In Vitro and In Vivo Biotransformation Profiling of 6PPD-Quinone toward Their Detection in Human Urine.

The antioxidant N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its oxidized quinone product 6PPD-quinone (6PPD-Q) in rubber have attracted attention due to the ecological risk that they pose. Both 6PPD and 6PPD-Q have been detected in various environments that humans cohabit. However, to date, a clear understanding of the biotransformation of 6PPD-Q and a potential biomarker for exposure in humans are lacking. To address this issue, this study presents a comprehensive analysis of the extensive biotransformation of 6PPD-Q across species, encompassing both in vitro and in vivo models. We have tentatively identified 17 biotransformation metabolites in vitro, 15 in mice in vivo, and confirmed the presence of two metabolites in human urine samples. Interestingly, different biotransformation patterns were observed across species. Through semiquantitative analysis based on peak areas, we found that almost all 6PPD-Q underwent biotransformation within 24 h of exposure in mice, primarily via hydroxylation and subsequent glucuronidation. This suggests a rapid metabolic processing of 6PPD-Q in mammals, underscoring the importance of identifying effective biomarkers for exposure. Notably, monohydroxy 6PPD-Q and 6PPD-Q-O-glucuronide were consistently the most predominant metabolites across our studies, highlighting monohydroxy 6PPD-Q as a potential key biomarker for epidemiological research. These findings represent the first comprehensive data set on 6PPD-Q biotransformation in mammalian systems, offering insights into the metabolic pathways involved and possible exposure biomarkers.

H/D Exchange Coupled with 2H-labeled Stable Isotope-Assisted Metabolomics Discover Transformation Products of Contaminants of Emerging Concern.

Stable isotope-assisted metabolomics (SIAM) is a powerful tool for discovering transformation products (TPs) of contaminants. Nevertheless, the high cost or lack of isotope-labeled analytes limits its application. In-house H/D (hydrogen/deuterium) exchange reactions enable direct 2H labeling to target analytes with favorable reaction conditions, providing intuitive and easy-to-handle approaches for environmentally relevant laboratories to obtain cost-effective 2H-labeled contaminants of emerging concern (CECs). We first combined the use of in-house H/D exchange and 2H-SIAM to discover potential TPs of 6PPD (N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine), providing a new strategy for finding TPs of CECs. 6PPD-d9 was obtained by in-house H/D exchange with favorable reaction conditions, and the impurities were carefully studied. Incomplete deuteride, for instance, 6PPD-d8 in this study, constitutes a major part of the impurities. Nevertheless, it has few adverse effects on the 2H-SIAM pipeline in discovering TPs of 6PPD. The 2H-SIAM pipeline annotated 9 TPs of 6PPD, and commercial standards further confirmed the annotated 6PPDQ (2-anilino-5-(4-methylpentan-2-ylamino)cyclohexa-2,5-diene-1,4-dione) and PPPD (N-phenyl-p-phenylenediamine). Additionally, a possible new formation mechanism for 6PPDQ was proposed, highlighting the performance of the strategy. In summary, this study highlighted a new strategy for discovering the TPs of CECs and broadening the application of SIAM in environmental studies.

Uptake, Metabolism, and Accumulation of Tire Wear Particle-Derived Compounds in Lettuce.

