Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure.

Oxidative stress leads to the activation of the Nuclear factor-erythroid-2-related factor 2 (Nrf2) pathway. While most studies have focused on the activation of the Nrf2 pathway after single chemical treatment, little is known about the dynamic regulation of the Nrf2 pathway in the context of repeated exposure scenarios. Here we employed single cell live imaging to quantitatively monitor the dynamics of the Nrf2 pathway during repeated exposure, making advantage of two HepG2 fluorescent protein reporter cell lines, expressing GFP tagged Nrf2 or sulfiredoxin 1 (Srxn1), a direct downstream target of Nrf2. High throughput live confocal imaging was used to measure the temporal dynamics of these two components of the Nrf2 pathway after repeated exposure to an extensive concentration range of diethyl maleate (DEM) and tert-butylhydroquinone (tBHQ). Single treatment with DEM or tBHQ induced Nrf2 and Srxn1 over time in a concentration-dependent manner. The Nrf2 response to a second treatment was lower than the response to the first exposure with the same concentration, indicating that the response is adaptive. Moreover, a limited fraction of individual cells committed themselves into the Nrf2 response during the second treatment. Despite the suppression of the Nrf2 pathway, the second treatment resulted in a three-fold higher Srxn1-GFP response compared to the first treatment, with all cells participating in the response. While after the first treatment Srxn1-GFP response was linearly related to Nrf2-GFP nuclear translocation, such a linear relationship was less clear for the second exposure. siRNA-mediated knockdown demonstrated that the second response is dependent on the activity of Nrf2. Several other, clinically relevant, compounds (i.e., sulphorophane, nitrofurantoin and CDDO-Me) also enhanced the induction of Srxn1-GFP upon two consecutive repeated exposure. Together the data indicate that adaptation towards pro-oxidants lowers the Nrf2 activation capacity, but simultaneously primes cells for the enhancement of an antioxidant response which depends on factors other than just Nrf2. These data provide further insight in the overall dynamics of stress pathway activation after repeated exposure and underscore the complexity of responses that may govern repeated dose toxicity.

Comprehensive Landscape of Nrf2 and p53 Pathway Activation Dynamics by Oxidative Stress and DNA Damage.

A quantitative dynamics pathway map of the Nrf2-mediated oxidative stress response and p53-related DNA damage response pathways as well as the cross-talk between these pathways has not systematically been defined. To allow the dynamic single cell evaluation of these pathways, we have used BAC-GFP recombineering to tag for each pathway's three key components: for the oxidative stress response, Keap1-GFP, Nrf2-GFP, and Srxn1-GFP; for the DNA damage response, 53bp1-GFP, p53-GFP, and p21-GFP. The dynamic activation of these individual components was assessed using quantitative high throughput confocal microscopy after treatment with a broad concentration range of diethyl maleate (DEM; to induce oxidative stress) and etoposide (to induce DNA damage). DEM caused a rapid activation of Nrf2, which returned to baseline levels at low concentrations but remained sustained at high concentrations. Srxn1-GFP induction and Keap1-GFP translocation to autophagosomes followed later, with upper boundaries reached at high concentrations, close to the onset of cell death. Etoposide caused rapid accumulation of 53bp1-GFP in DNA damage foci, which was later followed by the concentration dependent nuclear accumulation of p53-GFP and subsequent induction of p21-GFP. While etoposide caused activation of Srxn1-GFP, a modest activation of DNA damage reporters was observed for DEM at high concentrations. Interestingly, Nrf2 knockdown caused an inhibition of the DNA damage response at high concentrations of etoposide, while Keap1 knockdown caused an enhancement of the DNA damage response already at low concentrations of etoposide. Knockdown of p53 did not affect the oxidative stress response. Altogether, the current stress response landscapes provide insight in the time course responses of and cross-talk between oxidative stress and DNA-damage and defines the tipping points where cell injury may switch from adaptation to injury.

Nuclear magnetic resonance- and mass spectrometry-based metabolomics to study maleic acid toxicity from repeated dose exposure in rats.

