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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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Indoxyl sulfate (IS) and p-cresyl sulfate (PCS), protein-bound uremic toxins, can induce oxidative stress and cause renal disease progression. However, the different cytotoxic effects on renal cells between IS and PCS are not stated. Due to uremic toxins are generally found in CKD patients, the mechanisms of uremic toxins-induced renal injury are required to study. Curcumin has anti-oxidant, anti-inflammatory and anti-apoptotic effects which may be potential used to protect against renal damage. In contrast, curcumin also exert cytotoxic effects on various cells. In addition, curcumin may reduce or enhance cytotoxicity combined with different chemicals treatments. However, whether curcumin may influence uremic toxins-induced renal injury is unclear. The goal of this study is to compare the different cytotoxic effects on renal cells between IS and PCS treatment, as well as the synergistic or antagonistic effects by combination treatments with curcumin and PCS. Our experimental result shows the PCS exerts a stronger antiproliferative effect on renal tubular cells than IS treatment. In addition, our study firstly demonstrates that curcumin enhances PCS-induced cell cytotoxicity through caspase-dependent apoptotic pathway and cell cycle alteration., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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., Competing Interests: Declaration of Competing Interest ZM declare the following financial interests/personal relationships, which may be considered as potential competing interests: Receipt of grants/research supports: Amgen, Baxter,Fresenius Medical Care, GlaxoSmithKline, Merck Sharp and Dohme-Chibret, Genzyme/ Sanofi, Lilly, Otsuka, and Government support for CKD REIN PROJECT. Receipt of honoraria or consultation fees: To charities from Astra Zeneca., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 (K m  = 0.19 ± 0.02 μM [with atypical kinetic behavior at lower substrate concentrations; see text discussion], V max  = 789.5 ± 101.7 nmol/mg/min, K si  = 2458.0 ± 332.8 μM, mean ± 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 (K m  = 81.5 ± 31.4 μM, V max  = 230.6 ± 17.7 nmol/mg/min, K si  = 986.0 ± 434.4 μM) compared to the wild type. The sulfonation of p-cresol was characterized by Michaelis-Menten kinetics in liver cytosols (K m  = 14.8 ± 3.4 μM, V max  = 1.5 ± 0.2 nmol/mg/min) and substrate inhibition in kidney cytosols (K m  = 0.29 ± 0.02 μM, V max  = 0.19 ± 0.05 nmol/mg/min, K si  = 911.7 ± 278.4 μM). Of the 14 investigated therapeutic inhibitors, mefenamic acid (K i  = 2.4 ± 0.1 nM [liver], K i  = 1.2 ± 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Liquid detergent has an increasing demand in North America, Western Europe, and Southeast Asia countries owing to its convenience to use and efficiency to clean. Alpha methyl ester sulfonates (α-MES), an anionic surfactant derived from palm oil based methyl ester, was reported to have lower manufacturing cost, good detergency with less dosage, excellent biodegradability, higher tolerance to hard water, and lower eco-toxicity as compared to linear alkylbenzene sulfonates (LABS). LABS was known as the workhorse of the detergent industry in the 20 th century. Although palm-based α-MES was successfully used as the sole surfactant in powder detergent, there are still some unsettled technical issues related to phase stability and viscosity when using this anionic surfactant in heavy-duty laundry liquid detergent formulations. This paper will review not only the market overview of detergents, the application and performance of green surfactants in laundry detergents but also will highlight the technical issues related to the application of palm-based α-MES in laundry liquid detergent and some of the possible methods to overcome the formulation adversities.

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.

Mixed micelles of linear alkylbenzene sulfonic acid (LAS) and ether sulfate-based surfactants (SLE n S) can be added in household products and cleaning agents. SLE n S with higher ethylene oxide (EO) units in the head groups have economic and environmental advantages. This work aims to assess the influence of the number of EO units in the ecotoxicity of seven variants of SLE n S-LAS micelles (0-50 EO units) in soils. Ecotoxicological tests were carried out to assess emergence and growth of four plants species and reproduction of collembolans. Most of the variants inhibited plants growth at the highest concentrations (1237.5 μg SLE n S kg -1 of soil dw ). For reproduction, lower number of EO units resulted in EC 50 from 924.2 (95 % CL: 760.7-1063.4) to 963.2 (95 % CL: 676.9-1249.6) μg SLE n S kg -1 of soil dw , whereas for higher number of EO units (50 and 30) no inhibition was reported. Based on these results, we suggest that a higher number of EO units contribute to less hazardous formulations, confirming that different designs of surfactants may contribute to changes in the responses of terrestrial organisms. Therefore, we demonstrate that standardized ecotoxicological assays may contribute to more sustainable and effective formulations, when used upstream, prior to manufacture and marketing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Elimination of pulmonary Pseudomonas aeruginosa (PA) infections is challenging to accomplish with antibiotic therapies, mainly due to resistance mechanisms. Quorum sensing inhibitors (QSIs) interfering with biofilm formation can thus complement antibiotics. For simultaneous and improved delivery of both active agents to the infection sites, self-assembling nanoparticles of a newly synthesized squalenyl hydrogen sulfate (SqNPs) were prepared. These nanocarriers allowed for remarkably high loading capacities of hydrophilic antibiotic tobramycin (Tob) and a novel lipophilic QSI at 30 % and circa 10 %, respectively. The drug-loaded SqNPs showed improved biofilm penetration and enhanced efficacy in relevant biological barriers (mucin/human tracheal mucus, biofilm), leading to complete eradication of PA biofilms at circa 16-fold lower Tob concentration than Tob alone. This study offers a viable therapy optimization and invigorates the research and development of QSIs for clinical use., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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., Competing Interests: The authors have no conflicts of interest.