Tire wear particle (TWP)-derived compounds may be of high concern to consumers when released in the root zone of edible plants. We exposed lettuce plants to the TWP-derived compounds diphenylguanidine (DPG), hexamethoxymethylmelamine (HMMM), benzothiazole (BTZ), N-phenyl-N'-(1,3-dimethylbutyl)-p-phenylenediamine (6PPD), and its quinone transformation product (6PPD-q) at concentrations of 1 mg L-1 in hydroponic solutions over 14 days to analyze if they are taken up and metabolized by the plants. Assuming that TWP may be a long-term source of TWP-derived compounds to plants, we further investigated the effect of leaching from TWP on the concentration of leachate compounds in lettuce leaves by adding constantly leaching TWP to the hydroponic solutions. Concentrations in leaves, roots, and nutrient solution were quantified by triple quadrupole mass spectrometry, and metabolites in the leaves were identified by Orbitrap high resolution mass spectrometry. This study demonstrates that TWP-derived compounds are readily taken up by lettuce with measured maximum leaf concentrations between ∼0.75 (6PPD) and 20 µg g-1 (HMMM). Although these compounds were metabolized in the plant, we identified several transformation products, most of which proved to be more stable in the lettuce leaves than the parent compounds. Furthermore, continuous leaching from TWP led to a resupply and replenishment of the metabolized compounds in the lettuce leaves. The stability of metabolized TWP-derived compounds with largely unknown toxicities is particularly concerning and is an important new aspect for the impact assessment of TWP in the environment.

Interspecies Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semiquantification.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire antidegradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations that surpass those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semiquantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkii lewisi), and nonfry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might detoxify 6PPD-Q more effectively. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semiquantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being valuable to environmental monitoring and risk assessment.

Urine Excretion, Organ Distribution, and Placental Transfer of 6PPD and 6PPD-Quinone in Mice and Potential Developmental Toxicity through Nuclear Receptor Pathways.

The rubber antioxidant 6PPD has gained significant attention due to its highly toxic transformation product, 6PPD-quinone (6PPDQ). Despite their detection in urines of pregnant women, the placental transfer and developmental toxicity of 6PPD and 6PPDQ are unknown. Here, we treated C57Bl/6 mice with 4 mg/kg 6PPD or 6PPDQ to investigate their urine excretion and placental transfer. Female and male mice exhibited sex difference in excretion profiles of 6PPD and 6PPDQ. Urine concentrations of 6PPDQ were one order of magnitude lower than those of 6PPD, suggesting lower excretion and higher bioaccumulation of 6PPDQ. In pregnant mice treated with 6PPD or 6PPDQ from embryonic day 11.5 to 15.5, 6PPDQ showed ∼1.5-8 times higher concentrations than 6PPD in placenta, embryo body, and embryo brain, suggesting higher placental transfer of 6PPDQ. Using in vitro dual-luciferase reporter assays, we revealed that 6PPDQ activated the human retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα) at concentrations as low as 0.3 µM, which was ∼10-fold higher than the concentrations detected in human urines. 6PPD activated the RXRα at concentrations as low as 1.2 µM. These results demonstrate the exposure risks of 6PPD and 6PPDQ during pregnancy and emphasize the need for further toxicological and epidemiological investigations.

Tire-Derived Transformation Product 6PPD-Quinone Induces Mortality and Transcriptionally Disrupts Vascular Permeability Pathways in Developing Coho Salmon.

Urban stormwater runoff frequently contains the car tire transformation product 6PPD-quinone, which is highly toxic to juvenile and adult coho salmon (Onchorychus kisutch). However, it is currently unclear if embryonic stages are impacted. We addressed this by exposing developing coho salmon embryos starting at the eyed stage to three concentrations of 6PPD-quinone twice weekly until hatch. Impacts on survival and growth were assessed. Further, whole-transcriptome sequencing was performed on recently hatched alevin to address the potential mechanism of 6PPD-quinone-induced toxicity. Acute mortality was not elicited in developing coho salmon embryos at environmentally measured concentrations lethal to juveniles and adults, however, growth was inhibited. Immediately after hatching, coho salmon were sensitive to 6PPD-quinone mortality, implicating a large window of juvenile vulnerability prior to smoltification. Molecularly, 6PPD-quinone induced dose-dependent effects that implicated broad dysregulation of genomic pathways governing cell-cell contacts and endothelial permeability. These pathways are consistent with previous observations of macromolecule accumulation in the brains of coho salmon exposed to 6PPD-quinone, implicating blood-brain barrier disruption as a potential pathway for toxicity. Overall, our data suggests that developing coho salmon exposed to 6PPD-quinone are at risk for adverse health events upon hatching while indicating potential mechanism(s) of action for this highly toxic chemical.

