Development, validation and application of a sensitive LC–MS/MS method for the quantification of thalidomide in human serum, cells and cell culture medium

Roche, Sandra; Sewell, Louise; Meiller, Justine; Pedersen, Kasper; Rajpal, Rajesh; O’Gorman, Peter; Clynes, Martin; O’Connor, Robert

doi:10.1016/j.jchromb.2012.06.008

Cited by 13 publications

(11 citation statements)

References 46 publications

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“…4d ). Assuming comparable cellular concentrations 27 , 28 , 29 , this would imply that solvent-exposed bulky groups at C4 (and methyl groups at C5 and C6) interfere with Ikaros binding. The aniline and methyl substituents at C4, on the other hand, appear to contribute to Ikaros degradation, likely through direct interactions.…”

Section: Crbn Functions As a Dcaf For The Crl4 Crbn mentioning

confidence: 99%

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Fischer

Böhm

Lydeard

et al. 2014

Nature

841

830

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In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. This dual activity implies that small molecules can principally modulate a ligase to up- or down-regulate the ubiquitination of proteins.

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Section: Crbn Functions As a Dcaf For The Crl4 Crbn mentioning

confidence: 99%

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Fischer

Böhm

Lydeard

et al. 2014

Nature

841

830

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“…Briefly, the cells were washed with 1 mL DPBS(−) by centrifugation at 200×g for 5 min. 17) They were then washed 3 more times under the same conditions. Next, the cell pellets were collected and lysed with 50 µL of 70% methanol.…”

Section: Exposure Of Mouse Melanoma Cells To Cse Mvk Camentioning

confidence: 99%

Intracellular Metabolism of α,β-Unsaturated Carbonyl Compounds, Acrolein, Crotonaldehyde and Methyl Vinyl Ketone, Active Toxicants in Cigarette Smoke: Participation of Glutathione Conjugation Ability and Aldehyde–Ketone Sensitive Reductase Activity

et al. 2016

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The major toxicants in cigarette smoke, α,β-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,β-unsaturated ketone, methyl vinyl ketone (MVK), are known to form Michael-type adducts with glutathione (GSH) and consequently cause intracellular GSH depletion, which is involved in cigarette smoke-induced cytotoxicity. We have previously clarified that exposure to cigarette smoke extract (CSE) of a mouse melanoma cell culture medium causes rapid reduction of intracellular GSH levels, and that the GSH-MVK adduct can be detected by LC/MS analysis while the GSH-CA adduct is hardly detected. In the present study, to clarify why the GSH-CA adduct is difficult to detect in the cell medium, we conducted detailed investigation of the structures of the reaction products of ACR, CA, MVK and CSE in the GSH solution or the cell culture medium. The mass spectra indicated that in the presence of the cells, the GSH-CA and GSH-ACR adducts were almost not detected while their corresponding alcohols were detected. On the other hand, both the GSH-MVK adducts and their reduced products were detected. In the absence of the cells, the reaction of GSH with all α,β-unsaturated carbonyls produced only their corresponding adducts. These results show that the GSH adducts of α,β-unsaturated aldehydes, CA and ACR, are quickly reduced by certain intracellular carbonyl reductase(s) and excreted from the cells, unlike the GSH adduct of α,β-unsaturated ketone, MVK. Such a difference in reactivity to the carbonyl reductase might be related to differences in the cytotoxicity of α,β-unsaturated aldehydes and ketones.

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“…Briefly, the cells were washed with 1 mL DPBS (−) by centrifugation at 200×g for 5 min. 17) These cells were washed 3 times under the same conditions. Next, the cell pellets were collected and lysed with 50 µL of 70% methanol.…”

Section: Methyl Vinyl Ketone a Toxic Ingredient In Cigarette Smoke Ementioning

confidence: 99%

Methyl Vinyl Ketone, a Toxic Ingredient in Cigarette Smoke Extract, Modifies Glutathione in Mouse Melanoma Cells

Horiyama

Takahashi

Hatai

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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Cigarette smoke contains many harmful chemicals, which contribute to the pathogenesis of smokingrelated diseases such as chronic obstructive pulmonary disease, cancer and cardiovascular disease. The cytotoxicity of cigarette smoke is well documented, but the definitive mechanism behind its toxicity remains unknown. Ingredients in cigarette smoke are known to deplete intracellular glutathione (GSH), the most abundant cellular thiol antioxidant, and to cause oxidative stress. In the present study, we investigated the mechanism of cigarette smoke extract (CSE)-induced cytotoxicity in B16-BL6 mouse melanoma (B16-BL6) cells using liquid chromatography-tandem mass spectrometry. CSE and ingredients in cigarette smoke, methyl vinyl ketone (MVK) and crotonaldehyde (CA), reduced cell viability in a concentration-dependent manner. Also, CSE and the ingredients (m/z 70, each) irreversibly reacted with GSH (m/z 308) to form GSH adducts (m/z 378) in cells and considerably decreased cellular GSH levels at concentrations that do not cause cell death. Mass spectral data showed that the major product formed in cells exposed to CSE was the GSH-MVK adduct via Michael-addition and was not the GSH-CA adduct. These results indicate that MVK included in CSE reacts with GSH in cells to form the GSH-MVK adduct, and thus a possible reason for CSE-induced cytotoxicity is a decrease in intracellular GSH levels. Key words cigarette smoke extract (CSE); glutathione (GSH); methyl vinyl ketone (MVK); B16-BL6 mouse melanoma (B16-BL6) cell; LC/MS; LC/MS/MSCigarette smoking has been known as a major risk factor implicated in increased incidence of cardiovascular disease, cancer and chronic obstructive pulmonary disease.1) Cigarette smoke contains more than 4800 identified chemical compounds, many of which are toxic and harmful to the human body.2) Oxidants and aldehydes, major constituents in the gaseous phase of cigarette smoke, are thought to mediate oxidative stress which has been implicated in the pathogenesis of smoking-related diseases. [3][4][5] Reactive α,β-unsaturated carbonyl compounds such as acrolein and crotonaldehyde, are abundant in the gas phase of cigarette smoke and are major mediators of cigarette smokeinduced macrophage activation. 6) These compounds are also thought to contribute to oxidative stress-induced inflammation and vascular dysfunction. 7) Furthermore, acrolein and crotonaldehyde have been reported to directly react with the thiol groups, 8) especially glutathione (GSH), via a Michael-type addition reaction, resulting in the formation of nonreducible GSH-aldehyde derivatives, thereby depleting the total available GSH pool.9,10) Because GSH plays a key role in cellular antioxidant defense against oxidant injury, GSH depletion leads to cigarette smoke-induced cytotoxicity.11,12) Similarly, methyl vinyl ketone (MVK), an α,β-unsaturated carbonyl compound, has been shown to react with free GSH in nucleophilic Michael-type addition.13) The MVK-induced cell apoptosis was associated with depletion of GSH, disruption of the mi...

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Development, validation and application of a sensitive LC–MS/MS method for the quantification of thalidomide in human serum, cells and cell culture medium

Cited by 13 publications

References 46 publications

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Intracellular Metabolism of α,β-Unsaturated Carbonyl Compounds, Acrolein, Crotonaldehyde and Methyl Vinyl Ketone, Active Toxicants in Cigarette Smoke: Participation of Glutathione Conjugation Ability and Aldehyde–Ketone Sensitive Reductase Activity

Methyl Vinyl Ketone, a Toxic Ingredient in Cigarette Smoke Extract, Modifies Glutathione in Mouse Melanoma Cells

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