Two new polyketides, purpurofuranone (1) and purpuropyranone (2), were isolated along with the known polyketides, cillifuranone (3) and taiwapyrone (4), from a mutant BD-3n-1 derived from the diethyl sulfate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. The structures of 1 and 2 were elucidated by spectroscopic methods especially on the basis of X-ray diffraction and calculated optical rotations data. The plausible biosynthesis of 1 - 4 was also proposed and discussed. In preliminary MTT assay, 1 - 4 showed no notable inhibitory effects on the tested four human cancer cell lines.

The p-cresyl sulfate accumulates in kidney disease and may be involved in renal injury. α-Lipoic acid (α-LA) acts as an antioxidant in cell injury. We investigated the effects of α-LA treatment on p-cresyl sulfate-induced renal tubular injury. p-Cresyl sulfate induced cell death, and increased Bax/Bcl-2, cleaved caspase-3, Beclin-1, and LC3BII/LC3BI in human renal proximal tubular epithelial (HK-2) cells, which was counteracted by α-LA treatment. p-Cresyl sulfate-induced apoptosis was reduced by autophagy inhibitor 3-methyladenine, and p-cresyl sulfate induced autophagy was reduced by pan-caspase inhibitor Z-VAD-FMK. Moreover, p-cresyl sulfate treatment increased the expression of ER stress proteins and decreased the expression of baculoviral IAP repeat-containing proteins 6; these effects were prevented by α-LA treatment. Apoptosis and autophagy were associated with the phosphorylation of mitogen-activated protein kinase and nuclear translocation of the nuclear factor-κB p65 subunit. Pretreatment inhibitors of p38 and JNK, and knockdown of ATF4 gene reduced apoptosis- and autophagy-related protein expressions in p-cresyl sulfate treated HK-2 cells. These results demonstrate that α-lipoic acid attenuated p-cresyl sulfate-induced cell death by suppression of apoptosis and autophagy via regulation of ER stress in HK-2 cells., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

At intestinal level, after acute or chronic exposure to iNOS-derived NO, a toxic mechanism of action leads to inflammation and degenerative diseases. The aim of this study was to investigate the effect of glucuronide and sulfate metabolites of the extra virgin olive oil phenols tyrosol (Tyr) and hydroxytyrosol (HT), in comparison with their parent compounds, on the release of NO following exposure to a pro-inflammatory stimulus, the bacterial lipopolysaccharide (LPS). Human colon adenocarcinoma cells (Caco-2), differentiated as normal enterocytes, were treated with pathological concentrations of LPS, in order to stimulate iNOS pathway, which involves NF-ĸB activation through IĸBα phosphorylation and subsequent degradation induced by Akt or MAPKs. All the tested metabolites inhibited NO release induced by LPS, acting as inhibitors of iNOS expression, with an efficacy comparable to that of the parent compounds. HT and Tyr metabolites were effective in the inhibition of IĸBα degradation. No one of the compounds was able to inhibit Akt activation, whereas they modulated p38 and ERK1/2 MAPK. Obtained data show that HT and Tyr metabolites are able to prevent a pathological NO overproduction at intestinal level, where they concentrate, thus significantly contributing to the protective activity exerted by their parent compounds against inflammation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

p-Cresyl sulfate (pCS), a uremic toxin, can cause renal damage and dysfunction. Studies suggest that renal dysfunction increases the prevalence of renal cancer. However, the effect of pCS on the proliferation and migration of renal cancer is unclear. Clear cell renal cell carcinoma (ccRCC) expresses mutant von Hippel-Lindau gene and is difficult to treat. Hypoxia-inducible factor-1α and 2-α (HIF-1α and HIF-2α) as well as microRNA-21 (miR-21) can regulate the proliferation and migration of ccRCC cells. However, the association between HIF-α and miR-21 in ccRCC remains unclear. Therefore, the effects of pCS on ccRCC cells were investigated for HIF-α and miR-21 signals. Our results showed that pCS induced overexpression of HIF-1α and promoted the proliferation and regulated epithelial-mesenchymal transition-related proteins, including E-cadherin, fibronectin, twist and vimentin in ccRCC cells. pCS treatment increased miR-21 expression. Specifically, inhibition of miR-21 blocked pCS-induced proliferation and migration. Taken together, the present results demonstrate that pCS directly induced the proliferation and migration of ccRCC cells through mechanisms involving miR-21/HIF-1α signaling pathways.

Objectives: Andrographlide sulphonate E, namely sodium 9-dehydro-17-hydro-andrographolide-19-yl sulphate, was one of the major ingredients of Xiyanping injection. The present study aimed to demonstrate its suitability as a reference standard for use of quality control of this traditional Chinese medicine preparation made from andrographlide that has been widely used to treat various infectious diseases., Methods: The stable crystals were prepared for unambiguous elucidation of the chemical structure by comprehensive spectral and thermal analysis. The anti-inflammatory effects were investigated using in vitro and in vivo methods, and the potential allergenic risk related with safety was evaluated by in silico molecular docking analysis., Key Findings: The dihydrated sulphonate derivative could be present as orthorhombic crystals with stable three-dimensional supramolecular structure, providing it the favourable physico-chemical stability as reference substance. It exhibited potent anti-inflammatory activity both in vitro and in vivo, suggesting the potency responsible for clinic efficacy of Xiyanping. Molecular docking further demonstrated its low risk of allergic reaction, as well as the proposed mechanism of anaphylactic effect of andrographolide analogues., Conclusions: Dihydrated sodium 9-dehydro-17-hydro-andrographolide-19-yl sulphate may be the ideal reference standard for use in quality control of Xiyanping., (© 2018 Royal Pharmaceutical Society.)

