Selenium-containing chrysin (SeChry) and 3,7,3′,4′-tetramethylquercetin (SePQue) derivatives were synthesized by a microwave-based methodology. In addition to their improvement in terms of DPPH ...scavenging and potential GPx-like activities, when tested in a panel of cancer cell lines both selenium-derivatives revealed consistently to be more cytotoxic when compared with their oxo and thio-analogues, evidencing the key role of selenocabonyl moiety for these activities. In particular, SeChry elicited a noteworthy cytotoxic activity with mean IC50 values 18- and 3-fold lower than those observed for chrysin and cisplatin, respectively. Additionally, these seleno-derivatives evidenced an ability to overcome cisplatin and multidrug resistance. Notably, a differential behavior toward malignant and nonmalignant cells was observed for SeChry and SePQue, exhibiting higher selectivity indexes when compared with the chalcogen-derivatives and cisplatin. Our preliminary investigation on the mechanism of cytotoxicity of SeChry and SePQue in MCF-7 human mammary cancer cells demonstrated their capacity to efficiently suppress the clonal expansion along with their ability to hamper TrxR activity leading to apoptotic cell death.
Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor widely used in combined antiretroviral therapy and to prevent mother-to-child transmission of the human immunodeficiency virus type ...1, is associated with several adverse side effects. Using 12-mesyloxy-nevirapine, a model electrophile of the reactive metabolites derived from the NVP Phase I metabolite, 12-hydroxy-NVP, we demonstrate that the nucleophilic core and
-terminal residues of histones are targets for covalent adduct formation. We identified multiple NVP-modification sites at lysine (e.g., H2BK47, H4K32), histidine (e.g., H2BH110, H4H76), and serine (e.g., H2BS33) residues of the four histones using a mass spectrometry-based bottom-up proteomic analysis. In particular, H2BK47, H2BH110, H2AH83, and H4H76 were found to be potential hot spots for NVP incorporation. Notably, a remarkable selectivity to the imidazole ring of histidine was observed, with modification by NVP detected in three out of the 11 histidine residues of histones. This suggests that NVP-modified histidine residues of histones are prospective markers of the drug's bioactivation and/or toxicity. Importantly, NVP-derived modifications were identified at sites known to determine chromatin structure (e.g., H4H76) or that can undergo multiple types of post-translational modifications (e.g., H2BK47, H4H76). These results open new insights into the molecular mechanisms of drug-induced adverse reactions.
The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using ...the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with
N
-acetyl-
l
-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.
Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV-1 transmission in developing ...countries. Despite its clinical efficacy, NVP administration is associated with a variety of toxic responses that include hepatotoxicity and skin rash. Although the reasons for the adverse effects of NVP administration are still unclear, increasing evidence supports the involvement of metabolic activation to reactive electrophiles. In particular, Phase II activation of the NVP metabolite 12-hydroxy-NVP is thought to mediate NVP binding to bionucleophiles, which may be at the onset of toxicity. In the present study, we investigated the nature and specific locations of the covalent adducts produced in human serum albumin and human hemoglobin by reaction in vitro with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP. Multiple sites of modification were identified by two different mass spectrometry-based methodologies, liquid chromatography−electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF-TOF-MS). These two distinct methodologies, which in some instances afforded complementary information, allowed the identification of multiple adducts involving cysteine, lysine, tryptophan, histidine, serine, and the N-terminal valine of hemoglobin. Tryptophan, which is not a common site of covalent protein modification, was the NVP-modified amino acid residue detected in the two proteins and consistently identified by both LC-ESI-MS/MS and MALDI-TOF-TOF-MS. The propensity of tryptophan to react with the NVP-derived electrophile is further emphasized by the fact that human serum albumin possesses a single tryptophan residue, which suggests a remarkable selectivity that may be useful for biomonitoring purposes. Likewise, the NVP adduct with the terminal valine of hemoglobin, detected by LC-ESI-MS/MS after N-alkyl Edman degradation, appears as an easily assessed marker of NVP binding to proteins. Our results demonstrate the merits and complementarity of the two MS-based methodologies for the characterization of protein binding by NVP and suggest a series of plausible biomarkers of NVP toxicity that should be useful in the monitoring of toxicity effects in patients administered NVP.
Carbamazepine (CBZ) is one of the most widely used antiepileptic drugs by both adults and children. Despite its widespread use, CBZ is associated with central nervous system toxicity and severe ...hypersensitivity reactions, which raise concerns about its chronic use. While the precise mechanisms of CBZ-induced adverse events are still unclear, metabolic activation to the epoxide (CBZ-EP) has been thought to play a significant role.
