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Behl C, Moosmann B. Free Radic Biol Med 2002;33:182-191. Various neurodegenerative disorders and syndromes are associated with oxidative stress. The deleterious consequences of excessive oxidations and the pathophysiological role of [...]

Neuroprotective effects have been described for many can-nabinoids in several neurotoxicity models. However, the exact mechanisms have not been clearly understood yet. In the present study, antioxidant neuroprotective effects of cannabinoids and the involvement of the cannabinoid receptor 1 (CB1) were analysed in detail employing cell-free biochemical assays and cultured cells. As it was reported for oestrogens that the phenolic group is a lead structure for antioxidant neuroprotective effects, eight compounds were classified into three groups. Group A: phenolic compounds that do not bind to CB1. Group B: non-phenolic compounds that bind to CB1. Group C: phenolic compounds that bind to CB1. In the biochemical assays employed, a requirement of the phenolic lead structure for antioxidant activity was shown. The effects paralleled the protective potential of group A and C compounds against oxidative neuronal cell death using the mouse hippocampal HT22 cell line and rat primary cerebellar cell cultures. To elucidate the role of CB1 in neuroprotection, we established stably transfected HT22 cells containing CB1 and compared the protective potential of cannabinoids with that observed in the control transfected HT22 cell line. Furthermore, oxidative stress experiments were performed in cultured cerebellar granule cells, which were derived either from CB1 knock-out mice or from control wild-type littermates. The results strongly suggest that CB1 is not involved in the cellular antioxidant neuroprotective effects of cannabinoids. [ABSTRACT FROM AUTHOR]

Abstract Background Accumulation of aberrant proteins to form Lewy bodies (LBs) is a hallmark of Parkinson's disease (PD). Ubiquitination-mediated degradation of aberrant, misfolded proteins is critical for maintaining normal cell function. Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, particularly affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death. Results To gain insight into the mechanism whereby cell stress alters parkin-mediated ubiquitination and LB formation, we investigated the effect of oxidative stress. We found significant increases in oxidation (sulfonation) and subsequent aggregation of parkin in SH-SY5Y cells exposed to the mitochondrial complex I inhibitor 1-methyl-4-phenlypyridinium (MPP+), representing an in vitro cell-based PD model. Exposure of these cells to direct oxidation via pathological doses of H2O2 induced a vicious cycle of increased followed by decreased parkin E3 ligase activity, similar to that previously reported following S-nitrosylation of parkin. Pre-incubation with catalase attenuated H2O2 accumulation, parkin sulfonation, and parkin aggregation. Mass spectrometry (MS) analysis revealed that H2O2 reacted with specific cysteine residues of parkin, resulting in sulfination/sulfonation in regions of the protein similar to those affected by parkin mutations in hereditary forms of PD. Immunohistochemistry or gel electrophoresis revealed an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors, as well as in postmortem human brain from patients with PD with LBs. Conclusion These findings show that oxidative stress alters parkin E3 ligase activity, leading to dysfunction of the ubiquitin-proteasome system and potentially contributing to LB formation.

Low-molecular-mass organic chemicals are widely discussed as potential indicators of life in extraterrestrial habitats. However, demarcation lines between biotic chemicals and abiotic chemicals have been difficult to define. Here, we have analyzed the potential utility of the quantum chemical property, HOMO-LUMO gap (HLG), as a novel proxy variable of life, since a significant trend towards incrementally smaller HLGs has been described in the genetically encoded amino acids. The HLG is a zeroth-order predictor of chemical reactivity. Comparing a set of 134 abiotic organic molecules recovered from meteorites, with 570 microbial and plant secondary metabolites thought to be exclusively biotic, we found that the average HLG of biotic molecules was significantly narrower (-10.4 ± 0.9 eV versus -12.4 ± 1.6 eV), with an effect size of g = 1.87. Limitation to hydrophilic molecules (XlogP < 2) improved the separation of biotic from abiotic compounds (g = 2.52). The "hydrophilic reactivity" quadrant defined by |HLG| < 11.25 eV and XlogP < 2 was populated exclusively by 183 biotic compounds and 6 abiotic compounds, 5 of which were nucleobases. We conclude that hydrophilic molecules with small HLGs represent valuable indicators of biotic activity, and we discuss the evolutionary plausibility of this inference.

Inhibition of mitochondrial complex I (NADH dehydrogenase) is the primary mechanism of the antidiabetic drug metformin and various unrelated natural toxins. Complex I inhibition can also be induced by antidiabetic PPAR agonists, and it is elicited by methionine restriction, a nutritional intervention causing resistance to diabetes and obesity. Still, a comprehensible explanation to why complex I inhibition exerts antidiabetic properties and engenders metabolic inefficiency is missing. To evaluate this issue, we have systematically reanalyzed published transcriptomic datasets from MPP-treated neurons, metformin-treated hepatocytes, and methionine-restricted rats. We found that pathways leading to NADPH formation were widely induced, together with anabolic fatty acid biosynthesis, the latter appearing highly paradoxical in a state of mitochondrial impairment. However, concomitant induction of catabolic fatty acid oxidation indicated that complex I inhibition created a "futile" cycle of fatty acid synthesis and degradation, which was anatomically distributed between adipose tissue and liver in vivo. Cofactor balance analysis unveiled that such cycling would indeed be energetically futile (-3 ATP per acetyl-CoA), though it would not be redox-futile, as it would convert NADPH into respirable FADH 2 without any net production of NADH. We conclude that inhibition of NADH dehydrogenase leads to a metabolic shift from glycolysis and the citric acid cycle (both generating NADH) towards the pentose phosphate pathway, whose product NADPH is translated 1:1 into FADH 2 by fatty acid cycling. The diabetes-resistant phenotype following hepatic and intestinal complex I inhibition is attributed to FGF21- and GDF15-dependent fat hunger signaling, which remodels adipose tissue into a glucose-metabolizing organ., (© 2024. The Author(s).)