Maleic acid (MA), a chemical intermediate used in many consumer and industrial products, was intentionally adulterated in a variety of starch-based foods and instigated food safety incidents in Asia. We aim to elucidate possible mechanisms of MA toxicity after repeated exposure by (1) determining the changes of metabolic profile using 1 H nuclear magnetic resonance spectroscopy and multivariate analysis, and (2) investigating the occurrence of oxidative stress using liquid chromatography tandem mass spectrometry by using Sprague-Dawley rat urine samples. Adult male rats were subjected to a 28 day subchronic study (0, 6, 20 and 60 mg kg-1 ) via oral gavage. Urine was collected twice a day on days 0, 7, 14, 21 and 28; organs underwent histopathological examination. Changes in body weight and relative kidney weights in medium- and high-dose groups were significantly different compared to controls. Morphological alterations were evident in the kidneys and liver. Metabolomic results demonstrated that MA exposure increases the urinary concentrations of 8-hydroxy-2'-deoxyguanosine, 8-nitroguanine and 8-iso-prostaglandin F2α ; levels of acetoacetate, hippurate, alanine and acetate demonstrated time- and dose-dependent variations in the treatment groups. Findings suggest that MA consumption escalates oxidative damage, membrane lipid destruction and disrupt energy metabolism. These aforementioned changes in biomarkers and endogenous metabolites elucidate and assist in characterizing the possible mechanisms by which MA induces nephro- and hepatotoxicity.

Salt Selection Matters: Differential Renal Toxicity With MDV1634.Maleate Versus MDV1634.2HCl.

Salt forms of pharmaceutical compounds can have unique pharmacokinetic and toxicity properties. MDV1634 was evaluated for neurology indication and also demonstrated blood pressure (BP)-lowering effects in nonclinical studies. During the chemistry manufacturing campaign, 2 salt forms, dihydrochloride (2HCl) and maleate (MAL), which improved chemical stability and water solubility of the free base were identified. MDV1634.MAL showed better chemical attributes and was evaluated in toxicology studies for further development. A 28-day oral toxicity study in dogs with MDV1634.MAL demonstrated partially reversible renal toxicity. Although MAL salt is generally regarded as safe, renal toxicity is sometimes observed in rats and dogs. To evaluate contribution of each salt form to renal toxicity and BP lowering, an additional 28-day study was conducted with MDV1634.2HCL and MDV1634.MAL, which included toxicokinetics, continuous BP measurement in a subset of dogs, and sensitive urinary biomarker evaluation for temporal monitorability and reversibility of potential renal findings. In the repeat study, both salt forms showed similar exposures during the dosing period, but renal tubular toxicity was observed only with MDV1634.MAL and not with MDV1634.2HCl. The renal findings with MDV1634.MAL included early urinary biomarker changes (increase in albumin, clusterin, ß2 microglobulin, and neutrophil gelatinase-associated lipocalin); elevations in serum blood urea nitrogen and creatinine; and microscopic findings of partially reversible tubular basophilia, single cell necrosis, pigmentation, and mineralization. The renal findings in contrast to the BP findings were MAL-specific and considered not related to MDV1634, thereby under scoring the importance of salt forms in pharmaceutical development.

Combined iron sucrose and protoporphyrin treatment protects against ischemic and toxin-mediated acute renal failure.

Tissue preconditioning, whereby various short-term stressors initiate organ resistance to subsequent injury, is well recognized. However, clinical preconditioning of the kidney for protection against acute kidney injury (AKI) has not been established. Here we tested whether a pro-oxidant agent, iron sucrose, combined with a protoporphyrin (Sn protoporphyrin), can induce preconditioning and protect against acute renal failure. Mice were pretreated with iron sucrose, protoporphyrin, cyanocobalamin, iron sucrose and protoporphyrin, or iron sucrose and cyanocobalamin. Eighteen hours later, ischemic, maleate, or glycerol models of AKI were induced, and its severity was assessed the following day (blood urea nitrogen, plasma creatinine concentrations; post-ischemic histology). Agent impact on cytoprotective gene expression (heme oxygenase 1, hepcidin, haptoglobin, hemopexin, α1-antitrypsin, α1-microglobulin, IL-10) was assessed as renal mRNA and protein levels. AKI-associated myocardial injury was gauged by plasma troponin I levels. Combination agent administration upregulated multiple cytoprotective genes and, unlike single agent administration, conferred marked protection against each tested model of acute renal failure. Heme oxygenase was shown to be a marked contributor to this cytoprotective effect. Preconditioning also blunted AKI-induced cardiac troponin release. Thus, iron sucrose and protoporphyrin administration can upregulate diverse cytoprotective genes and protect against acute renal failure. Associated cardiac protection implies potential relevance to both AKI and its associated adverse downstream effects.