Objective: The study aimed to explore the effects of p-cresyl sulfate (PCS) of damaging vascular endothelial cells and promoting the formation of atherosclerosis in mice., Materials and Methods: The apolipoprotein E (ApoE)-/- mice were fed normally and with a high-fat diet; the ApoE-/- mice fed with high-fat diet were divided into two groups and treated with blank control and PCS, respectively. The aortic arch in each group was taken and underwent the oil red O staining, and the serum PCS content in each group was detected. The basic components of plaque were observed, including foam cells, lipid deposition, and cholesterol crystal. Moreover, human umbilical vein endothelial cells were cultured and divided into control group, PCS treatment group (PCS), PCS treatment with TLR4 overexpression group (PCS+TLR4+), and PCS treatment with TLR4 knock-out group (PCS+TLR4-). The degree of endothelial cell damage was detected using a cluster of differentiation CD42b-/CD31+ endothelial microparticles (EMPs), and expressions of Toll-like receptor 4 (TLR4), triggering receptor expressed on myeloid cells-1 (TREM-1), phosphorylated-endothelial nitric oxide synthase (p-eNOS), and tumor necrosis factor-α (TNF-α) in cells were detected via Polymerase Chain Reaction (PCR) and Western blotting., Results: The serum PCS concentration in high-fat ApoE-/- mice was increased, and the aortic arch sections of ApoE-/- mice treated with PCS displayed the evident atherosclerotic plaques. Experimental results of human umbilical vein endothelial cells showed that the activity of human umbilical vein endothelial cells treated with PCS declined, the expression levels of TLR4, TREM-1, and TNF-α were increased, while that of p-eNOS was decreased. After the TLR4 knockout, the above effects of PCS were reversed., Conclusions: PCS damages vascular endothelial cells through TRL4/TREM-1, thereby accelerating the formation of atherosclerosis.

Objective: To study the protective effect of simvastatin on arterial plaque instability induced by p-cresyl sulfate (PCS)., Materials and Methods: Apolipoprotein E (ApoE)-/- mice were selected as objects of this study. All mice were randomly divided into three groups: 1) the control group, 2) the PCS group and 3) the PCS + simvastatin group. After successful modeling, the levels of plasma cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-β (TNF-β) were detected. The gross specimen of coronary artery was stained. Meanwhile, oil red O staining and Sirius red staining were performed for coronary arterial sections to observe the lipid and collagen components. The expression levels of smooth muscle cells and macrophages were observed by immunohistochemistry. In addition, the expression levels of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and monocyte chemoattractant protein-1 (MCP-1) in tissues were detected by Western blotting., Results: Simvastatin could improve atherosclerotic plaque growth and atherosclerotic plaque instability induced by PCS. Moreover, simvastatin could also improve the changes of MMPs and TIMPs caused by PCS as well as the inflammatory status in mice., Conclusions: Simvastatin can improve the inflammatory status in mice, eventually improving the arterial plaque instability caused by PCS.

Infection is a major cause of mortality in chronic kidney disease (CKD) patients. Although immune dysfunction is a risk factor for infection in CKD patients, its causes are not fully elucidated. In the present study, we evaluated whether p-cresyl sulfate (pCS), an intestinal bacteria-derived uremic toxin, was involved in immune dysfunction in CKD. We used osmotic pumps to establish adenine-induced renal dysfunction mice with a chronically high blood pCS concentration. Analysis of lymphocyte subsets revealed that pCS significantly reduced peripheral B cells in renal dysfunction mice. In vitro, pCS inhibited interleukin (IL)-7-induced proliferation of CD43 + B-cell progenitors and suppressed IL-7-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) in these cells. Cell cycle analysis showed that pCS significantly decreased the percentage of CD43 + B-cell progenitors in S phase and increased that in G1 phase. These results suggest that pCS suppressed IL-7-induced STAT5 signaling and inhibited B-cell progenitor proliferation, leading to reduction of peripheral B cells in adenine-induced renal dysfunction mice. Therefore, pCS decreases peripheral B cells by inhibiting proliferation of CD43 + B-cell progenitors and is a likely cause of immune dysfunction in CKD patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In chronic kidney disease patients, oxidative stress is generally associated with disease progression and pathogenesis of its comorbidities. Phenyl sulfate is a protein-bound uremic solute, which accumulates in chronic kidney disease patients, but little is known about its nature. Although many reports revealed that protein-bound uremic solutes induce reactive oxygen species production, the effects of these solutes on anti-oxidant level have not been well studied. Therefore, we examined the effects of protein-bound uremic solutes on glutathione levels. As a result, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decreased glutathione levels in porcine renal tubular cells. Next we examined whether phenyl sulfate-treated cells becomes vulnerable to oxidative stress. In phenyl sulfate-treated cells, hydrogen peroxide induced higher rates of cell death than in control cells. Buthionine sulfoximine, which is known to decrease glutathione level, well mimicked the effect of phenyl sulfate. Finally, we evaluated a mixture of indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate at concentrations comparable to the serum concentrations of hemodialysis patients, and we confirmed its decreasing effect on glutathione level. In conclusion, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decrease glutathione levels, rendering the cells vulnerable to oxidative stress.