Oxidative modifications of amino acid side chains in proteins are a hallmark of oxidative stress, and they are usually regarded as structural damage. However, amino acid oxidation may also have a protective effect and may serve regulatory or structural purposes. Here, we have attempted to characterize the global redox role of the 20 proteinogenic amino acids in animals by analyzing their usage frequency in 5 plausible evolutionary paradigms of increased oxidative burden: (i) peroxisomal proteins versus all proteins, (ii) mitochondrial proteins versus all proteins, (iii) mitochondrially encoded respiratory chain proteins versus all mitochondrial proteins, (iv) proteins from long-lived animals versus those from short-lived animals, and (v) proteins from aerobic, free-living animals versus those from facultatively anaerobic animals. We have found that avoidance of cysteine in the oxidative condition was the most pronounced and significant variation in the majority of comparisons. Beyond this preeminent pattern, only local signals were observed, primarily increases in methionine and glutamine as well as decreases in serine and proline. Hence, certain types of cysteine oxidation appear to enforce its proteome-wide evolutionary avoidance despite its essential role in disulfide bond formation and metal ligation. The susceptibility to oxidation of all other amino acids appears to be generally unproblematic, and sometimes advantageous.

Consumption of opioids is a growing global health problem. The gold standard for drugs of abuse screening is immunochemical assays. However, this method comes with some disadvantages when screening for a wide variety of opioids. Detection of the binding of a compound at the human μ-opioid receptor (MOR) offers a promising alternative target. Here, we set up a urine assay to allow for detection of compounds that bind at the MOR, thus allowing the assay to be utilized as a screening tool for opioid intake. The assay is based on the incubation of MOR-containing cell membranes with the selective MOR-ligand DAMGO and urine. After filtration, the amount of DAMGO in the eluate is analyzed by liquid chromatography tandem mass spectroscopy (LC-MS/MS). The absence of DAMGO in the eluate corresponds to a competing MOR ligand in the urine sample, thus indicating opiate/opioid intake by the suspect. Sensitivity and specificity were determined by the analysis of 200 consecutive forensic routine casework urine samples. A pronounced displacement of DAMGO was observed in 29 of the 35 opiate/opioid-positive samples. Detection of fentanyl intake proved to be the most challenging aspect. Applying a cut-off value of, e.g., 10% DAMGO binding would lead to a sensitivity of 83% and a specificity of 95%. Consequently, the novel assay proved to be a promising screening tool for opiate/opioid presence in urine samples. The nontargeted approach and possible automation of the assay make it a promising alternative to conventional methods.

Lipid peroxidation is a biochemically adverse phenomenon with key involvement in many different diseases including premature infant blindness, nonalcoholic steatohepatitis, or Parkinson's disease. Moreover, lipid peroxidation may be the most important universal driver of the biological aging process. Canonic lipid peroxidation is a free radical chain reaction consisting of three kinetically independent steps, initiation, propagation, and termination. During the bulk propagation phase, only lipids and oxygen are consumed as substrates and maintain the chain reaction. In native biological membranes, however, lipid peroxidation takes place in direct vicinity to high concentrations of inserted membrane proteins with their exposed hydrophobic amino acid side chains. In the following, we review the evidence that redox-active intramembrane amino acid residues have a profound impact on the course and extent of lipid peroxidation in vivo . Specifically, tyrosine and tryptophan are concluded to be chain-breaking antioxidants that effectuate termination, whereas cysteine is a chain-transfer catalyst that accelerates propagation and thereby promotes lipid peroxidation. Methionine, in turn, is highly accumulated in mitochondrial membrane proteins of animal species with high metabolic rates and imminent danger of lipid peroxidation, though its specific role has not been entirely defined. Potentially, it interferes with initiation on the membrane protein surface. Nevertheless, all four residues are distinguished by their clear relevance to lipid peroxidation as deduced from either experimental or genetic and comparative data. The latter have uncovered distinct evolutionary pressures in favor or against each residue in lipid membranes and have shed light on formerly unacknowledged chemical mechanisms.