Dibutyl Maleate and Dibutyl Fumarate Enhance Contact Sensitization to Fluorescein Isothiocyanate in Mice.

Di-n-butyl phthalate (DBP), a phthalate ester, has been shown to have an adjuvant effect on fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse models. Di-n-butyl maleate (DBM), widely used as a plasticizer for industrial application, has been reported to cause dermatitis in humans. DBM is a butyl alcohol ester of di-carboxylic acid that represents a part of the DBP structure, while di-n-butyl fumarate (DBF) is a trans isomer of DBM. We examined whether DBM or DBF exhibits an adjuvant effect like DBP does. When BALB/c mice were epicutaneously sensitized with FITC in the presence of DBM or DBF, the FITC-specific CHS response was enhanced, as we have observed for DBP. As to underlying mechanisms, DBM and DBF facilitated the trafficking of FITC-presenting CD11c(+) dendritic cells (DCs) from skin to draining lymph nodes and increased the cytokine production by draining lymph nodes. In conclusion, DBM and DBF may have an effect that aggravates contact dermatitis through a skin sensitization process.

Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice.

Selol is a semi-synthetic compound containing selenite that is effective against cancerous cells and safer for clinical applications in comparison with other inorganic forms of selenite. Recently, we have developed a formulation of poly(methyl vinyl ether-co-maleic anhydride)-shelled selol nanocapsules (SPN), which reduced the proliferative activity of lung adenocarcinoma cells and presented little deleterious effects on normal cells in in vitro studies. In this study, we report on the antitumor activity and systemic effects induced by this formulation in chemically induced lung adenocarcinoma-bearing mice. The in vivo antitumor activity of the SPN was verified by macroscopic quantification, immunohistochemistry and morphological analyses. Toxicity analyses were performed by evaluations of the kidney, liver, and spleen; analyses of hemogram and plasma levels of alanine aminotransferase, aspartate transaminase, urea, and creatinine; and DNA fragmentation and cell cycle activity of the bone marrow cells. Furthermore, we investigated the potential of the SPN formulation to cause hemolysis, activate the complement system, provoke an inflammatory response and change the conformation of the plasma proteins. Our results showed that the SPN reduced the area of the surface tumor nodules but not the total number of tumor nodules. The biochemical and hematological findings were suggestive of the low systemic toxicity of the SPN formulation. The surface properties of the selol nanocapsules point to characteristics that are consistent with the treatment of the tumors in vivo: low hemolytic activity, weak inflammatory reaction with no activation of the complement system, and mild or absent conformational changes of the plasma proteins. In conclusion, this report suggests that the SPN formulation investigated herein exhibits anti-tumoral effects against lung adenocarcinoma in vivo and is associated with low systemic toxicity and high biocompatibility.

Resistance selection and molecular mechanisms of cypermethrin resistance in red hairy caterpillar (Amsacta albistriga walker).

Amsacta albistriga is one of the important pests of oilseed crops in India. This pest has developed high resistance to organophosphate (OP) insecticide in field. Therefore, cypermethrin insecticide was used as an alternative for this pest. After 20 generations of selection with cypermethrin, the LD50 value for A. albistriga was increased by 21.5-folds. The synergism ratio of piperonyl butoxide (PBO) and triphenyl phosphate (TPP) was increased by 10- and 9.6-fold in resistant strains and comparatively, 3.9 and 4.2-fold in susceptible strains. Detoxification enzyme analysis and native PAGE electrophoresis of esterase isoenzyme further revealed that esterase and mixed function oxidase may be involved in cypermethrin resistance in CypRes strain. In addition to enzyme analysis overexpression of CYP4M44, CYP9A77 and CYP6B47 (ortholog) can confer metabolic resistance in the CypRes strain. These data provide a foundation for further study of cypermethrin resistance mechanism observed in A. albistriga.

Cytotoxicity of four denture adhesives on human gingival fibroblast cells.