An accumulation of protein-bound uremic toxins (PBUTs) is one of major reasons for development of uremia-related complications. We examined the PBUT removal ability of a hexadecyl-immobilized cellulose bead (HICB)-containing column for patients undergoing hemodialysis. Adsorption of indoxyl sulfate (IS), a representative PBUT, to HICBs was examined in vitro. The HICB column was used in patients undergoing hemodialysis for direct hemoperfusion with a regular hemodialyzer. The serum IS, indole acetic acid (IAA), phenyl sulfate (PhS), and p-cresyl sulfate (PCS) levels were measured before and after passing the column. HICBs adsorbed protein-free (free) IS in a dose- and time-dependent manner in vitro (55.4 ± 1.4% adsorption of 1 millimolar, 251 µg/mL, IS for 1 h). In clinical studies, passing the HICB-containing column decreased the serum level of free IS, IAA, PhS, and PCS levels significantly (by 34.4 ± 30.0%, 34.8 ± 25.4%, 28.4 ± 18.0%, and 34.9 ± 22.1%, respectively), but not protein-bound toxins in maintenance hemodialysis patients. HICBs absorbed some amount of free PBUTs, but the clinical trial to use HICB column did not show effect to reduce serum PBUTs level in hemodialysis patients. Adsorption treatment by means of direct hemoperfusion with regular hemodialysis may become an attractive blood purification treatment to increase PBUT removal when more effective materials to adsorb PBUTs selectively will be developed., (© 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Cd 2+ resistance and bioaccumulation capacity were selected from parental Zygosaccharomyces rouxii (CRZ-0) while maintaining NaCl tolerance using protoplast mutagenesis technology. Ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis, followed by preliminary screening and rescreening, was used to select the mutant strain CRZ-9. CRZ-9 grew better than CRZ-0 in YPD medium with 20 or 50 mg L -1 of Cd 2+ . Scanning electron microscopy observations and flow cytometry tests indicated that CRZ-9 was more effective at eliminating reactive oxygen species (ROS) generated by Cd 2+ , which led to less cellular structural damage and lower lethality. Furthermore, compared with CRZ-0, CRZ-9 exhibited increased potential for application with higher Cd 2+ removal ratio, wider working pH range, and lower biomass dosage in Cd 2+ bioaccumulation. The mutant strain CRZ-9 possessed improved Cd 2+ resistance and bioaccumulation capacity and therefore is a promising strain to remove Cd 2+ from wastewater.

Organic anion transporters (OATs) are involved in the uptake of uremic toxins such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS), which play a role in endothelial dysfunction in patients with chronic kidney diseases (CKD). In this study, we investigated the role of OAT1 and OAT3 in the uptake of PCS and IS into human endothelial cells. PCS was synthesized via p-cresol sulfation and characterized using analytical methods. The cells were treated with PCS and IS in the absence and presence of probenecid (Pb), an OAT inhibitor. Cell viability was assessed using the MTT assay. The absorbed toxins were analyzed using chromatography, OAT expression using immunocytochemistry and western blot, and monocyte chemoattractant protein-1 (MCP-1) expression using enzyme-linked immunosorbent assay. Cell viability decreased after toxin treatment in a dose-dependent manner. PCS and IS showed significant internalization after 60 min treatment, while no internalization was observed in the presence of Pb, suggesting that OATs are involved in the transport of both toxins. Immunocytochemistry and western blot demonstrated OAT1 and OAT3 expression in endothelial cells. MCP-1 expression increased after toxins treatment but decreased after Pb treatment. PCS and IS uptake were mediated by OATs, and OAT blockage could serve as a therapeutic strategy to inhibit MCP-1 expression., (© 2017 S. Karger AG, Basel.)

Cardiovascular complications are the main cause of increased mortality in patients with chronic kidney disease. Besides traditional risk factors, accumulated uremic toxins contribute to the accelerated atherosclerosis, which is accompanied by the progression of chronic kidney disease. Increased clearance of toxins with low molecular weight does not significantly improve survival of hemodialysis patients, and therefore the role and influence of other toxins, including protein-bound solutes (PBS) is intensively investigated. In our work, PBS is discussed on the example of indoxyl sulphate and pcresol sulphate. These substances are highly bound to proteins and removed from the circulation during dialysis to a small extent only. In our article, pathophysiological effects, the impact on the progression of chronic kidney disease and the survival of patients were discussed. We also assessed available methods of removing toxins from the system.

Chronic kidney disease (CKD) has emerged as a global public health problem. Although the incidence and prevalence of CKD vary from one country to another, the estimated worldwide prevalence is 8-16%. The complications associated with CKD include progression to end-stage renal disease (ESRD), mineral and bone disorders, anaemia, cognitive decline and elevated all-cause and cardiovascular (CV) mortality. As a result of progressive nephron loss, patients with late-stage CKD are permanently exposed to uraemic toxins. These toxins have been classified into three groups as a function of the molecular mass: small water-soluble molecules, middle molecules and protein-bound uraemic toxins. The compounds can also be classified according to their origin (i.e. microbial or not) or their protein-binding ability. The present review will focus on the best-characterized protein-bound uraemic toxins, namely indoxylsulfate (IS), indole acetic acid (IAA) and p-cresylsulfate (PCS, a cresol metabolite). Recent research suggests that these toxins accelerate the progression of CV disease, kidney disease, bone disorders and neurological complications. Lastly, we review therapeutic approaches that can be used to decrease toxin levels., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Immune system dysfunction is a common condition in chronic kidney disease (CKD). The present study investigated the effect of p-Cresyl sulfate (pCS) on human cell line U937 monocyte-derived macrophages (MDM) activity. MDM (1×10 6 cells/mL) were incubated with pCS (10, 25, or 50μg/mL), with or without lipopolysaccharide (LPS; 25ng/mL) and then evaluated NO production, phagocytosis and antigen-presenting molecules expression (HLA-ABC, HLA-DR, CD80 and CD86). All analyses were performed by flow cytometry. All pCS concentrations were able to increase NO production (49±12.1%, 39.8±7.75%, 43.7±11.9%, respectively) compared to untreated cells (4.35±3.34%) after 6h incubation but only the lowest concentration increased this production after 12h (82.9±8.6%, 61±7.2%, 40.8±11.7%). Combined with LPS, the same results were observed. Regarding to phagocytosis, all concentrations were able to induce bead engulfment (35.4±2.71%, 30±3.04%, 23.28±4.58%). In addition, pCS (50μg/mL) was able to increase HLA-ABC and CD80 expression, showed a slight effect on HLA-DR expression and, no difference in basal CD86 levels. pCS can induce an increased oxidative burst and phagocytosis by human macrophages while no modulation of HLA-DR or CD86 expression was induced. Together, these results suggest that pCS induces macrophage activation but interfere in antigen processing, leading to a failure in adaptive immune response in CKD., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Fusarium oxysporum strain BM-201 was treated with ultraviolet (UV) radiation to obtain a high pectinase-producing strain. Mutant UV-10-41 was obtained and then treated by diethyl sulfate. Next, the mutant UV-diethyl sulfate-43 derived from UV-10-41 was selected as high pectinase-producing strain. Mutant UV-diethyl sulfate-43 was incubated on slant for 10 generations, demonstrating that the pectinase-producing genes were stable. Pectinase activity reached 391.2 U/mL, which is 73.6% higher than that of the original strain., Competing Interests: The authors declare no conflict of interest.

