitoring their different intracellular pH levels.