Radioactive ligand binding assays are the most commonly applied method for the determination of binding affinities of compounds at a particular receptor. While they are highly sensitive and high-throughput capable they come with major disadvantages due to the radioactive ligands utilized. Here we present the development of a mass-spectrometry-based binding assay for the determination of binding affinities at the human μ-opioid receptor using non-labelled DAMGO ([D-Ala 2 , N-MePhe 4 , Gly 5 -ol]-enkephalin). The runtime of the LC-MS/MS method was 5.5 min per data point and allowed for the highly sensitive detection of 38.5 fg DAMGO on column. The assay shows low non-specific binding and the equilibrium dissociation constant of DAMGO was 0.57 nM. The assay was applied to determine the K i values of 17 opiates/opioids and the results were in good agreement with the data from radioactive receptor binding assays published in the literature. Additionally, the K i value of six 2-benzylbenzimidazoles, including the widely abused designer opioid isotonitazene, were determined ranging from 0.654 to 72.9 nM. Consequently, the developed assay provides a suitable alternative to radioactive binding assays as it allows for a reliable and rapid determination of receptor binding affinities of e.g. newly emerging designer opioids., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Thiyl radicals are exceptionally interesting reactive sulfur species (RSS), but rather rarely considered in a biological or medical context. We here review the reactivity of protein thiyl radicals in aqueous and lipid phases and provide an overview of their most relevant reaction partners in biological systems. We deduce that polyunsaturated fatty acids (PUFAs) are their preferred reaction substrates in lipid phases, whereas protein side chains arguably prevail in aqueous phases. In both cellular compartments, a single, dominating thiyl radical-specific antioxidant does not seem to exist. This conclusion is rationalized by the high reaction rate constants of thiyl radicals with several highly concentrated substrates in the cell, precluding effective interception by antioxidants, especially in lipid bilayers. The intractable reactivity of thiyl radicals may account for a series of long-standing, but still startling biochemical observations surrounding the amino acid cysteine: (i) its global underrepresentation on protein surfaces, (ii) its selective avoidance in aerobic lipid bilayers, especially the inner mitochondrial membrane, (iii) the inverse correlation between cysteine usage and longevity in animals, (iv) the mitochondrial synthesis and translational incorporation of cysteine persulfide, and potentially (v) the ex post introduction of selenocysteine into the genetic code.

Prooxidative therapy is a well-established concept in infectiology and parasitology, in which prooxidative drugs like artemisinin and metronidazole play a pivotal clinical role. Theoretical considerations and earlier studies have indicated that prooxidative therapy might also represent a promising strategy in oncology. Here, we have investigated a novel class of prooxidative drugs, namely chain-transfer agents, as cytostatic agents in a series of human tumor cell lines in vitro. We have found that different chain-transfer agents of the lipophilic thiol class (like dodecane-1-thiol) elicited half-maximal effective concentrations in the low micromolar range in SY5Y cells (human neuroblastoma), Hela cells (human cervical carcinoma), HEK293 cells (immortalized human kidney), MCF7 cells (human breast carcinoma), and C2C12 cells (mouse myoblast). In contrast, HepG2 cells (human hepatocellular carcinoma) were resistant to toxicity, presumably through their high detoxification capacity for thiol groups. Cytotoxicity was undiminished by hypoxic culture conditions, but substantially lowered after cellular differentiation. Compared to four disparate, clinically used reference compounds in vitro (doxorubicin, actinomycin D, 5-fluorouracil, and hydroxyurea), chain-transfer agents emerged as comparably potent on a molar basis and on a maximum-effect basis. Our results indicate that chain-transfer agents possess a promising baseline profile as cytostatic drugs and should be explored further for anti-tumor chemotherapy.

The sirtuin (SIRT) protein family has been of major research interest over the last decades because of their involvement in aging, cancer, and cell death. SIRTs have been implicated in gene and metabolic regulation through their capacity to remove acyl groups from lysine residues in proteins in an NAD + -dependent manner, which may alter individual protein properties as well as the histone-DNA interaction. Since SIRTs regulate a wide range of different signaling cascades, a fine-tuned homeostasis of these proteins is imperative to guarantee the function and survival of the cell. So far, however, how exactly this homeostasis is established has remained unknown. Here, we provide evidence that neuronal SIRT degradation in Parkinson's disease (PD) models is executed by autophagy rather than the proteasome. In neuronal Lund human mesencephalic (LUHMES) cells, all seven SIRTs were substrates for autophagy and showed an accelerated autophagy-dependent degradation upon 1-methyl-4-phenylpyridinium (MPP + ) mediated oxidative insults in vitro, whereas the proteasome did not contribute to the removal of oxidized SIRTs. Through blockade of endogenous H 2 O 2 generation and supplementation with the selective radical scavenger phenothiazine (PHT), we could identify H 2 O 2 -derived species as the responsible SIRT-oxidizing agents. Analysis of all human SIRTs suggested a conserved regulatory motif based on cysteine oxidation, which may have triggered their degradation via autophagy. High amounts of H 2 O 2 , however, rapidly carbonylated selectively SIRT2, SIRT6, and SIRT7, which were found to accumulate carbonylation-prone amino acids. Our data may help in finding new strategies to maintain and modify SIRT bioavailability in neurodegenerative disorders., (© 2021. The Author(s).)

