Interplay between the oxidation process and cytotoxic effects of antimonene nanomaterials.

Pnictogen nanomaterials have recently attracted researchers' attention owing to their promising properties in the field of electronic, energy storage, and nanomedicine applications. Moreover, especially in the case of heavy pnictogens, their chemistry allows for nanomaterial synthesis using both top-down and bottom-up approaches, yielding materials with remarkable differences in terms of morphology, size, yield, and properties. In this study, we carried out a comprehensive structural and spectroscopic characterization of antimony-based nanomaterials (Sb-nanomaterials) obtained by applying different production methodologies (bottom-up and top-down routes) and investigating the influence of the synthesis on their oxidation state and stability in a biological environment. Indeed, in situ XANES/EXAFS studies of Sb-nanomaterials incubated in cell culture media were carried out, unveiling a different oxidation behavior. Furthermore, we investigated the cytotoxic effects of Sb-nanomaterials on six different cell lines: two non-cancerous (FSK and HEK293) and four cancerous (HeLa, SKBR3, THP-1, and A549). The results reveal that hexagonal antimonene (Sb-H) synthesized using a colloidal approach oxidizes the most and faster in cell culture media compared to liquid phase exfoliated (LPE) antimonene, suffering acute degradation and anticipating well-differentiated toxicity from its peers. In addition, the study highlights the importance of the synthetic route for the Sb-nanomaterials as it was observed to influence the chemical evolution of Sb-H into toxic Sb oxide species, playing a critical role in its ability to rapidly eliminate tumor cells. These findings provide insights into the mechanisms underlying the dark cytotoxicity of Sb-H and other related Sb-nanomaterials, underlining the importance of developing therapies based on controlled and on-demand nanomaterial oxidation.

Supplementation with a New Standardized Extract of Green and Black Tea Exerts Antiadipogenic Effects and Prevents Insulin Resistance in Mice with Metabolic Syndrome.

Insulin resistance is one of the main characteristics of metabolic syndrome (MetS) and the main cause of the development of type II diabetes. The high prevalence of this syndrome in recent decades has made it necessary to search for preventive and therapeutic agents, ideally of natural origin, with fewer side effects than conventional pharmacological treatments. Tea is widely known for its medicinal properties, including beneficial effects on weight management and insulin resistance. The aim of this study was to analyze whether a standardized extract of green and black tea (ADM® Complex Tea Extract (CTE)) prevents the development of insulin resistance in mice with MetS. For this purpose, C57BL6/J mice were fed for 20 weeks with a standard diet (Chow), a diet with 56% kcal from fat and sugar (HFHS) or an HFHS diet supplemented with 1.6% CTE. CTE supplementation reduced body weight gain, adiposity and circulating leptin levels. Likewise, CTE also exerted lipolytic and antiadipogenic effects in 3T3-L1 adipocyte cultures and in the C. elegans model. Regarding insulin resistance, CTE supplementation significantly increased plasma adiponectin concentrations and reduced the circulating levels of insulin and the HOMA-IR. Incubation of liver, gastrocnemius muscle and retroperitoneal adipose tissue explants with insulin increased the pAkt/Akt ratio in mice fed with Chow and HFHS + CTE but not in those fed only with HFHS. The greater activation of the PI3K/Akt pathway in response to insulin in mice supplemented with CTE was associated with a decrease in the expression of the proinflammatory markers Mcp-1, IL-6, IL-1ß or Tnf-α and with an overexpression of the antioxidant enzymes Sod-1, Gpx-3, Ho-1 and Gsr in these tissues. Moreover, in skeletal muscle, mice treated with CTE showed increased mRNA levels of the aryl hydrocarbon receptor (Ahr), Arnt and Nrf2, suggesting that the CTE's insulin-sensitizing effects could be the result of the activation of this pathway. In conclusion, supplementation with the standardized extract of green and black tea CTE reduces body weight gain, exerts lipolytic and antiadipogenic effects and reduces insulin resistance in mice with MetS through its anti-inflammatory and antioxidant effects.

Immunomodulatory and Antioxidant Properties of a Novel Potential Probiotic Bacillus clausii CSI08.