Synthesis and In Situ Behavior of 1,4- and 2,5-(13C) Isotopomers of p-Phenylenediamine in Reconstructed Human Epidermis Using High Resolution Magic Angle Spinning NMR.

p-Phenylenediamine (PPD) has been classified as a strong skin allergen, but when it comes to toxicological concerns, benzoquinone diamine (BQDI), the primary oxidation derivative of PPD, is frequently considered and was shown to covalently bind nucleophilic residues on model peptides. However, tests in solution are far from providing a reliable model, as the cutaneous metabolism of PPD is not covered. We now report the synthesis of two 13C substituted isotopomers of PPD, 1,4-(13C)p-phenylenediamine 1 and 2,5-(13C)p-phenylenediamine 2, and the investigation of their reactivity in reconstructed human epidermis (RHE) using the high resolution magic angle spinning (HRMAS) NMR technique. RHE samples were first treated with 1 or 2 and incubated for 1 to 48 h. Compared to the control, spectra clearly showed only the signals of 1 or 2 gradually decreasing with time to disappear after 48 h of incubation. However, the culture media of RHE incubated with 1 for 1 and 24 h, respectively, showed the presence of both monoacetylated- and diacetylated-PPD as major products. Therefore, the acetylation reaction catalyzed by N-acetyltransferase (NAT) enzymes appeared to be the main process taking place in RHE. With the aim of increasing the reactivity by oxidation, 1 and 2 were treated with 0.5 equiv of H2O2 prior to their application to RHE and incubated for different times. Under these conditions, new peaks having close chemical shifts to those of PPD-cysteine adducts previously observed in solution were detected. Under such oxidative conditions, we were thus able to detect and quantify cysteine adducts in RHE (maximum of 0.2 nmol/mg of RHE at 8 h of incubation) while no reaction with other nucleophilic amino acid residues could be observed.

Investigations on detoxification mechanisms of novel para-phenylenediamine analogues through N-acetyltransferase 1 (NAT-1).

Para-phenylenediamine (PPD) is one of the most used chemicals in oxidative hair dyes. However, its use has been associated with adverse effects on health, including contact dermatitis and other systemic toxicities. Novel PPD derivatives have been proposed as a safer replacement for PPD. This can be achieved if these molecules minimally permeate the skin and/or are easily metabolised by enzymes in the skin (e.g., N-acetyltransferase-1 (NAT-1)) into innocuous compounds before gaining systemic entry. This study investigated the detoxification pathway mediated by NAT-1 enzymes on 6 synthesized PPD analogues (namely, P1-P6) with different chemical properties, to study the role of functional groups on detoxification mechanisms in HaCaT skin cells. These compounds were carefully designed with different chemical properties (whereby the ortho position of PPD was substituted by nucleophile and electrophile groups to promote N-acetylation reactions, metabolism and clearance). Compounds P2-P4 N-acetylated at 54-49 nmol/mg/min, which is 1.6 times higher than N-acetylation of PPD, upregulated NAT-1 activity from 8-7% at 50 µM to 22-11% at 100 µM and showed 4 times higher rate of elimination (k equal to 0.141 ± 0.016-0.124 ± 0.01 h-1) and 3 times faster rate of clearance (0.172 ± 0.007-0.158 ± 0.005 h-1mgprotein-1) than PPD (0.0316 ± 0.0019 h-1, 0.0576 ± 0.003 h-1mg protein-1, respectively). The data suggest that nucleophile substituted compounds detoxify at a faster rate than PPD. Our metabolic and detoxification mechanistic studies revealed significantly higher rates of N-acetylation, NAT-1 activity and higher detoxification of P2-P4 in keratinocytes, suggesting the importance of nucleophilic groups at the ortho position in PPD to reduce toxicity of aniline-based dyes on human skin cells.

Multicommutated Flow Analysis System for Determination of Horseradish Peroxidase and Its Inhibitors.