New findings on the chemistry of the amino acids, their role in protein folding, and their sequential primordial introduction have uncovered concealed causalities in genetic code evolution. The genetically encoded amino acids successively provided (i) membrane anchors, (ii) halophilic protein folds, (iii) mesophilic protein folds, (iv) metal ligation, and (v) antioxidation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Macroautophagy is a conserved degradative process for maintaining cellular homeostasis and plays a key role in aging and various human disorders. The microtubule-associated protein 1A/1B light chain 3B (MAP1LC3B or LC3B) is commonly analyzed as a key marker for autophagosomes and as a proxy for autophagic flux. Three paralogues of the LC3 gene exist in humans: LC3A, LC3B and LC3C. The molecular function, regulation and cellular localization of LC3A and LC3C have not been investigated frequently, even if a similar function to that described for LC3B appears likely. Here, we have selectively decapacitated LC3B by three separate strategies in primary human fibroblasts and analyzed the evoked effects on LC3A, LC3B and LC3C in terms of their cellular distribution and co-localization with p62, a ubiquitin and autophagy receptor. First, treatment with pharmacological sirtuin 1 (SIRT1) inhibitors to prevent the translocation of LC3B from the nucleus into the cytosol induced an increase in cytosolic LC3C, a heightened co-localization of LC3C with p62, and an increase LC3C-dependent autophagic flux as assessed by protein lipidation. Cytosolic LC3A, however, was moderately reduced, but also more co-localized with p62. Second, siRNA-based knock-down of SIRT1 broadly reproduced these findings and increased the co-localization of LC3A and particularly LC3C with p62 in presumed autophagosomes. These effects resembled the effects of pharmacological sirtuin inhibition under normal and starvation conditions. Third, siRNA-based knock-down of total LC3B in cytosol and nucleus also induced a redistribution of LC3C as if to replace LC3B in the nucleus, but only moderately affected LC3A. Total protein expression of LC3A, LC3B, LC3C, GABARAP and GABARAP-L1 following LC3B decapacitation was unaltered. Our data indicate that nuclear trapping and other causes of LC3B functional loss in the cytosol are buffered by LC3A and actively compensated by LC3C, but not by GABARAPs. The biological relevance of the potential functional compensation of LC3B decapacitation by LC3C and LC3A warrants further study.

Cysteine is arguably the best-studied biological amino acid, whose thiol group frequently participates in catalysis or ligand binding by proteins. Still, cysteine's unusual biological distribution has remained mysterious, being strikingly underrepresented in transmembrane domains and on accessible protein surfaces, particularly in aerobic life forms ("cysteine anomaly"). Noting that lipophilic thiols have been used for decades as radical chain transfer agents in polymer chemistry, we speculated that the rapid formation of thiyl radicals in hydrophobic phases might provide a rationale for the cysteine anomaly. Hence, we have investigated the effects of dodecylthiol and related compounds in isolated biomembranes, cultivated human cells and whole animals (C. elegans). We have found that lipophilic thiols at micromolar concentrations were efficient accelerators, but not inducers of lipid peroxidation, catalyzed fatty acid isomerization to trans-fatty acids, and evoked a massive cellular stress response related to protein and DNA damage. These effects were specific for lipophilic thiols and were absent with thioethers, alcohols or hydrophilic compounds. Catalytic chain transfer activity by thiyl radicals appears to have deeply influenced the structural biology of life as reflected in the cysteine anomaly. Chain transfer agents represent a novel class of biological cytotoxins that selectively accelerate oxidative damage in vivo., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Modern geroscience is divided as regards the validity of the free radical theory of aging. Thermodynamic arguments and observations from comparative zoology support it, whereas results from experimental manipulations in representative animal species sometimes strongly contradict it. From a comparison of the multi-step aging process with a linear metabolic pathway (glycolysis), we here argue that the identification of the rate-limiting kinetic steps of the aging cascade is essential to understand the overall flux through the cascade, i.e., the rate of aging. Examining free radical reactions as a case in point, these reactions usually occur as chain reactions with three kinetically independent steps: initiation, propagation, and termination, each of which can be rate-limiting. Revisiting the major arguments in favor and against a role of free radicals in aging, we find that the majority of arguments in favor point to radical propagation as relevant and rate-limiting, whereas almost all arguments in disfavor are based on experimental manipulations of radical initiation or radical termination which turned out to be ineffective. We conclude that the overall lack of efficacy of antioxidant supplementation (which fosters termination) and antioxidant enzyme overexpression (which inhibits initiation) in longevity studies is attributable to the fact that initiation and termination are not the rate-limiting steps of the aging cascade. The biological and evolutionary plausibility of this interpretation is discussed. In summary, radical propagation is predicted to be rate-limiting for aging and should be explored in more detail.