Chemical mutagenesis is an efficient tool used in mutation-breeding programs to improve the vital characters of the floricultural crops. This study aimed to estimate the effects of different concentrations of two chemical mutagens; sodium azide (SA) and diethyl sulfate (DES). The vegetative growth and flowering characteristics in two generations (M1 and M2) of calendula plants were investigated. Seeds were treated with five different concentrations of SA and DES (at the same rates) of 1000, 2000, 3000, 4000, and 5000 ppm, in addition to a control treatment of 0 ppm. Results showed that lower concentrations of SA mutagen had significant effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements in plants of both generations. Calendula plants tended to flower earlier under low mutagen concentrations (1000 ppm), whereas higher concentrations delayed flowering significantly. Positive results on seed germination, plant height, number of branches, plant fresh weight, and leaf area were observed in the M2-generation at lower concentrations of SA (1000 ppm), as well as at 4000 ppm DES on number of leaves and inflorescences. The highest total soluble protein was detected at the concentrations of 1000 ppm SA and 2000 ppm DES. DES showed higher average of acid phosphatase activity than SA. Results indicated that lower concentrations of SA and DES mutagens had positive effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements. Thus, lower mutagen concentrations could be recommended for better floral and physio-chemical performance.

Chronic kidney disease (CKD) is characterized by vascular remodeling and the retention of uremic toxins, several of which are independently associated with the high cardiovascular mortality rate in CKD patients. Whether the association between these uremic toxins and cardiovascular mortality is due to induction of vascular dysfunction and resulting vascular remodeling remains to be determined. This study evaluates the effects of para-cresyl sulfate (PCS), a newly identified uremic toxin, on vascular function and remodeling. PCS acutely induced oxidative stress in both endothelial and vascular smooth muscle cells, with a maximal effect at 0.15 mM, corresponding to the mean "uremic" concentration found in dialysis patients. PCS significantly increased within 30 min phenylephrine-induced contraction of mouse thoracic aorta, through direct activation of rho-kinase, independently of oxidative stress induction, as demonstrated by the capacity of rho-kinase inhibitor Y-27632 to abolish this effect. After exposure of the aorta to PCS for 48 h, we observed inward eutrophic remodeling, a hallmark of uremic vasculopathy characterized by a reduction of the area of both lumen and media, with unchanged media/lumen ratio. In conclusion, elevated PCS concentrations such as those observed in CKD patients, by promoting both vascular dysfunction and vascular remodeling, may contribute to the development of hypertension and to cardiovascular mortality in CKD., (© 2015 Wiley Periodicals, Inc.)

A growing number of publications supports a biologic effect of the protein-bound uremic retention solutes indoxyl sulfate and p-cresyl sulfate. However, the use of unrealistically high free concentrations of these compounds and/or inappropriately low albumin concentrations may blur the interpretation of these results. Here, we performed a systematic review, selecting only studies in which, depending on the albumin concentration, real or extrapolated free concentrations of indoxyl sulfate and p-cresyl sulfate remained in the uremic range. The 27 studies retrieved comprised in vitro and animal studies. A quality score was developed, giving 1 point for each of the following criteria: six or more experiments, confirmation by more than one experimental approach, neutralization of the biologic effect by counteractive reagents or antibodies, use of a real-life model, and use of dose-response analyses in vitro and/or animal studies. The overall average score was 3 of 5 points, with five studies scoring 5 of 5 points and six studies scoring 4 of 5 points, highlighting the superior quality of a substantial number of the retrieved studies. In the 11 highest scoring studies, most functional deteriorations were related to uremic cardiovascular disease and kidney damage. We conclude that our systematic approach allowed the retrieval of methodologically correct studies unbiased by erroneous conditions related to albumin binding. Our data seem to confirm the toxicity of indoxyl sulfate and p-cresyl sulfate and support their roles in vascular and renal disease progression., (Copyright © 2014 by the American Society of Nephrology.)

This study provides data to define an efficient biocide-free strategy based on zosteric acid to counteract biofilm formation on the membranes of submerged bioreactor system plants. 16S rRNA gene phylogenetic analysis showed that gammaproteobacteria was the prevalent taxa on fouled membranes of an Italian wastewater plant. Pseudomonas was the prevalent genus among the cultivable membrane-fouler bacteria and Pseudomonas putida was selected as the target microorganism to test the efficacy of the antifoulant. Zosteric acid was not a source of carbon and energy for P. putida cells and, at 200 mg/L, it caused a reduction of bacterial coverage by 80%. Biofilm experiments confirmed the compound caused a significant decrease in biomass (-97%) and thickness (-50%), and it induced a migration activity of the peritrichous flagellated P. putida over the polycarbonate surface not amenable to a biofilm phenotype. The low octanol-water partitioning coefficient and the high water solubility suggested a low bioaccumulation potential and the water compartment as its main environmental recipient and capacitor. Preliminary ecotoxicological tests did not highlight direct toxicity effects toward Daphnia magna. For green algae Pseudokirchneriella subcapitata an effect was observed at concentrations above 100 mg/L with a significant growth of protozoa that may be connected to a concurrent algal growth inhibition.