A fully mechanized multicommutated flow analysis (MCFA) system dedicated to determining horseradish peroxidase (HRP) activity was developed. Detection was conducted using a flow-through optoelectronic detector-constructed of paired LEDs operating according to the paired emitter-detector diode (PEDD) principle. The PEDD-MCFA system is dedicated to monitoring the enzyme-catalyzed oxidation of p-phenylenediamine (pPD) by a hydrogen peroxide. Under optimized conditions, the presented bioanalytical system was characterized by a linear response range (33.47-200 U/L) with a detection limit at 10.54 U/L HRP activity and 1.66 mV·L/U sensitivity, relatively high throughput (12 signals recordings per hour), and acceptable precision (RSD below 6%). Additionally, the utility of the developed PEDD-MCFA system for the determination of HRP inhibitors allowing the detection of selected thiols at micromolar levels, is demonstrated. The practical utility of the flow system was illustrated by the analysis of some dietary supplements containing L-cysteine, N-acetylcysteine, and L-glutathione.

Targeting ferroptosis in rhabdomyosarcoma cells.

Recent data suggest that rhabdomyosarcoma (RMS) cells might be vulnerable to oxidative stress-induced cell death. Here, we show that RMS are susceptible to cell death induced by Erastin, an inhibitor of the glutamate/cystine antiporter xc- that can increase reactive oxygen species (ROS) production via glutathione (GSH) depletion. Prior to cell death, Erastin caused GSH depletion, ROS production and lipid peroxidation. Importantly, pharmacological inhibitors of lipid peroxidation (i.e., Ferrostatin-1, Liproxstatin-1), ROS scavengers (i.e., α-Tocopherol, GSH) and the iron chelator Deferoxamine inhibited ROS accumulation, lipid peroxidation and cell death, consistent with ferroptosis. Interestingly, the broad-spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I as well as the PKCα- and ß-selective inhibitor Gö6976 significantly reduced Erastin-induced cell death. Similarly, genetic knockdown of PKCα significantly protected RMS cells from Erastin-induced cell death. Furthermore, the broad-spectrum nicotinamide adenine dinucleotide phosphate-oxidase (NOX) inhibitor Diphenyleneiodonium and the selective NOX1/4 isoform inhibitor GKT137831 significantly decreased Erastin-stimulated ROS, lipid ROS and cell death. These data provide new insights into the molecular mechanisms of ferroptosis in RMS, contributing to the development of new redox-based treatment strategies.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z' ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH3 fragment ions ("twins"). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH3 ions requires consideration for accurate and confident assignments of z' ions in MALDI-ISD FT-ICR mass spectra.

Fluorescence Immunoassay Based on the Alkaline Phosphatase Triggered in Situ Fluorogenic Reaction of o-Phenylenediamine and Ascorbic Acid.

Inspired by the special reducing capability of ascorbic acid (AA), ascorbic acid 2-phosphate (AA2P) has been extensively utilized as a substrate in current alkaline phosphatase (ALP) activity assays owing to the ALP-triggered transformation of AA2P into AA. However, such assays usually require AA-related complicated and laborious synthesis and/or signal generation procedures. Herein, we report an interesting in situ fluorogenic interaction between o-phenylenediamine (OPD) and AA, which inspires us to put forward a novel and simple AA2P/OPD-participated fluorescence turn-on ALP activity assay for the first time, and then the corresponding ALP-based fluorescence enzyme-linked immunosorbent assay (ELISA) has also been developed by means of the conventional ELISA platforms. According to the convenient and facile detection process with clear response mechanism, our fluorogenic reaction-based assay exhibits good sensitivity, selectivity, and excellent sensing performance, which ensures fluorescence ELISA to potentially be applied in clinical diagnosis by employing a well-studied biomarker of hepatocellular carcinoma, α-fetoprotein (AFP) as the model analyte. Such original ELISA via in situ formation of fluorophore from scratch gives a new sight to develop other potential immunoassay platforms in early clinical diagnosis by controlling the target antigens in the near future.