Retrotransposon activation occurs in a variety of neurological disorders including multiple sclerosis and Alzheimer's Disease. While the origins of disease-related retrotransposon activation have remained mostly unidentified, this phenomenon may well contribute to disease progression by inducing inflammation, disrupting transcription and, potentially, genomic insertion. Here, we report that the inhibition of mitochondrial respiratory chain complex I by pharmacological agents widely used to model Parkinson's disease leads to a significant increase in expression of the ORF1 protein of the long interspersed nucleotide element 1 (LINE1) retrotransposon in human dopaminergic LUHMES cells. These findings were recapitulated in midbrain lysates from accordingly treated wild-type mice that mimic Parkinson's disease. Retrotransposon activation was paralleled by a loss of DNA cytosine methylation, providing a potential mechanism of retrotransposon mobilization. Loss of DNA methylation as well as retrotransposon activation were suppressed by the mitochondrial antioxidant phenothiazine, indicating that the well-established production of oxidants by inhibited complex I was causing these effects. Retrotransposon activation in some brain disorders may be less of a primary disease trigger rather than a consequence of mitochondrial distress, which is very common in neurodegenerative diseases., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Life most likely developed under hyperthermic and anaerobic conditions in close vicinity to a stable geochemical source of energy. Epitomizing this conception, the first cells may have arisen in submarine hydrothermal vents in the middle of a gradient established by the hot and alkaline hydrothermal fluid and the cooler and more acidic water of the ocean. To enable their escape from this energy-providing gradient layer, the early cells must have overcome a whole series of obstacles. Beyond the loss of their energy source, the early cells had to adapt to a loss of external iron-sulfur catalysis as well as to a formidable temperature drop. The developed solutions to these two problems seem to have followed the principle of maximum parsimony: Cysteine was introduced into the genetic code to anchor iron-sulfur clusters, and fatty acid unsaturation was installed to maintain lipid bilayer viscosity. Unfortunately, both solutions turned out to be detrimental when the biosphere became more oxidizing after the evolution of oxygenic photosynthesis. To render cysteine thiol groups and fatty acid unsaturation compatible with life under oxygen, numerous counter-adaptations were required including the advent of glutathione and the addition of the four latest amino acids (methionine, tyrosine, tryptophan, selenocysteine) to the genetic code. In view of the continued diversification of derived antioxidant mechanisms, it appears that modern life still struggles with the initially developed strategies to escape from its hydrothermal birthplace. Only archaea may have found a more durable solution by entirely exchanging their lipid bilayer components and rigorously restricting cysteine usage.

Cerebral hypoperfusion in the first hours after subarachnoid haemorrhage (SAH) is a major determinant of poor neurological outcome. However, the underlying pathophysiology is only partly understood. Here we induced neutropenia in C57BL/6N mice by anti-Ly6G antibody injection, induced SAH by endovascular filament perforation, and analysed cerebral cortical perfusion with laser SPECKLE contrast imaging to investigate the role of neutrophils in mediating cerebral hypoperfusion during the first 24 h post-SAH. SAH induction significantly increased the intracranial pressure (ICP), and significantly reduced the cerebral perfusion pressure (CPP). At 3 h after SAH, ICP had returned to baseline and CPP was similar between SAH and sham mice. However, in SAH mice with normal neutrophil counts cortical hypoperfusion persisted. Conversely, despite similar CPP, cortical perfusion was significantly higher at 3 h after SAH in mice with neutropenia. The levels of 8-iso-prostaglandin-F2α in the subarachnoid haematoma increased significantly at 3 h after SAH in animals with normal neutrophil counts indicating oxidative stress, which was not the case in neutropenic SAH animals. These results suggest that neutrophils are important mediators of cortical hypoperfusion and oxidative stress early after SAH. Targeting neutrophil function and neutrophil-induced oxidative stress could be a promising new approach to mitigate cerebral hypoperfusion early after SAH.

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC MS/MS), liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4'-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings., (© 2018 John Wiley & Sons, Ltd.)

Purpose: In recent years e-liquids used in electronic cigarettes have become an attractive alternative to smoking tobacco. A new trend is the use of e-liquids containing synthetic cannabinoids (SCs) instead of smoking cannabis or herbal mixtures laced with SCs. In the frame of a systematic monitoring of the online market of 'legal high' products, e-liquids from online retailers who also sell herbal blends were bought., Methods: The products were analyzed by gas chromatography-mass spectrometry. In some of the e-liquids an unknown compound was detected which was identified as the SC 5F-Cumyl-PINACA (1-(5-fluoropentyl)- N -(2-phenylpropan-2-yl)-1 H -indazole-3-carboxamide) by nuclear magnetic resonance analysis. To investigate the phase I metabolism of this new class of compounds, 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA were incubated with pooled human liver microsomes (pHLM). Cumyl-PINACA was additionally ingested orally (0.6 mg) by a volunteer in a controlled self-experiment. To assess the relative potency of Cumyl-PINACA a set of SCs were characterized using a cAMP assay., Results: Metabolism of 5F-Cumyl-PINACA and Cumyl-PINACA showed similarities with AM-2201 and JWH-018. The main metabolites were formed by hydroxylation at the N -pentyl side chain. The main metabolites detected in the volunteer's urine sample were the same as in the pHLM assay. All SCs tested with the cAMP assay were full agonists at the CB 1 receptor. Cumyl-PINACA was the most potent SC among the tested compounds and showed an EC 50 value of 0.06 nM., Conclusions: The increasing popularity of e-liquids particularly among young people, and the extreme potency of the added SCs, pose a serious threat to public health. To our knowledge, this is the first report describing the tentative identification of human in vivo metabolites of Cumyl-PINACA and 5F-Cumyl-PINACA., Competing Interests: The authors declare that they have no conflict of interest.This article does not contain any studies with animals performed by any of the authors. Although this study included a self-administration experiment, such scientific human experiments do not necessitate approval by the university ethics committee in Germany.