OBJECTIVE: The purpose of this study was to compare the cytotoxicity of four denture adhesives on human gingival fibroblast cells. MATERIALS AND METHODS: Immortalized human gingival fibroblasts were cultured with one of four different denture adhesives, Polident, Protefix, Staydent or Denfix-A, which was placed in insert dishes (10% w/v concentration) for 48 h. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometric apoptosis assay were used to evaluate cell viability and apoptosis rates. The fibroblasts were also examined under a scanning electron microscope. RESULTS: The MTT assay showed that all denture adhesives resulted in a significantly lower cell viability compared to the control cells propagated in normal culture medium (p < 0.05), with Staydent demonstrating the lowest cell viability. According to the flow cytometric apoptosis assay, Staydent and Protefix showed significantly higher apoptosis rates than the control group (p < 0.05), whereas Polident and Denfix-A did not demonstrate any significant differences (p > 0.05). Staydent showed the highest apoptosis rate. Scanning electron microscopy showed that the cells of the Staydent group underwent cytoplasmic membrane shrinkage, with cell free areas containing residual fragments of the membrane of dead cells. CONCLUSIONS: The four denture adhesives evaluated in this study imparted cytotoxic effects on human gingival fibroblast cells. Staydent showed the highest toxicity.

Role of PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in THP-1 cells.

We previously demonstrated an age-related decrease in receptor for activated C-kinase (RACK-1) expression and functional deficit in Langerhans cells' responsiveness. This defect specifically involves the translocation of protein kinase C (PKC)-ß. The purpose of this study was to investigate the role of RACK-1 and PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in the human promyelocytic cell line THP-1 and primary human dendritic cells (DC). Dinitrochlorobenzene, p-phenylenediamine and diethyl maleate were used as contact allergens. The selective cell-permeable inhibitor of PKC-ß and the broad PKC inhibitor GF109203X completely prevented chemical allergen- or lipopolysaccharide (LPS)-induced CD86 expression and significantly modulated IL-8 release (50 % reduction). The selective cell-permeable inhibitor of PKC-ε (also known to bind to RACK-1) failed to modulate allergen- or LPS-induced CD86 expression or allergen-induced IL-8 release, while modulating LPS-induced IL-8 release. The use of a RACK-1 pseudosubstrate, which directly activates PKC-ß, resulted in dose-related increase in CD86 expression and IL-8 release. Similar results were obtained with human DC, confirming the relevance of results obtained in THP-1 cells. Overall, our findings demonstrate the role of PKC-ß and RACK-1 in allergen-induced CD86 expression and IL-8 production, supporting a central role of PKC-ß in the initiation of chemical allergen-induced DC activation.

Cytotoxic effects of denture adhesives on primary human oral keratinocytes, fibroblasts and permanent L929 cell lines.

BACKGROUND: To date, there have been very little data on the cytotoxic responses of different cell lines to denture adhesives. OBJECTIVE: To determine the cytotoxicity of three denture adhesives on primary human oral keratinocytes (HOKs), fibroblasts (HOFs) and permanent mouse fibroblasts cell lines (L929). METHODS: Three commercial denture adhesives (two creams and one powder) were prepared for indirect contact using the agar diffusion test, as well as extracts in MTT assay. The results of the MTT assay were statistically analysed by one-way anova and Tukey's test (p < 0.05). RESULTS: All of the tested denture adhesives showed mild to moderate cytotoxicity to primary HOKs (p < 0.001), whereas none of three was toxic to L929 cells (p > 0.05) in both assays. For primary HOFs cultures, slight cytotoxicity was observed for one of the products from the agar diffusion test and undiluted eluates of all tested adhesives with MTT assay (p < 0.01). CONCLUSION: Denture adhesives are toxic to the primary HOKs and HOFs cultures, whereas non-toxic to L929 cells. The results suggest that primary human oral mucosal cells may provide more valuable information in toxicity screening of denture adhesives.

Assessment of genotoxic effect of maleic acid and EDTA: a comparative in vitro experimental study.