Spore-forming bacteria of the Bacillus genus have demonstrated potential as probiotics for human use. Bacillus clausii have been recognized as efficacious and safe agents for preventing and treating diarrhea in children and adults, with pronounced immunomodulatory properties during several in vitro and clinical studies. Herein, we characterize the novel strain of B. clausii CSI08 (Munispore®) for probiotic attributes including resistance to gastric acid and bile salts, the ability to suppress the growth of human pathogens, the capacity to assimilate wide range of carbohydrates and to produce potentially beneficial enzymes. Both spores and vegetative cells of this strain were able to adhere to a mucous-producing intestinal cell line and to attenuate the LPS- and Poly I:C-triggered pro-inflammatory cytokine gene expression in HT-29 intestinal cell line. Vegetative cells of B. clausii CSI08 were also able to elicit a robust immune response in U937-derived macrophages. Furthermore, B. clausii CSI08 demonstrated cytoprotective effects in in vitro cell culture and in vivo C. elegans models of oxidative stress. Taken together, these beneficial properties provide strong evidence for B. clausii CSI08 as a promising potential probiotic.

Singlet oxygen and radical-mediated mechanisms in the oxidative cellular damage photosensitized by the protease inhibitor simeprevir.

Hepatitis C, a liver inflammation caused by the hepatitis C virus (HCV), is treated with antiviral drugs. In this context, simeprevir (SIM) is an NS3/4A protease inhibitor used in HCV genotypes 1 and 4. It is orally administered and achieves high virological cure rates. Among adverse reactions associated with SIM treatment, photosensitivity reactions have been reported. In the present work, it is clearly shown that SIM is markedly phototoxic, according to the in vitro NRU assay using BALB/c 3T3 mouse fibroblast. This result sheds light on the nature of the photosensitivity reactions induced by SIM in HCV patients, suggesting that porphyrin elevation in patients treated with SIM may not be the only mechanism responsible for SIM-associated photosensitivity. Moreover, lipid photoperoxidation and protein photooxidation assays, using human skin fibroblasts (FSK) and human serum albumin (HSA), respectively, reveal the capability of this drug to promote photodamage to cellular membranes. Also, DNA photo lesions induced by SIM are noticed through comet assay in FSK cells. Photochemical and photobiological studies on the mechanism of SIM-mediated photodamage to biomolecules indicate that the key transient species generated upon SIM irradiation is the triplet excited state. This species is efficiently quenched by oxygen giving rise to singlet oxygen, which is responsible for the oxidation of lipids and DNA (Type II mechanism). In the presence of HSA, the photobehavior is dominated by binding to site 3 of the protein, to give a stable SIM@HSA complex. Inside the complex, quenching of the triplet excited state is less efficient, which results in a longer triplet lifetime and in a decreased singlet oxygen formation. Hence, SIM-mediated photooxidation of the protein is better explained through a radical (Type I) mechanism.

Cytotoxic sub-nanometer aqueous platinum clusters as potential antitumoral agents.

Ligand-free sub-nanometer metal clusters (MCs) of Pt, Ir, Rh, Au and Cu, are prepared here in neat water and used as extremely active (nM) antitumoral agents for HeLa and A2870 cells. The preparation just consists of adding the biocompatible polymer ethylene-vinyl alcohol (EVOH) to an aqueous solution of the corresponding metal salt, to give liters of a MC solution after filtration of the polymer. Since the MC solution is composed of just neat metal atoms and water, the intrinsic antitumoral activity of the different sub-nanometer metal clusters can now fairly be evaluated. Pt clusters show an IC50 of 0.48 µM for HeLa and A2870 cancer cells, 23 times higher than that of cisplatin and 1000 times higher than that of Pt NPs, and this extremely high cytotoxicity also occurs for cisplatin-resistant (A2870 cis) cells, with a resistance factor of 1.4 (IC50 = 0.68 µM). Rh and Ir clusters showed an IC50 ∼ 1 µM. Combined experimental and computational studies support an enhanced internalization and cytotoxic activation.

Identification of an ASC oligomerization inhibitor for the treatment of inflammatory diseases.

The ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) protein is an scaffold component of different inflammasomes, intracellular multiprotein platforms of the innate immune system that are activated in response to pathogens or intracellular damage. The formation of ASC specks, initiated by different inflammasome receptors, promotes the recruitment and activation of procaspase-1, thereby triggering pyroptotic inflammatory cell death and pro-inflammatory cytokine release. Here we describe MM01 as the first-in-class small-molecule inhibitor of ASC that interferes with ASC speck formation. MM01 inhibition of ASC oligomerization prevents activation of procaspase-1 in vitro and inhibits the activation of different ASC-dependent inflammasomes in cell lines and primary cultures. Furthermore, MM01 inhibits inflammation in vivo in a mouse model of inflammasome-induced peritonitis. Overall, we highlight MM01 as a novel broad-spectrum inflammasome inhibitor for the potential treatment of multifactorial diseases involving the dysregulation of multiple inflammasomes.

In vitro assessment of the photo(geno)toxicity associated with Lapatinib, a Tyrosine Kinase inhibitor.

The epidermal growth factor receptors EGFR and HER2 are the main targets for tyrosine kinase inhibitors (TKIs). The quinazoline derivative lapatinib (LAP) is used since 2007 as dual TKI in the treatment of metastatic breast cancer and currently, it is used as an oral anticancer drug for the treatment of solid tumors such as breast and lung cancer. Although hepatotoxicity is its main side effect, it makes sense to investigate the ability of LAP to induce photosensitivity reactions bearing in mind that BRAF (serine/threonine-protein kinase B-Raf) inhibitors display a considerable phototoxic potential and that afloqualone, a quinazoline-marketed drug, causes photodermatosis. Metabolic bioactivation of LAP by CYP3A4 and CYP3A5 leads to chemically reactive N-dealkylated (N-LAP) and O-dealkylated (O-LAP) derivatives. In this context, the aim of the present work is to explore whether LAP and its N- and O-dealkylated metabolites can induce photosensitivity disorders by evaluating their photo(geno)toxicity through in vitro studies, including cell viability as well as photosensitized protein and DNA damage. As a matter of fact, our work has demonstrated that not only LAP, but also its metabolite N-LAP have a clear photosensitizing potential. They are both phototoxic and photogenotoxic to cells, as revealed by the 3T3 NRU assay and the comet assay, respectively. By contrast, the O-LAP does not display relevant photobiological properties. Remarkably, the parent drug LAP shows the highest activity in membrane phototoxicity and protein oxidation, whereas N-LAP is associated with the highest photogenotoxicity, through oxidation of purine bases, as revealed by detection of 8-Oxo-dG.

Photomutagenicity of chlorpromazine and its N-demethylated metabolites assessed by NGS.

The human genome is constantly attacked by endogenous and exogenous agents (ultraviolet light, xenobiotics, reactive oxygen species), which can induce chemical transformations leading to DNA lesions. To combat DNA damage, cells have developed several repair mechanisms; however, if the repair is defective, DNA lesions lead to permanent mutations. Single-cell gel electrophoresis (COMET assay) is a sensitive and well-established technique for quantifying DNA damage in individual cells. Nevertheless, this tool lacks relationship with mutagenesis. Therefore, to identify errors that give rise to mutations it would be convenient to test an alternative known procedure, such as next generation sequencing (NGS). Thus, the present work aims to evaluate the photomutagenicity of neuroleptic drug chlorpromazine (CPZ), and its N-demethylated metabolites using COMET assay and to test NGS as an alternative method to assess photomutagenesis. In this context, upon exposure to UVA radiation, COMET assay reveals CPZ-photosensitized DNA damage partially repaired by cells. Conversely with this result, metabolites demethylchlorpromazine (DMCPZ) and didemethylchlorpromazine (DDMCPZ) promote extensive DNA-photodamage, hardly repaired under the same conditions. Parallel assessment of mutagenesis by NGS is consistent with these results with minor discrepancies for DDMCPZ. To our knowledge, this is the first example demonstrating the utility of NGS for evaluating drug-induced photomutagenicity.

Chemical tuning for potential antitumor fluoroquinolones.