In this study, to discuss the importance of the cell cycle distribution in cell-based in vitro toxicity mechanism studies, diethyl sulfate (DES) was selected as a model chemical that induced the alteration of the cell cycle distribution in human bronchial epithelial cell line 16HBE 14o- (HBE) cells. Cells were treated with various concentrations of DES, cell proliferation and apoptosis were then determined. The results showed that DES concentration-dependently inhibited HBE cells proliferation and induced apoptosis. When cells were treated with 2.0 mM of DES for 20 or 28 h, significant S and G2/M phase accumulation was observed. Then, the relative cellular levels of Cdk4, p-Cdk2 (Thr160), Cyclins A and B1 in DES-treated HBE cells at 20 and 28 h were determined by two ways. The differences of the cell cycle distribution between DES and control groups were ignored in one way and eliminated by using flow cytometric cell sorting in the other. The results obtained by the two ways were quite different, which indicated that the cell cycle distribution might result in confounding if it was significantly different between the treated and control groups. Therefore, we propose that the cell cycle distribution should be given more consideration in cell-based in vitro toxicological studies.

Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.

In this study, we investigated the effects of diethyl sulfate (DES) on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0mM) for 12, 24 or 36h. Cell proliferation and apoptosis were determined by MTT assay and flow cytometer, respectively. The results showed that DES inhibited cell proliferation in a dose- and time-dependent manner, and induced significant apoptosis in 16HBE cells. Apoptosis related proteins measurement results revealed that DES-induced apoptosis was concurrent with the increasing of Bax and cleavage fragment caspase-3 and the decreasing of Bcl-2 and full length procaspase-3. When cells were incubated with 2.0mM of DES for several time intervals, S and G2/M phase accumulation was observed. Further analysis indicated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation. Western blotting results demonstrated that after DES treatment p-chk1 (Ser345) and p-chk2 (Thr68) levels decreased in G1 cells, and increased in S and G2/M cells. In addition, the increasing of chk1 and chk2 were also induced by DES treatment. With the increase in the dose of DES, p53 levels first increased (0.5-4.0mM) and then decreased (8.0mM). Down-regulation of p53 by RNA interference increased 4.0mM of DES-induced apoptosis but did not affect 2.0mM DES-induced cell cycle arrest. In conclusion, DES inhibits 16HBE cells proliferation in a dose- and time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Watanabe et al. report that Nox4 NADPH oxidase catalytic moiety and the subunit p22(phox) mediate the increase in oxidative stress and human tubular epithelial cell injury induced by p-cresyl sulfate, a protein-bound uremic toxin. These findings could be instrumental for the design of novel therapeutic intervention utilizing small-molecule inhibitors specifically targeting Nox oxidases to prevent or slow down the progression of chronic kidney disease and the associated disorders due to uremic toxicity.

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-β1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate.

Background: Uremic toxins are considered to have a determinant pathological role in the progression of chronic kidney disease. The aim of this study was to define the putative pathological roles of the renal renin-angiotensin-aldosterone system (RAAS) and renal tubular epithelial-to-mesenchymal transition (EMT) in kidney fibrosis induced by (indoxyl sulfate) IS and (p-cresol sulfate) PCS., Methods: Mouse proximal renal tubular cells (PKSV-PRs) treated with IS or PCS were used. Half-nephrectomized B-6 mice were treated with IS or PCS for 4 weeks. In the losartan treatment study, the study animal was administrated with IS+losartan or PCS+losartan for 4 weeks., Results: IS and PCS significantly activated the intrarenal RAAS by increasing renin, angiotensinogen, and angiotensin 1 (AT1) receptor expression, and decreasing AT2 receptor expression in vitro and in vivo. IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression. The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. IS and PCS induced the phenotype of EMT-like transition in renal tubules by increasing the expression of fibronectin and α-smooth muscle actin and decreasing the expression of E-cadherin. Losartan significantly attenuated the expression of TGF-β1 and Snail, and decreased kidney fibrosis induced by IS and PCS in vivo., Conclusion: Activating the renal RAAS/TGF-β pathway has an important pathological role in chronic kidney injury caused by IS and PCS. IS and PCS may increase Snail expression and induce EMT-like transition.

Dunaliella salina, a unicellular green alga of environmental tolerance, was employed as test organism to investigate the toxicity effects of trichlorfon and dimehypo widely used in agriculture and veterinary as pesticides. The influences of trichlorfon and dimehypo on cell growth, β-carotene level, cell morphology changes, and activities of superoxide dismutase (Sod) and catalase (Cat) were investigated. At the concentrations less than 0.050 g L(-1) trichlorfon or 0.0005 g L(-1) dimehypo, cell responses were similar to control. When treated with 0.075-0.100 g L(-1) trichlorfon or 0.001-0.004 g L(-1) dimehypo, cell growth and β-carotene levels declined at first and then revived. When concentrations were higher than 0.125 g L(-1) trichlorfon or 0.005 g L(-1) dimehypo, both cell growth and β-carotene levels decreased until they were undetectable. The 10-d IC50 of trichlorfon and dimehypo on D. salina were 0.179 g L(-1) and 0.032 g L(-1). Both pollutants could stimulate the increase of Cat activity at a low concentration. Tolerance of D. salina to trichlorfon was obviously higher than that of dimehypo., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