OBJECTIVES: This study aims to evaluate and compare the genotoxic and apoptotic effect of aqueous solutions of ethylenediaminetetraacetic acid (EDTA) with that of maleic acid (MA) using Chinese hamster lung fibroblast (V79) cells growing in vitro. MATERIALS AND METHODS: Exponentially growing V79 cells were treated with various concentrations of EDTA or MA alone for 30 min, and genotoxic effect was analyzed by micronucleus as well as comet assays and the type of cell death by apoptotic cell measurements using microscopic and flow cytometric methods. For all the experiments, H2O2 was used as a positive control. RESULTS: Treatment of V79 cells with H2O2 resulted in significantly (P < 0.001) increased micronuclei and levels of DNA damage, whereas, EDTA/MA alone treated cells did not show significant increase of MN frequencies and comet parameters even at their higher concentrations when compared with that of untreated control. V79 cells treated with EDTA/MA for 30 min showed a nonsignificant increase in the percentage of apoptotic and necrotic cells at their lower concentrations (0.025 and 0.05 % for EDTA and MA, respectively). However, at higher concentrations, i.e., >IC50 (0.1 and 0.5 %) for EDTA and MA resulted in increased number of apoptotic and necrotic cells when compared with the untreated group. CONCLUSIONS: This study clearly demonstrates that MA and EDTA are not potentially genotoxic agents and MA induced lesser apoptotic/necrotic death than that of EDTA at their clinically relevant doses. CLINICAL RELEVANCE: MA may have a better clinical acceptability with comparable smear layer removal ability. Hence, the results presented here might be an additional supporting evidence for the use of MA in endodontic practice.

An assessment of the ocular safety of excipient maleic acid following intravitreal injection in rabbits.

Maleic acid was formulated in 0.7% saline and injected intravitreally in rabbits in order to evaluate ocular safety and tolerability. Maleic acid was formulated within a narrow pH range (2-3), administered in a fixed volume (100 µl), and concentrations ranged from 0.00 to 2.00 mg/eye (0.00 to 12.30 mM vitreous). Ocular evaluations were conducted at 2, 4, and 8 days post injection. Ocular irritation responses were observed at doses from 0.50 mg/eye (3.07 mM vitreous) to 2.00 mg/eye (12.30 mM vitreous) and included conjunctival redness and scleral swelling. Chemosis was observed at 2.00 mg/eye (12.30 mM vitreous). Funduscopic evaluations revealed enlarged retinal blood vessels and optic disk swelling at doses ≥1.50 mg/eye (9.22 mM vitreous), retinal folds and retinal discoloration at 2.00 mg/eye (12.30 mM vitreous). Histopathologic evaluations on days 4 and 8 post injection revealed retinal degeneration at doses ≥1.0 mg/eye (6.15 mM vitreous), conjunctival inflammation at doses ≥1.5 mg/eye (9.22 mM vitreous), and retinal pigment epithelial hypertrophy, optic nerve demyelination, anterior chamber fluid, and conjunctival fibrosis at 2.00 mg/eye (12.30 mM vitreous) maleic acid. The data suggest that maleic acid formulations at ≥1.00 mg/eye (6.15 mM vitreous) were not suitable for intraocular indications.

Genetic damage, but limited evidence of oxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK(+/-)) cell cultures.

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK(+/-)) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039-2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 10(8) NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK(+/-)) cells, but not with other oxidative markers.

Structure-activity comparison of the cytotoxic properties of diethyl maleate and related molecules: identification of diethyl acetylenedicarboxylate as a thiol cross-linking agent.

Many α,ß-unsaturated carbonyl compounds are used in biochemical and medical research. Their biological effects are due in large part to their electrophilic properties, whereby they undergo reaction with nucleophilic sites in proteins and nucleic acids. Here, we describe a structure-activity comparison of the cytotoxic properties of diethyl maleate (DEM) and closely related chemical analogs. All molecules that contained an α,ß-unsaturated carbonyl group were cytotoxic to human colorectal carcinoma cells, causing apoptotic cell death. However, related molecules lacking this chemical moiety were not cytotoxic. One of the molecules screened, diethyl acetylenedicarboxylate (DAD), was considerably more cytotoxic than DEM and other analogues. Induction of cell death by DAD was significantly decreased following preincubation of cells with N-acetylcysteine, suggesting that its reactivity with thiols in cells might account for its cytotoxicity. By use of a model thiol compound, it was found that DAD can undergo addition reactions with two equivalents of thiol. When the reactivity of DAD with proteins was explored, it was determined that DAD induces oligomerization of Gpx3p, a yeast glutathione peroxidase with highly reactive cysteine residues in its active site. Our results suggest that DAD functions as a protein-thiol cross-linker, providing a potential chemical explanation for its cytotoxic potency.