Synthetic cannabinoids (SCs) are a structurally diverse class of new psychoactive substances. Most SCs used for recreational purposes are based on indole or indazole core structures. EG-018 (naphthalen-1-yl(9-pentyl-9H-carbazol-3-yl)methanone), EG-2201 ((9-(5-fluoropentyl)-9H-carbazol-3-yl)(naphthalen-1-yl)methanone), and MDMB-CHMCZCA (methyl 2-(9-(cyclohexylmethyl)-9H-carbazole-3-carboxamido)-3,3-dimethylbutanoate) are 3 representatives of a structural subclass of SCs, characterized by a carbazole core system. In vitro and in vivo phase I metabolism studies were conducted to identify the most suitable metabolites for the detection of these substances in urine screening. Detection and characterization of metabolites were performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-QToF-MS). Eleven in vivo metabolites were detected in urine samples positive for metabolites of EG-018 (n = 8). A hydroxypentyl metabolite, most probably the 4-hydroxypentyl isomer, and an N-dealkylated metabolite mono-hydroxylated at the carbazole core system were most abundant. In vitro studies of EG-018 and EG-2201 indicated that oxidative defluorination of the 5-fluoropentyl side chain of EG-2201 as well as dealkylation led to common metabolites with EG-018. This has to be taken into account for interpretation of analytical findings. A differentiation between EG-018 and EG-2201 (n = 1) uptake is possible by the detection of compound-specific in vivo phase I metabolites evaluated in this study. Out of 30 metabolites detected in urine samples of MDMB-CHMCZCA users (n = 20), a metabolite mono-hydroxylated at the cyclohexyl methyl tail is considered the most suitable compound-specific consumption marker while a biotransformation product of mono-hydroxylation in combination with hydrolysis of the terminal methyl ester function provides best sensitivity due to its high abundance., (Copyright © 2018 John Wiley & Sons, Ltd.)

Selective degeneration of differentiated neurons in the brain is the unifying feature of neurodegenerative disorders such as Parkinson's disease (PD) or Alzheimer's disease (AD). A broad spectrum of evidence indicates that initially subtle, but temporally early calcium dysregulation may be central to the selective neuronal vulnerability observed in these slowly progressing, chronic disorders. Moreover, it has long been evident that excitotoxicity and its major toxic effector mechanism, neuronal calcium overload, play a decisive role in the propagation of secondary neuronal death after acute brain injury from trauma or ischemia. Under physiological conditions, neuronal calcium homeostasis is maintained by a fine-tuned interplay between calcium influx and releasing mechanisms (Ca 2+ -channels), and calcium efflux mechanisms (Ca 2+ -pumps and -exchangers). Central functional components of the calcium efflux machinery are the Plasma Membrane Calcium ATPases (PMCAs), which represent high-affinity calcium pumps responsible for the ATP-dependent removal of calcium out of the cytosol. Beyond a growing body of experimental evidence, it is their high expression level, their independence of secondary ions or membrane potential, their profound redox regulation and autoregulation, their postsynaptic localization in close proximity to the primary mediators of pathological calcium influx, i.e. NMDA receptors, as well as evolutionary considerations which all suggest a pivotal role of the PMCAs in the etiology of neurodegeneration and make them equally challenging and alluring candidates for drug development. This review aims to summarize the recent literature on the role of PMCAs in the pathogenesis of neurodegenerative disorders., (Copyright © 2017 Elsevier B.V. All rights reserved.)

All extant life employs the same 20 amino acids for protein biosynthesis. Studies on the number of amino acids necessary to produce a foldable and catalytically active polypeptide have shown that a basis set of 7-13 amino acids is sufficient to build major structural elements of modern proteins. Hence, the reasons for the evolutionary selection of the current 20 amino acids out of a much larger available pool have remained elusive. Here, we have analyzed the quantum chemistry of all proteinogenic and various prebiotic amino acids. We find that the energetic HOMO-LUMO gap, a correlate of chemical reactivity, becomes incrementally closer in modern amino acids, reaching the level of specialized redox cofactors in the late amino acids tryptophan and selenocysteine. We show that the arising prediction of a higher reactivity of the more recently added amino acids is correct as regards various free radicals, particularly oxygen-derived peroxyl radicals. Moreover, we demonstrate an immediate survival benefit conferred by the enhanced redox reactivity of the modern amino acids tyrosine and tryptophan in oxidatively stressed cells. Our data indicate that in demanding building blocks with more versatile redox chemistry, biospheric molecular oxygen triggered the selective fixation of the last amino acids in the genetic code. Thus, functional rather than structural amino acid properties were decisive during the finalization of the universal genetic code., Competing Interests: Conflict of interest statement: Some of the chemical compounds used in this work have been patented by the Max Planck Society, naming author B.M. as one of the inventors (EP 1113795 B1).