An Investigation into Retigabine (Ezogabine) Associated Dyspigmentation in Rat Eyes by MALDI Imaging Mass Spectrometry.

Retigabine (RTG) is an antiepileptic drug approved as an adjunctive treatment for refractory partial-onset seizures in adults. In April 2013, the Food and Drug Administration issued a warning that RTG could cause changes in retinal pigmentation and discoloration of skin, resulting in a blue appearance. As part of a larger preclinical effort to gain a mechanistic understanding as to the origins of retinal pigment changes associated with RTG, we conducted a long-term repeat dosing study in rats. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) was used to determine the distribution of RTG and its metabolites in the rat eye following 13 and 39 weeks of dosing. IMS revealed the presence of RTG, a previously characterized N-acetyl metabolite of RTG (NAMR), and several species structurally related through the dimerization of RTG and NAMR. These species were highly localized to the melanin-containing layers of the uveal tract of the rat eye including the choroid, ciliary body, and iris, suggesting that the formation of these dimers occurs from melanin bound RTG and NAMR. Furthermore, several of the RTG-related dimers have UV absorbance which give them a purple color in solution. We propose that the melanin binding of RTG and NAMR effectively concentrates the two compounds to enable mixed condensation reactions to occur when the binding provides the proper geometry in the redox environment of the uveal tissues. High lateral resolution images illustrate that the blood-retinal barrier effectively restricts retinal access to RTG-related compounds. The spatial information provided by MALDI IMS was critical in contextualizing the homogenate concentrations of key RTG-related compounds and helped provide a basis for the mechanism of dimer formation.

Pharmacokinetics of a Pediatric Tribendimidine Dose-Finding Study To Treat Hookworm Infection in African Children.

Tribendimidine is a broad-spectrum anthelminthic available in China, which is currently being pursued for U.S. Food and Drug Administration approval for soil-transmitted helminth infections. Pharmacokinetic (PK) studies with tribendimidine in children, the main target group for treatment programs, have not been conducted to date. In the framework of a dose-ranging study in hookworm-infected school-aged children in Côte d'Ivoire, children were treated with either 100, 200, or 400 mg tribendimidine. Dried blood spot samples were collected up to 22 h after treatment. The active metabolite, deacetylated amidantel (dADT) and its metabolite acylated dADT (adADT) were quantified using liquid chromatography tandem mass spectrometry. PK parameters were calculated using a noncompartmental model, and univariate logistic regression was applied using maximal blood concentrations (Cmax) and area under the blood concentration-time curve for 0 to 22 h (AUC0-22) as predictors of drug efficacy. Dried blood spot samples of 101 children were analyzed. We observed a less than proportional and proportional exposure in dADT's median Cmax and AUC0-22, respectively, following administration of 100 mg (Cmax = 853 ng/ml; AUC0-22 = 3,019 h · ng/ml) and 400 mg (Cmax = 2,275 ng/ml; AUC0-22 = 12,530 h · ng/ml) tribendimidine. There were large, dose-independent variations in the time to Cmax (Tmax) and ratios of dADT to adADT. We did not detect an influence of Cmax or AUC0-22 of dADT or adADT on drug efficacy or adverse events. Since our study population was bearing hookworm infection of mainly low intensity, additional studies with heavy intensity infections might be required to confirm this observation.

Lipid Peroxidation-Dependent Cell Death Regulated by GPx4 and Ferroptosis.