Hepatic transcriptome and proteome responses against diethyl maleate-induced glutathione depletion in the rat.

Hepatic transcriptome and proteome responses against glutathione depletion were investigated by Affymetrix GeneChip Microarray and 2-dimensional gel electrophoresis (2D-DIGE), followed by MALDI-TOF-MS analysis and utilizing a glutathione-depleted rat model treated with diethyl maleate (DEM). Hepatic glutathione content decreased to 1.29 µmol/g liver (25.5% compared to control) after DEM treatment, and there were no apparent hepatotoxic signs estimated by blood chemistry examinations. A total of 247 and 213 annotated gene probe sets exhibited greater than twofold up- and down-regulation compared with controls, respectively. The up-regulated gene list contained a number of glutathione depletion-responsive genes reported previously, such as Trib3, Srxn1, Myc, Asns, Igfbp1, Txnrd1, or Hmox1, suggesting that these genes are robust mRNA biomarkers for evaluating hepatic glutathione depletion. In the 2D-DIGE analysis, proteins for a total of 361 spots were identified by MALDI-TOF-MS analysis. Of the identified proteins, 5 and 14 proteins showed up- and down-regulation, respectively. Some proteins exhibited differential expression in the protein level but not in the mRNA level, including L-FABP, MAWDBP, aldo-keto reductase family 1 member A1, catalase and ATP synthase subunit beta, suggesting that these proteins would be potential protein biomarkers for evaluating glutathione depletion. Moreover, up-regulation of FABP1 protein along with up-regulation of PPARα-regulated gene transcripts (i.e., Acot2 and Acot4) is indicative of PPARα activation, which may contribute to hepatocellular protection against glutathione depletion-induced oxidative stress. The up-regulation of L-FABP1 was detected by proteome data but not by transcriptome data, demonstrating the advantage of utilizing transcriptomics and proteomics combination to investigate glutathione depletion-induced molecular dynamics.

Final report of the Amended Safety Assessment of PVM/MA copolymer and its related salts and esters as used in cosmetics.

Polyvinyl methyl ether/maleic acid (PVM/MA) copolymer, and its related salts and esters, are used in cosmetics, mainly as binders, film formers, and hair fixatives. Animal and human data relevant to the use of these ingredients in cosmetic products were reviewed by the CIR Expert Panel. The Panel concluded that these ingredients are safe for use in cosmetic products.

Polymer nanoparticles regulate macrophage repolarization for antitumor treatment.

We demonstrate an intrinsic antitumor effect of polymer nanoparticles (P-NPs), which could re-program tumor-associated macrophages to pro-inflammatory phenotype. The intrinsic effect of P-NPs on macrophage repolarization and its combination with other therapies provide new ideas for drug delivery, macrophage regulation and immunotherapy in cancer treatment.

Sensitization to dimethyl fumarate with multiple concurrent patch test reactions.

BACKGROUND: Chairs and sofas imported from China to Europe were shown to contain dimethyl fumarate (DMF), a sensitizing, volatile chemical. Many of the sensitized patients also had positive patch test reactions to acrylates. OBJECTIVES: To analyse the occurrence and strength of DMF sensitization and the appearance of concomitant reactions. METHODS: Patch testing with DMF in concentrations of 0.1-0.00001% was carried out in 37 patients. Diethyl fumarate (DEF), diethyl maleate (DEM), dimethyl maleate (DMM), ethyl acrylate (EA), methyl acrylate (MA), and methyl methacrylate (MMA) were also tested with a dilution series at equimolar concentrations. RESULTS: The lowest concentration of DMF eliciting a reaction varied between 0.0001% and 0.1% and all but four patients reacted concurrently to DEF. DEM elicited positive patch test reactions in 21/37 patients and DMM reactions were seen in all 9 patients tested. EA elicited positive reactions in 13/37 patients and a positive MA reaction was seen in 7/37 patients, 2 of whom also reacted to MMA. CONCLUSIONS: The strength of the sensitization to DMF showed variation and concurrent reactions were common. Concurrent reactions to (meth)acrylates were seen in patients, who reacted to lower (0.001% or less) DMF concentration probably elicited by cross-reactivity.