The function of centrosome that serves as the main microtubule organizing center is essential to ensure the genomic integrity during the cell division cycle. Centrosome abnormalities are frequently observed in many tumors and cells exposed to genotoxic agents. Here, we investigated the centrosome abnormalities induced by diethyl sulfate (DES) in Chinese hamster lung (CHL) fibroblasts and the underlying molecular mechanisms. Our results showed that DES exposure at 0.3, 1 and 3mM for 48 h caused centrosome amplification in a dose dependent manner. This effect was associated with transient S and G2/M phase delay and up-regulating of Cdk2, Cyclin A expressions. Furthermore, inhibition of Cdk2 activities reversed the centrosome amplification induced by DES. These results reveal that centrosome is one of the key subcellular targets of DES. Centrosome abnormalities might be important mechanisms behind the aneuploidy induction and carcinogenicity of DES., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Troglitazone (TGZ), an orally active hypoglycemic agent, was found to be associated with severe drug-induced liver failure and was withdrawn from the market in 2000. Although the exact mechanism is not clear, it has been postulated that the formation of its major sulfo-conjugated metabolite (TGZS) plays an important role in its toxicity. TGZS inhibits bile salt export pump (BSEP) that causes accumulation of bile salts in liver. High concentration of bile salts causes cell death and mitochondrial dysfunction via detergent properties. One question arises whether TGZS has direct toxicity effect on human liver cells in addition to BSEP inhibition. In this study, both TGZ and chemically synthesized TGZS were incubated with normal human hepatocytes (THLE-2 cells) for measuring their cytotoxicity in vitro using the MTT assay. Glutathione (GSH) and protein carbonyl (PC) assays were further performed to measure the oxidative stress generated by these two compounds during incubation with THLE-2 cells. The results from this study indicated that TGZS (EC(50)=21.74+/-5.38 microM) was more toxic than TGZ (EC(50)=41.12+/-4.3 microM) in THLE-2 cells. The GSH and PC data further confirmed that TGZS produced greater oxidative stress in THLE-2 cells as compared to TGZ. In conclusion, our study demonstrated for the first time that TGZS has direct toxicity effect on human liver cells and may be partially responsible for the hepatotoxicity of TGZ., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Sodium cetearyl sulfate is the sodium salt of a mixture of cetyl and stearyl sulfate. The other ingredients in this safety assessment are also alkyl salts, including ammonium coco-sulfate, ammonium myristyl sulfate, magnesium coco-sulfate, sodium cetyl sulfate, sodium coco/hydrogenated tallow sulfate, sodium coco-sulfate, sodium decyl sulfate, sodium ethylhexyl sulfate, sodium myristyl sulfate, sodium oleyl sulfate, sodium stearyl sulfate, sodium tallow sulfate, sodium tridecyl sulfate, and zinc coco-sulfate. These ingredients are surfactants used at concentrations from 0.1% to 29%, primarily in soaps and shampoos. Many of these ingredients are not in current use. The Cosmetic Ingredient Review (CIR) Expert Panel previously completed a safety assessment of sodium and ammonium lauryl sulfate. The data available for sodium lauryl sulfate and ammonium lauryl sulfate provide sufficient basis for concluding that sodium cetearyl sulfate and related alkyl sulfates are safe in the practices of use and concentration described in the safety assessment.

Recent regulatory pressures (e.g., REACh, CEPA) requiring bioaccumulation assessments and the need for reduced animal use have increased the necessity for the development of in vitro-based methods to estimate bioaccumulation. Our study explored the potential use of subcellular and cellular hepatic systems to determine the biotransformation potential of two surfactants: octaethylene glycol monohexadecyl ether (C16EO8) and diethylene glycol monotetradecyl ether sulfate (C14EO2S). The subcellular systems tested were liver homogenates and microsomes from the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Cellular systems consisted of primary hepatocytes from the common carp (C. carpio) and PLHC-1 cells, hepatocarcinoma cells from the desert topminnow (Poeciliopsis lucida) cell line. Each in vitro system was exposed to radiolabeled test compounds and assayed for biotransformation using liquid scintillation and thin layer chromatographic methods. First-order kinetics were used to estimate rates of biotransformation. Bioconcentration of test materials in fish were predicted using an in vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model commonly used to predict bioaccumulation in fish. Both subcellular and cellular tests using microsomes, liver homogenates and hepatocytes respectively showed biotransformation of the parent surfactants. Biotransformation rates were fastest for hepatocytes, followed by microsomes and homogenates. Rates were too low from homogenate tests to extrapolate to in vivo-based biotransformation rates using the extrapolation model. Trout microsomes metabolized C16EO8 faster than carp microsomes, yet rates were approximately the same for C14EO2S. Predicted BCF values incorporating in vitro biotransformation rates from hepatocytes were similar to measured in vivo or USEPA's bioconcentration model (BCFWIN) predicted values. Predicted BCF values using microsomal-based rates from trout and carp studies were only slightly less than default BCF values which assumes a linear logKow to BCF relationship with no biotransformation. However, hepatocyte-based results showed substantially decreased BCFs compared to the default BCF values. These results indicate that BCF estimates based on in vitro metabolic rates can provide reasonable estimates of in vivo BCF values, therefore, supporting the use of in vitro approaches within a tiered approach to assess bioconcentration.

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity characterized as polyuric renal failure and mediated via metabolites arising from oxidation of the succinimide ring. Recent findings have suggested that the stereochemical nature of NDPS metabolites may be an important factor in NDPS metabolite-induced nephrotoxicity. The purpose of the present study was to determine the role of stereochemistry in the in vivo nephrotoxicity induced by R-(+)- and S-(-)-N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (R- and S-NDHS) and the in vitro nephrotoxicity induced by their enantiomeric sulfate conjugates, R-(-)- and S-(+)-N-(3,5-dichlorophenyl)-2-hydroxysuccinimide-O-sulfate (R- and S-NSC). Male Fischer 344 rats (four rats/group) were administered intraperitoneally (i.p.) an enantiomer of NDHS (0.05, 0.1 or 0.2 mmol/kg) or vehicle, and renal function monitored for 48 h. R-NDHS (0.1 or 0.2 mmol/kg) had little effect on renal function. In contrast, S-NDHS (0.1 mmol/kg) induced marked nephrotoxicity. The nephrotoxic potential of R- and S-NSC (0.5, 0.75 or 1.0mM) was determined using freshly isolated rat renal cortical cells (IRCC, 3-4 x 10(6)cells/ml). Cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH) at the end of a 1h incubation period. The LDH release observed in these studies was similar between R- and S-NSC. These results indicate that stereochemistry is an important factor for NDPS metabolite nephrotoxicity and that the role of stereochemistry, at least for NSC, occurs at extra-renal sites.