Benzodiazepines have been introduced as medical drugs in the 1960s. They replaced the more toxic barbiturates, which were commonly used for treatment of anxiety or sleep disorders at the time. However, benzodiazepines show a high potential of misuse and dependence. Although being of great value as medicines, dependence to these drugs is a concern worldwide, in part due to overprescription and easy availability. Therefore, the phenomenon of benzodiazepines sold via Internet shops without restrictions at low prices is alarming and poses a serious threat to public health. Most of these compounds (with the exception of, e.g., phenazepam and etizolam) have never been licensed as medical drugs in any part of the world and are structurally derived from medically used benzodiazepines. Strategies of clandestine producers to generate new compounds include typical structural variations of medically used 1,4-benzodiazepines based on structure-activity relationships as well as synthesis of active metabolites and triazolo analogs of these compounds. As they were obviously designed to circumvent national narcotics laws or international control, they can be referred to as "designer benzodiazepines." The majority of these compounds, such as diclazepam, clonazolam, and nitrazolam, have been described in scientific or patent literature. However, little is known about their pharmacological properties and specific risks related to their use. This chapter describes the phenomenon of designer benzodiazepines and summarizes the available data on pharmacokinetics and pharmacodynamics as well as analytical approaches for their detection.

Indole or indazole-based synthetic cannabinoids (SCs) bearing substituents derived from valine or tert-leucine are frequently abused new psychoactive substances (NPS). The emergence of 5F-MDMB-PICA (methyl N-{[1-(5-fluoropentyl)-1H-indol-3-yl]carbonyl}-3-methylvalinate) on the German drug market is a further example of a substance synthesized in the context of scientific research being misused by clandestine laboratories by adding it to 'legal high' products. In this work, we present the detection of 5F-MDMB-PICA in several legal high products by gas chromatography-mass spectrometry (GC-MS) analysis. To detect characteristic metabolites suitable for a proof of 5F-MDMB-PICA consumption by urine analysis, pooled human liver microsome (pHLM) assays were performed and evaluated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) techniques to generate reference spectra of the in vitro phase I metabolites. The in vivo phase I metabolism was investigated by the analysis of more than 20 authentic human urine specimens and compared to the data received from the pHLM assay. Biotransformation of the 5-fluoropentyl side chain and hydrolysis of the terminal methyl ester bond are main phase I biotransformation steps. Two of the identified main metabolites formed by methyl ester hydrolysis or mono-hydroxylation at the indole ring system were evaluated as suitable urinary biomarkers and discussed regarding the interpretation of analytical findings. Exemplary analysis of one urine sample for 5F-MDMB-PICA phase II metabolites showed that two of the main phase I metabolites are subject to extensive glucuronidation prior to renal excretion. Therefore, conjugate cleavage is reasonable for enhancing sensitivity. Commercially available immunochemical pre-tests for urine proved to be unsuitable for the detection of 5F-MDMB-PICA consumption. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Since their first appearance on the Internet in 2012, designer benzodiazepines established as an additional, quickly growing compound class among new psychoactive substances. Data regarding pharmacokinetic parameters, metabolism, and detectability for new compounds are limited or often not available. One of these compounds, flubromazolam (8-bromo-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine), the triazolo-analogue of flubromazepam, has been offered on the Internet from 2014 on. The purpose of the present study was to assess the period of detectability in biological samples along with preliminary basic pharmacokinetic parameters of the designer benzodiazepine flubromazolam. To investigate these, one of the authors ingested a capsule containing 0.5 mg of the drug. Metabolism studies and suitability tests for the detection with immunochemical assays were performed with the samples obtained from the self-experiment and five authentic case samples. Flubromazolam and its mono-hydroxylated metabolite were detectable by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine for up to 6.5 and 8 days, respectively (lower limit of quantification (LLOQ) flubromazolam: 0.1 ng/mL). Peak serum concentrations were as low as 8 ng/mL (8 h post ingestion). Glucuronides were also detected. The terminal elimination half-life could be estimated in the range of 10-20 h. Immunochemical assays yielded negative results for serum samples and positive results for urine samples for up to five days post ingestion. The presented data demonstrate the detectability of a single uptake of 0.5 mg of flubromazolam in hair samples collected two weeks after drug uptake by LC-MS 3 (c max 0.6 pg/mg; LOD 0.01 pg/mg). The detected metabolites were in good agreement with those described in other studies. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background: The abuse of synthetic cannabinoids (SCs) as presumed legal alternative to cannabis poses a great risk to public health. For economic reasons many laboratories use immunoassays (IAs) to screen for these substances in urine. However, the structural diversity and high potency of these designer drugs places high demands on IAs regarding cross-reactivity of the antibodies used and detection limits., Methods: Two retrospective studies were carried out in order to evaluate the capability of two homogenous enzyme IAs for the detection of currently prevalent SCs in authentic urine samples. Urine samples were analyzed utilizing a 'JWH-018' kit and a 'UR-144' kit. The IA results were confirmed by an up-to-date liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) screening method covering metabolites of 45 SCs., Results: The first study (n=549) showed an 8% prevalence of SCs use (LC-MS/MS analysis) among inpatients of forensic-psychiatric clinics, whereas all samples were tested negative by the IAs. In a second study (n=200) the combined application of both IAs led to a sensitivity of 2% and a diagnostic accuracy of 51% when applying the recommended IA cut-offs. Overall, 10 different currently prevalent SCs were detected in this population. The results can be explained by an insufficient cross-reactivity of the antibodies towards current SCs in combination with relatively high detection limits of the IAs., Conclusions: In light of the presented study data it is strongly recommended not to rely on the evaluated IA tests for SCs in clinical or forensic settings. For IA kits of other providers similar results can be expected.