Phototoxic effects of 6,8 dihalogenated quinolones confers to this type of molecules a potential property as photochemotherapeutic agents. Two photodehalogenation processes seem to be involved in the remarkable photoinduced cellular damage. In this context, a new 6,8 dihalogenated quinolone 1 (1-methyl-6,8-difluoro-4-oxo-7-aminodimethyl-1,4-dihydroquinoline-3-carboxylic acid) was synthetized looking for improving the phototoxic properties of fluoroquinolones (FQ) and to determine the role of the photodegradation pathways in the FQ phototoxicity. With this purpose, fluorescence emissions, laser flash photolysis experiments and photodegradation studies were performed with compound 1 using 1-ethyl-6,8-difluoro-4-oxo-7-aminodimethyl-1,4-dihidroquinoline-3-carboxylic acid (2) and lomefloxacin (LFX) as reference compounds. The shortening of alkyl chain of the N(1) of the quinolone ring revealed a lifetime increase of the reactive aryl cation generated from photolysis of the three FQ and a significant reduction of the FQ photodegradation quantum yield. The fact that these differences were smaller when the same study was done using a hydrogen donor solvent (ethanol-aqueous buffer, 50/50 v/v) evidenced the highest ability of the reactive intermediate arising from 1 to produce intermolecular alkylations. These results were correlated with in vitro 3T3 NRU phototoxicity test. Thus, when Photo-Irritation-Factor (PIF) was determined for 1, 2 and LFX using cytotoxicity profiles of BALB/c 3T3 fibroblasts treated with each compound in the presence and absence of UVA light, a PIF more higher than 30 was obtained for 1 while the values for 2 and LFX were only higher than 8 and 10, respectively. Thereby, the present study illustrates an approach to modulate the photosensitizing properties of FQ with the purpose to improve the chemotherapeutic properties of antitumor quinolones. Moreover, the results obtained in this study also evidence that the key pathway responsible for the phototoxic properties associated with dihalogenated quinolones is the aryl cation generation.

Oxidatively Generated Lesions as Internal Photosensitizers for Pyrimidine Dimerization in DNA.

In this work, the attention is focused on UVA-photosensitized reactions triggered by a DNA chromophore-containing lesion, namely 5-formyluracil. This is a major oxidatively generated lesion that exhibits an enhanced light absorption in the UVB-UVA region. The mechanistic study combining photochemical and photobiological techniques shows that irradiation of 5-formyluracil leads to a triplet excited state capable of sensitizing formation of cyclobutane pyrimidine dimers in DNA via a triplet-triplet energy transfer. This demonstrates for the first time that oxidatively generated DNA damage can behave as an intrinsic sensitizer and result in an important extension of the active fraction of the solar spectrum with photocarcinogenic potential. Overall, this raises the question of an aggravated photomutagenicity of the 5-formyluracil lesion.

Photo(geno)toxicity changes associated with hydroxylation of the aromatic chromophores during diclofenac metabolism.

Diclofenac (DCF) can cause adverse reactions such as gastrointestinal, renal and cardiovascular disorders; therefore, topical administration may be an attractive alternative to the management of local pain in order to avoid these side effects. However, previous studies have shown that DCF, in combination with sunlight, displays capability to induce photosensitivity disorders. In humans, DCF is biotransformed into hydroxylated metabolites at positions 4' and 5 (4'OH-DCF and 5OH-DCF), and this chemical change produces non negligible alterations of the drug chromophore, resulting in a significant modification of its light-absorbing properties. In the present work, 5OH-DCF exhibited higher photo(geno)toxic potential than the parent drug, as shown by several in vitro assays (3T3 NRU phototoxicity, DNA ssb gel electrophoresis and COMET), whereas 4'OH-DCF did not display significant photo(geno)toxicity. This could be associated, at least partially with their more efficient UV-light absorption by 5OH-DCF metabolite and with a higher photoreactivity. Interestingly, most of the cellular DNA damage photosensitized by DCF and 5OH-DCF was repaired by the cells after several hours, although this effect was not complete in the case of 5OH-DCF.

Identification and validation of uterine stimulant methylergometrine as a potential inhibitor of caspase-1 activation.