This work reports first-hand evidence that CBZ reacts readily with biologically relevant thiyl radicals with no need for bioactivation. Using liquid chromatography coupled with high resolution mass spectrometry, multiple products from direct reaction of CBZ with glutathione (GSH) and N-acetyl-L-cysteine (NAC) were unequivocally identified, including the same product obtained upon ring-opening of CBZ-EP. The product profile is complex and consistent with radical-mediated mechanisms. Importantly, side products and adducts compatible with this non-enzymatic pathway were identified in liver extracts from CBZ-treated Wistar rats.
The reaction of CBZ with GSH and NAC is more extensive in the presence of oxygen. Taking into consideration that GSH conjugation is, in general, a detoxification pathway, these results suggest that under hyperoxia/oxidative stress conditions the bioavailability of the parent drug may be compromised. Additionally, this non-enzymatic process can be anticipated to play, at least in part, a role in the onset of CBZ-induced adverse reactions due to the concomitant generation of reactive oxygen species. Therefore, the search for causal relationships between the formation of non-enzymatically-driven CBZ products and the occurrence of CBZ-induced adverse events in human patients merits further research, aiming the translation of basic mechanistic findings into a clinical context that may ultimately lead to a safer CBZ prescription.
•CBZ reacts with biologically relevant thiols with no need for bioactivation.•The product profile is complex and consistent with radical-mediated mechanisms.•The reaction is more extensive in the presence of oxygen.•Products compatible with radical-mediated mechanisms were identified in liver extracts from CBZ-treated rats.•The concomitant generation of reactive oxygen species can have a role on CBZ-induced adverse reactions.
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•A furan-derived adduct was identified in liver histone 2B of furan-treated rats.•This adduct was detected prior to the identification of epigenetic changes.•This adduct represents an ...early compound-specific biomarker of exposure.•The first evidence for in vivo occurrence of carcinogen-modified histones is provided.•New insights into the mechanisms of chemical carcinogenesis are provided.
Furan is a rodent hepatocarcinogen ubiquitously found in the environment and heat-processed foods. Furan undergoes cytochrome P450 2E1-catalyzed bioactivation to cis-2-butene-1,4-dial (BDA), which has been shown to form an electrophilic conjugate (GSH-BDA) with glutathione. Both BDA and GSH-BDA yield covalent adducts with lysine residues in proteins. Dose- and time-dependent epigenetic histone alterations have been observed in furan-treated rats. While the covalent modification of histones by chemical carcinogens has long been proposed, histone-carcinogen adducts have eluded detection in vivo. In this study, we investigated if the covalent modification of histones by furan may occur in vivo prior to epigenetic histone alterations.
Using a “bottom-up” methodology, involving the analysis of tryptic peptides by liquid chromatography – high resolution mass spectrometry, we obtained evidence for a cross-link between GSH-BDA and lysine 107 of histone H2B isolated from the livers of male F344 rats treated with tumorigenic doses of furan. This cross-link was detected at the shortest treatment period (90 days) in the lowest dose group (0.92mg/kg body weight/day), prior to the identification of epigenetic changes, and occurred at a lysine residue that is a target for epigenetic modifications and crucial for nucleosome stability. Our results represent the first unequivocal proof of the occurrence of carcinogen-modified histones in vivo and suggest that such modification happens at the initial stages of furan-induced carcinogenesis. This type of alteration may be general in scope, opening new insights into the mechanisms of chemical carcinogenesis/toxicity and new opportunities for the development of early compound-specific biomarkers of exposure.
We report the development of a new microwave-based synthetic methodology mediated by Woollins' reagent that allowed an efficient conversion of caffeine into 6-selenocaffeine. A preliminary evaluation ...on the modulation of antioxidant activity upon selenation of caffeine, using the DPPH assay, indicated a mild antioxidant activity for 6-selenocaffeine, contrasting with caffeine, that exhibited no antioxidant activity under the same experimental conditions. Interestingly, whereas 6-selenocaffeine has revealed to have a low cytotoxic potential in both MCF10A and MCF-7 breast cells (24 h, up to 100 µM, MTT assay), a differential effect was observed when used in combination with the anticancer agents doxorubicin and oxaliplatin in MCF-7 breast cancer cells. The co-treatment of doxorubicin (1 µM) and 6-selenocaffeine (100 µM) resulted in a slight decrease in cellular viability when compared to doxorubicin (1 µM) alone. Conversely, the seleno-caffeine derivative at the same concentration markedly increased the viability of oxaliplatin (100 µM)-treated cells (p < 0.01). Overall, this work highlights an emerging methodology to synthesize organoselenium compounds and points out the differential roles of 6-selenocaffeine in the modulation of the cytotoxicity of anticancer agents.