Glutathione peroxidase 4 (Phospholipid hydroperoxide glutathione peroxidase, PHGPx) can directly reduce phospholipid hydroperoxide. Depletion of GPx4 induces lipid peroxidation-dependent cell death in embryo, testis, brain, liver, heart, and photoreceptor cells of mice. Administration of vitamin E in tissue specific GPx4 KO mice restored tissue damage in testis, liver, and heart. These results indicate that suppression of phospholipid peroxidation is essential for cell survival in normal tissues in mice. Ferroptosis is an iron-dependent non-apoptotic cell death that can elicited by pharmacological inhibiting the cystine/glutamate antiporter, system Xc- (type I) or directly binding and loss of activity of GPx4 (Type II) in cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression, but not in normal cells. Ferroptosis by Erastin (Type I) and RSL3 (RAS-selective lethal 3, Type II) treatment was suppressed by an iron chelator, vitamin E and Ferrostatin-1, antioxidant compound. GPx4 can regulate ferroptosis by suppression of phospholipid peroxidation in erastin and RSL3-induced ferroptosis. Recent works have identified several regulatory factors of erastin and RSL3-induced ferroptosis. In our established GPx4-deficient MEF cells, depletion of GPx4 induce iron and 15LOX-independent lipid peroxidation at 26 h and caspase-independent cell death at 72 h, whereas erastin and RSL3 treatment resulted in iron-dependent ferroptosis by 12 h. These results indicated the possibility that the mechanism of GPx4-depleted cell death might be different from that of ferroptosis induced by erastin and RSL3.

A sensitive and fast responsive fluorescent probe for imaging hypoxic tumors.

Nitroreductase activities are positively associated with the hypoxic level of tumors, making it an attractive target for tumor detection. Herein, we have developed a 2,5-bis(methylsulfinyl)-1,4-diaminobenzene based probe (BBP), which is a nitroreductase (NTR) responsive fluorescent probe and can rapidly detect NTRs with high sensitivity and specificity. The BBP showed not only a selective response to NTRs over other biological reductants, but also high sensitivity to NTRs and could detect as low as 20 ng mL-1 NTRs. Furthermore, the BBP responded rapidly to NTRs in as fast as 10 minutes, enabling real-time monitoring of the production levels of NTRs. Most importantly, the BBP could identify NTR activities in 2D cell monolayers, 3D tumor spheroids, and even solid tumors in mice. Particularly, the BBP could monitor the early tumor formation and treatment response via measuring NTR activities. Overall, the BBP appears to be an ideal imaging probe for the detection of solid tumors, and possesses great potential in a broad range of diagnostic and therapeutic applications in the clinic.

Development of a series of novel o-phenylenediamine-based indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

A novel series of o-phenylenediamine-based inhibitors of indoleamine 2,3-dioxygenase (IDO) has been identified. IDO is a heme-containing enzyme, overexpressed in the tumor microenvironment of many cancers, which can contribute to the suppression of the host immune system. Synthetic modifications to a previously described diarylether series resulted in an additional degree of molecular diversity which was exploited to afford compounds that demonstrated significant potency in the HeLa human cervical cancer IDO1 assay. .

t-BuOOH induces ferroptosis in human and murine cell lines.

Reactive oxygen species (ROS)-induced apoptosis has been extensively studied. Increasing evidence suggests that ROS, for instance, induced by hydrogen peroxide (H2O2), might also trigger regulated necrotic cell death pathways. Almost nothing is known about the cell death pathways triggered by tertiary-butyl hydroperoxide (t-BuOOH), a widely used inducer of oxidative stress. The lipid peroxidation products induced by t-BuOOH are involved in the pathophysiology of many diseases, such as cancer, cardiovascular diseases, or diabetes. In this study, we exposed murine fibroblasts (NIH3T3) or human keratinocytes (HaCaT) to t-BuOOH (50 or 200 µM, respectively) which induced a rapid necrotic cell death. Well-established regulators of cell death, i.e., p53, poly(ADP)ribose polymerase-1 (PARP-1), the stress kinases p38 and c-Jun N-terminal-kinases 1/2 (JNK1/2), or receptor-interacting serine/threonine protein kinase 1 (RIPK1) and 3 (RIPK3), were not required for t-BuOOH-mediated cell death. Using the selective inhibitors ferrostatin-1 (1 µM) and liproxstatin-1 (1 µM), we identified ferroptosis, a recently discovered cell death mechanism dependent on iron and lipid peroxidation, as the main cell death pathway. Accordingly, t-BuOOH exposure resulted in a ferrostatin-1- and liproxstatin-1-sensitive increase in lipid peroxidation and cytosolic ROS. Ferroptosis was executed independently from other t-BuOOH-mediated cellular damages, i.e., loss of mitochondrial membrane potential, DNA double-strand breaks, or replication block. H2O2 did not cause ferroptosis at equitoxic concentrations (300 µM) and induced a (1) lower and (2) ferrostatin-1- or liproxstatin-1-insensitive increase in lipid peroxidation. We identify that t-BuOOH and H2O2 produce a different pattern of lipid peroxidation, thereby leading to different cell death pathways and present t-BuOOH as a novel inducer of ferroptosis.