Inflammasomes are intracellular multiprotein complexes of the innate immune system. Upon an inflammatory insult, such as infection or intracellular damage, a nucleotide-binding oligomerization domain-like receptor (NLR) sensor protein and the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) are assembled to activate protease procaspase-1. This protease processes pro-IL-1ß and pro-IL-18 cytokines, which are released to induce the inflammatory response. De-regulation of inflammasome contributes to the progression of several diseases, such as Alzheimer's disease, diabetes, cancer, inflammatory and autoimmune disorders. We herein describe the identification of methylergometrine (MEM), a drug currently used as a smooth muscle constrictor during postpartum hemorrhage, as an inhibitor of the inflammasome complex in ASC-mediated procaspase-1 activation screening. MEM inhibits the activation of the nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP1) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasomes in cellular models upon different pro-inflammatory stimuli. Our results suggest that MEM has the potential to reposition in the treatment of inflammatory diseases with the advantages of established safety and clinical data.

Targeting inflammasome by the inhibition of caspase-1 activity using capped mesoporous silica nanoparticles.

Acute inflammation is a protective response of the body to harmful stimuli, such as pathogens or damaged cells. However, dysregulated inflammation can cause secondary damage and could thus contribute to the pathophysiology of many diseases. Inflammasomes, the macromolecular complexes responsible for caspase-1 activation, have emerged as key regulators of immune and inflammatory responses. Therefore, modulation of inflammasome activity has become an important therapeutic approach. Here we describe the design of a smart nanodevice that takes advantage of the passive targeting of nanoparticles to macrophages and enhances the therapeutic effect of caspase-1 inhibitor VX-765 in vivo. The functional hybrid systems consisted of MCM-41-based nanoparticles loaded with anti-inflammatory drug VX-765 (S2-P) and capped with poly-L-lysine, which acts as a molecular gate. S2-P activity has been evaluated in cellular and in vivo models of inflammation. The results indicated the potential advantage of using nanodevices to treat inflammatory diseases.

Enhanced photo(geno)toxicity of demethylated chlorpromazine metabolites.

Chlorpromazine (CPZ) is an anti-psychotic drug widely used to treat disorders such as schizophrenia or manic-depression. Unfortunately, CPZ exhibits undesirable side effects such as phototoxic and photoallergic reactions in humans. In general, the influence of drug metabolism on this type of reactions has not been previously considered in photosafety testing. Thus, the present work aims to investigate the possible photo(geno)toxic potential of drug metabolites, using CPZ as an established reference compound. In this case, the metabolites selected for the study are demethylchlorpromazine (DMCPZ), didemethylchlorpromazine (DDMCPZ) and chlorpromazine sulfoxide (CPZSO). The demethylated CPZ metabolites DMCPZ and DDMCPZ maintain identical chromophore to the parent drug. In this work, it has been found that the nature of the aminoalkyl side chain modulates the hydrophobicity and the photochemical properties (for instance, the excited state lifetimes), but it does not change the photoreactivity pattern, which is characterized by reductive photodehalogenation, triggered by homolytic carbon-chlorine bond cleavage with formation of highly reactive aryl radical intermediates. Accordingly, these metabolites are phototoxic to cells, as revealed by the 3T3 NRU assay; their photo-irritation factors are even higher than that of CPZ. The same trend is observed in photogenotoxicity studies, both with isolated and with cellular DNA, where DMCPZ and DDMCPZ are more active than CPZ itself. In summary, side-chain demethylation of CPZ, as a consequence of Phase I biotransformation, does not result a photodetoxification. Instead, it leads to metabolites that exhibit in an even enhanced photo(geno)toxicity.

Polypeptide modulators of caspase recruitment domain (CARD)-CARD-mediated protein-protein interactions.

The caspase recruitment domain (CARD) is present in a large number of proteins. Initially, the CARD was recognized as part of the caspase activation machinery. CARD-CARD interactions play a role in apoptosis and are responsible for the Apaf-1-mediated activation of procaspase-9 in the apoptosome. CARD-containing proteins mediate the inflammasome-dependent activation of proinflammatory caspase-1. More recently, new roles for CARD-containing proteins have been reported in signaling pathways associated with immune responses. The functional role of CARD-containing proteins and CARDs in coordinating apoptosis and inflammatory and immune responses is not completely understood. We have explored the putative cross-talk between apoptosis and inflammation by analyzing the modulatory activity on both the Apaf-1/procaspase-9 interaction and the inflammasome-mediated procaspase-1 activation of CARD-derived polypeptides. To this end, we analyzed the activity of individual recombinant CARDs, rationally designed CARD-derived peptides, and peptides derived from phage display.