Drug synthesis and/or formulation can generate genotoxic impurities. For instance, strong acid/alcohol interactions during the process of drug salt formation produce alkylating agents such as alkyl halides and alkyl esters of alkyl sulfonic acids. The genotoxicity of a few classic alkylating agents such as methyl and ethyl methanesulfonate have been previously well characterized, whereas the majority of compounds from this class have only been tested in the Salmonella reversion assay. Therefore, the goal of this study was to investigate clastogenicity and DEL recombination profiles of 22 halogenated alkanes and alkylesters of sulfuric and alkane-, aryl-sulfonic acids using a battery of cellular and molecular assays. The in-vitro micronucleus assay in CHO cells was used to measure clastogenicity and the deletion recombination (DEL) assay in S. cerevisiae provided a measure of DNA deletions. We also examined the compounds' reactivity towards 4-(p-nitrobenzyl)pyridine (NBP), a surrogate molecule for biological ring nitrogens. Methylating agents were most potent in all three assays and the alkyl chlorides evaluated in our study were negative in all three assays. Also, a strong correlation was found between the MN, DEL and NBP assays. In summary, this study contributes to a better understanding of the genotoxic properties of common alkyl halides and alkyl esters with alkylating activity and might provide guidance for managing risk of genotoxic process-related impurities of drug substances and products.

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung adenocarcinoma through formation of DNA adducts. Our previous research on susceptibility to tobacco-induced carcinogenesis focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen for phenotype measurements of DNA repair capacity (DRC) in mammalian cells. Here, we present a modified host-cell reactivation (HCR) assay to measure lymphocytic DRC for alkylating DNA damage as is induced by the tobacco-specific nitrosamine, NNK. We substituted dimethyl sulfate (DMS) to create alkylating damage in pCMVluc plasmid DNA and established the damage-repair dose-response curves in both normal and nucleotide excision repair-deficient lymphoblastoid cell lines and in phytohemagglutinin (PHA)-stimulated primary lymphocytes. We then successfully measured the DRC in PHA-stimulated lymphocytes from 48 patients with lung adenocarcinoma and 45 cancer-free controls and tested our hypothesis that lower DRC for alkylating damage is associated with an increased risk of lung adenocarcinoma. The cases exhibited a lower mean DRC than did the controls. A >3-fold increased risk (odds ratio = 3.21; 95% confidence interval = 1.25-8.21) was found for those with DRC levels below the control median. There was no correlation between the DRC measured with this DMS-HCR assay and that from the parallel BPDE-HCR assay. Interestingly, risk increased to >10-fold for those with sub-optimal DRC measured by both DMS- and BPDE-HCR assays. We conclude that variability in DRC is a risk factor for lung cancer and our results provide proof of principle for a new assay that can assess DRC for NNK-induced DNA damage.

Alkyl sulfates (AS), alkyl ethoxysulfates (AES) and linear alkyl benzene sulfonates (LAS) are all High Production Volume (HPV) and 'down-the-drain' chemicals used globally in detergent and personal care products, resulting in low levels ultimately released to the environment via wastewater effluent. Due to their surfactant properties, they preferentially sorb to sediments. Hence, assessment of their levels and potential perturbations on benthos are of interest. The relative levels of AS/AES decreased with distance from the wastewater treatment plant outfall. However, this was not evident for LAS. Short chained AES and especially AS dominated the homologue distribution for AES. There were no evident patterns in LAS homologue distribution. The overall mean margin of exposure (MoE) for AS/AES and LAS is approximately 40 (range: 3 to 100) suggesting no noteworthy perturbation on biota. The findings in this study are in concordance with previous preliminary hazard screening. Comparative sediment contamination analyses principally based on Chapman and Anderson [Chapman PM, Anderson, J. A decision-making framework for sediment contamination. Integr Environ Assess Mana. 2005; 1: 163-173.] and the U.S. Environmental Protection Agency RAPID assessment methods [USEPA. Rapid bioassessment protocols for use in wadeable streams and rivers: Periphyton, benthic, macroinvertebrates, and fish. 1999. Second Edition. U.S. Environmental Protection Agency Office of Water, Washington, D.C. EPA 841-B-99-002.] did not reveal significant correlations between the surfactant concentrations and ecological status of the sampling locations. Several Lines of Evidence (LoE) of the Weight-of-Evidence (WoE) lead to the conclusion of low aquatic risk associated to the monitored compounds.

HSAS (high-solubility alkyl sulfate) is a new anionic surfactant composed predominantly of methyl and ethyl branched hexadecyl and heptadecyl sulfate. Effects of HSAS on a wide range of fish, algae, and invertebrates were investigated in conventional laboratory toxicity tests as well as in exposures conducted as part of an experimental stream model ecosystem study. For invertebrates and fish, C(16.7)HSAS (average alkyl chain length 16.7) acute LC(50) values ranged from 0.23 (channel catfish) to 2.9 (Asiatic clam, Corbicula) mg/L in well and river waters. LC(50) values for those species tested in both waters were typically within a factor of 1.5 and all were within a factor of 2 of each other, suggesting bioavailability is similar in these waters. Chronic toxicity values ranged from 0.070 (fathead minnow) to 0.42 (amphipod, Hyalella) mg/L across fish and invertebrates with algal chronic toxicity values ranging from 0.5 (blue-green algae, Anabaena flos-aquae) to 7.8 (green algae, Scenedesmus) mg/L. The order of sensitivity to HSAS acute and chronic toxicity was fish = invertebrate > algae. Based on the chronic single species sensitivity distribution, the concentrations protective of 90 and 95% of species were estimated to be 0.058 and 0.036 mg/L, respectively. These compare well with the model ecosystem NOEC of 0.064 mg/L.