Among the recently emerged synthetic cannabinoids, MDMB-CHMICA (methyl N-{[1-(cyclohexylmethyl)-1H-indol-3-yl]carbonyl}-3-methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB-CHMICA was investigated using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-Q ToF-MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC-ESI-MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB-CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB-CHMICA at the CB 1 receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Recently, the pyrazole-containing synthetic cannabinoid N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-3-(4-fluorophenyl)-1H-pyrazole-5-carboxamide (3,5-AB-CHMFUPPYCA) has been identified as a 'research chemical' both in powdered form and as an adulterant present in herbal preparations. Urine is the most common matrix used for abstinence control and the extensive metabolism of synthetic cannabinoids requires implementation of targeted analysis. The present study describes the investigation of the in vitro phase I metabolism of 3,5-AB-CHMFUPPYCA and its regioisomer 5,3-AB-CHMFUPPYCA using pooled human liver microsomes. Metabolic patterns of both AB-CHMFUPPYCA isomers were qualitatively similar and dominated by oxidation of the cyclohexylmethyl side chain. Biotransformation to monohydroxylated metabolites of high abundance confirmed that these species might serve as suitable targets for urine analysis. Furthermore, since synthetic cannabinoids are commonly administered by smoking and because some metabolites can also be formed as thermolytic artefacts, the stability of both isomers was assessed under smoking conditions. Under these conditions, pyrolytic cleavage of the amide bond occurred that led to approximately 3 % conversion to heat-induced degradation products that were also detected during metabolism. These artefactual 'metabolites' could potentially bias in vivo metabolic profiles after smoking and might have to be considered for interpretation of metabolite findings during hair analysis. This might be relevant to the analysis of hair samples where detection of metabolites is generally accepted as a strong indication of drug use rather than a potential external contamination. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Purpose: Only a fraction of the currently established low-molecular weight antioxidants exhibit cytoprotective activity in living cells, which is considered a prerequisite for their potential clinical usefulness in Parkinson's disease or stroke. Post hoc structure-activity relationship analyses have predicted that increased lipophilicity and enhanced radical stabilization could contribute to such cytoprotective activity., Methods: We have synthesized a series of novel phenothiazine-type antioxidants exhibiting systematic variation in their lipophilicity and radical stabilization. Phenothiazine was chosen as lead structure for its superior activity at baseline. The novel compounds were evaluated for their neuroprotective potency in cell culture, and for their primary molecular targets., Results: Lipophilicity was associated with enhanced cytoprotective activity, but only to a certain threshold (logP ≈ 7). Benzannulation likewise produced improved cytoprotectants that exhibited very low EC 50 values of ~8 nM in cultivated neuronal cells. Inhibition of global protein oxidation was the best molecular predictor of cytoprotective activity, followed by the inhibition of membrane protein autolysis. In contrast, the inhibition of lipid peroxidation in isolated brain lipids and the suppression of intracellular oxidant accumulation were poor predictors of cytoprotective activity, primarily as they misjudged the cellular advantage of high lipophilicity., Conclusions: Lipophilicity, radical stabilization and molecular weight appear to form an uneasy triangle, in which a slightly faulty selection may readily abolish neuroprotective activity.

Secondary metastases are the leading cause of mortality in patients with breast cancer. Cytochrome P450 (CYP) 2J2 (CYP2J2) is upregulated in many human tumors and generates epoxyeicosanoids from arachidonic acid that promote tumorigenesis and metastasis, but at present there is little information on the genes that mediate these actions. In this study MDA-MB-468 breast cancer cells were stably transfected with CYP2J2 (MDA-2J2 cells) and Affymetrix microarray profiling was undertaken. We identified 182 genes that were differentially expressed in MDA-2J2 cells relative to control (MDA-CTL) cells (log[fold of control] ≥2). From gene ontology pathway analysis bone morphogenetic protein (BMP) receptor 1B (BMPR1B) emerged as an important upregulated gene in MDA-2J2 cells. Addition of the BMPR1B ligand BMP2 stimulated the migration of MDA-2J2 cells, but not MDA-CTL cells, from 3D-matrigel droplets. Migration of MDA-2J2 cells was prevented by the BMPR antagonist dorsomorphin. These findings indicate that over-expression of CYP2J2 in MDA-MB-468-derived breast cancer cells activates BMPR1B expression that may contribute to increased migration. Targeting BMPR1B may be a novel approach to inhibit the metastatic activity of breast cancers that contain high levels of CYP2J2., (Copyright © 2016 Elsevier Ltd. All rights reserved.)