In this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel ...electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with cell death, H
O
catabolic/metabolic process, stress response, and protein oligomerization. Validation by nonreducing WB was performed for peroxiredoxin-2 (PRDX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using antibodies against the sulfinylated/sulfonylated cysteine of these proteins to better evaluate their redox-oligomeric states under OSA and/or in response to PAP therapy. The results indicated that the redox-oligomeric state of GAPDH and PRDX2 involving overoxidation by sulfinic/sulfonic acids were differentially modulated in OSA RBC, which might be compromising RBC homeostasis. PAP therapy by restoring this modulation induced a higher oligomerization of overoxidized GAPDH and PRDX2 in some patients that could be associated with eryptosis and the chaperone "gain" of function, respectively. This varied response following PAP may result from the complex interplay between OSA and OSA metabolic comorbidity. Hence, information on the redox status of PRDX2 and GAPDH in RBC will help to better recognize OSA subtypes and predict the therapeutic response in these patients. GAPDH monomer combined with body mass index (BMI) and PRDX2 S-S dimer combined with homeostatic model assessment for insulin resistance (HOMA-IR) showed to be very promising biomarkers to predict OSA and OSA severity, respectively.
Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child transmission of the virus in ...developing countries. However, reports of severe NVP-induced hepatotoxicity and serious adverse cutaneous effects have raised concerns about its use. NVP metabolism involves oxidation of the 4-methyl substituent to 4-hydroxymethyl-NVP (12-hydroxy-NVP) and the formation of phenolic derivatives. Further metabolism, through either oxidation to quinoid derivatives or phase II esterification, may produce electrophilic derivatives capable of reacting with bionucleophiles to yield covalent adducts. These adducts could potentially be involved in the initiation of toxic responses. To gain insight into potentially reactive sites in proteins and prepare reliable and fully characterized NVP−amino acid adduct standards for subsequent assessment as biomarkers of NVP toxicity, we have used the model electrophile, 12-mesyloxy-NVP, as a synthetic surrogate for the NVP metabolite, 12-sulfoxy-NVP. Reactions of this model ester were conducted with glutathione and the nucleophilic amino acids arginine, cysteine, histidine, and tryptophan. Moreover, because adducts through the N-terminal valine of hemoglobin are convenient biomarkers of exposure to electrophilic toxicants, we also investigated the reaction with valine. We obtained very efficient (>80%) binding through the sulfur of both glutathione and N-acetylcysteine and moderate yields (10−14%) for binding through C2 of the indole ring of tryptophan and N1 of the imidazole ring of histidine. Reaction with arginine occurred through the α-amino group, possibly due to the high basicity of the guanidino group in the side chain. Reaction at the α-amino group of valine occurred to a significant extent (33%); the resulting adduct was converted to a thiohydantoin derivative, to obtain a standard useful for prospective biomonitoring studies. All adducts were characterized by a combination of 1H and 13C NMR spectroscopy and mass spectrometry techniques. The NVP conjugates with glutathione and N-acetylcysteine identified in this work were previously reported to be formed in vivo, although the corresponding structures were not fully characterized. Our results support the validity of 12-mesyloxy-NVP as a surrogate for 12-sulfoxy-NVP and suggest that NVP metabolism to 12-hydroxy-NVP, and subsequent esterification, could potentially be a factor in NVP toxicity. They further imply that multiple sites in proteins may be targets for modification by 12-hydroxy-NVP-derived electrophiles in vivo. Additionally, we obtained reliable, fully characterized standards for the assessment of protein modification by NVP in vivo, which should help clarify the potential role of metabolism in NVP-induced toxicity.
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•Immunohistochemistry with magnetic core nanoparticles to isolate viruses.•The use of MALDI-MS for rapid virus detection is explained in detail.•The use of ESI-MS/MS to pinpoint ...host-patient crosstalk is explained in detail.•The absolute quantitative MS is explained for large-scale protein quantitation.
The capabilities of bioanalytical mass spectrometry to (i) detect and differentiate viruses at the peptide level whilst maintaining high sample throughput and (ii) to provide diagnosis and prognosis for infected patients are presented as a tutorial in this work to aid analytical chemists and physicians to gain insights into the possibilities offered by current high-resolution mass spectrometry technology and bioinformatics. From (i) sampling to sample treatment; (ii) Matrix-Assisted Laser Desorption Ionization- to Electrospray Ionization -based mass spectrometry; and (iii) from clustering to peptide sequencing; a detailed step-by-step guide is provided and exemplified using SARS-CoV-2 Spike Y839 variant and the variant of concern SARS-CoV-2 Alpha (B.1.1.7 lineage), Influenza B, and Influenza A subtypes AH1N1pdm09 and AH3N2.
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