Spectrophotometric Quantification of Peroxidase with p-Phenylene-diamine for Analyzing Peroxidase-Encapsulating Lipid Vesicles.

A spectrophotometric assay for the determination of horseradish peroxidase (HRP) in aqueous solution with p-phenylenediamine (PPD, benzene-1,4-diamine) as electron donor substrate and hydrogen peroxide (H2O2) as oxidant was developed. The oxidation of PPD by HRP/H2O2 leads to the formation of Bandrowski's base ((3E,6E)-3,6-bis[(4-aminophenyl)imino]cyclohexa-1,4-diene-1,4-diamine), which can be quantified by following the increase in absorbance at 500 nm. The assay was applied for monitoring the activity of HRP inside ≈180 nm-sized lipid vesicles (liposomes), prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and purified by size exclusion chromatography. Because of the high POPC bilayer permeability of PPD and H2O2, the HRP-catalyzed oxidation of PPD occurs inside the vesicles once PPD and H2O2 are added to the vesicle suspension. In contrast, if instead of PPD the bilayer-impermeable substrate ABTS2- (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is used, the oxidation of ABTS2- inside the vesicles does not occur. Therefore, using PPD and ABTS2- in separate assays allows distinguishing between vesicle-trapped HRP and HRP in the external bulk solution. In this way, the storage stability of HRP-containing POPC vesicles was investigated in terms of HRP leakage and activity of entrapped HRP. It was found that pH 7.0 suspensions of POPC vesicles (2.2 mM POPC) containing on average about 12 HRP molecules per vesicle are stable for at least 1 month without any significant HRP leakage, if stored at 4 °C. Such high stability is beneficial not only for bioanalytical applications but also for exploring the kinetic properties of vesicle-entrapped HRP through simple spectrophotometric absorption measurements with PPD as a sensitive and cheap substrate.

A computational approach to study functional monomer-protein molecular interactions to optimize protein molecular imprinting.

Molecular imprinting has become a promising approach for synthesis of polymeric materials having binding sites with a predetermined selectivity for a given analyte, the so-called molecularly imprinted polymers (MIPs), which can be used as artificial receptors in various application fields. Realization of binding sites in a MIP involves the formation of prepolymerization complexes between a template molecule and monomers, their subsequent polymerization, and the removal of the template. It is believed that the strength of the monomer-template interactions in the prepolymerization mixture influences directly on the quality of the binding sites in a MIP and consequently on its performance. In this study, a computational approach allowing the rational selection of an appropriate monomer for building a MIP capable of selectively rebinding macromolecular analytes has been developed. Molecular docking combined with quantum chemical calculations was used for modeling and comparing molecular interactions among a model macromolecular template, immunoglobulin G (IgG), and 1 of 3 electropolymerizable functional monomers: m-phenylenediamine (mPD), dopamine, and 3,4-ethylenedioxythiophene, as well as to predict the probable arrangement of multiple monomers around the protein. It was revealed that mPD was arranged more uniformly around IgG participating in multiple H-bond interactions with its polar residues and, therefore, could be considered as more advantageous for synthesis of a MIP for IgG recognition (IgG-MIP). These theoretical predictions were verified by the experimental results and found to be in good agreement showing higher binding affinity of the mPD-based IgG-MIP toward IgG as compared with the IgG-MIPs generated from the other 2 monomers.