Identification of metabolites of 4,4′‐methylenedianiline in vascular smooth muscle cells by liquid chromatography‐electrospray tandem mass spectrometry

Chen, Kan; Dugas, Tammy R.; Cole, Richard B.

doi:10.1002/jms.1026

Cited by 11 publications

(10 citation statements)

References 19 publications

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“…Though we did observe DAPM metabolites in these cells, they were the same acetylated metabolites as we had previously reported - N -acetyl DAPM and one minor metabolite as N,N ′-diacetyl DAPM (8). While we expect that neither of these metabolites were responsible for DAPM-induced toxic effects, to try and unveil whether other metabolites not yet detected might also be formed, we compared the disappearance of DAPM in COX-2 deficient as compared to wildtype cells.…”

Section: Discussionsupporting

confidence: 85%

“…We observed 3 putative DAPM metabolites in these cells. In our prior studies (8), we had identified one as N -acetyl DAPM and one minor metabolite as N,N ′-diacetyl DAPM. On the assumption that a proximate toxicant of DAPM may be produced at levels below the detection limit or may be too rapidly conjugated to protein or other biomolecules so as to be difficult to detect, we compared the disappearance of DAPM in COX-2 deficient to that observed in wildtype cells (Figure 6A).…”

Section: Resultsmentioning

confidence: 99%

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Role of COX-2 in the Bioactivation of Methylenedianiline and in Its Proliferative Effects in Vascular Smooth Muscle Cells

et al. 2011

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4,4′-Methylenedianiline (DAPM) is an aromatic diamine used directly in the production of polyurethane forms and epoxy resins, or as a precursor to MDI in the manufacture of some polyurethanes. In our prior experiments, we showed that chronic, intermittent treatment of female rats with DAPM resulted in vascular medial hyperplasia of pulmonary arteries. In addition, treatment of vascular smooth muscle cells (VSMC) in culture with DAPM increased rates of proliferation in a manner that was inhibited by cotreatment with N-acetylcysteine but was not associated with oxidative stress. We thus hypothesized that NAC treatment inhibited DAPM toxicity by competing for binding reactive intermediates formed through DAPM metabolism. Because the peroxidase enzyme cyclooxygenase is constitutively expressed in VSMC, and because cyclooxygenase is known to metabolize similar aromatic amines to electrophilic intermediates, we further hypothesized that DAPM-induced VSMC proliferation was dependent upon COX-1/2-mediated bioactivation. To test this hypothesis, we treated VSMC with DAPM and measured cell proliferation, COX-2 expression, COX-1/2 activity and levels of covalent binding. DAPM treatment resulted in a dose-dependent increase in proliferation that was abolished by co-treatment with the COX-2-selective inhibitor celecoxib. In addition, DAPM exposure increased rates of proliferation in VSMC isolated from wildtype but not COX-2 (−/−) mice. Paradoxically, treatment with DAPM reduced cellular production of PGE2 and PGF2α, but dose-dependently increased COX-2 protein levels. Covalent binding of [14C]-DAPM to VSMC biomolecules was greater in wildtype than in COX-2 (−/−) cells. However, covalent binding of [14C]-DAPM was not altered by cotreatment with a nonselective inhibitor of cytochromes P450. These studies thus suggest that DAPM-induced VSMC proliferation may be due to bioactivation of DAPM, perhaps through the action of cyclooxygenase. The data furthermore suggest that DAPM’s mechanism of action may possibly involve inhibition or suicide inactivation of COX-2. In addition, because we observed an increase in DAPM-induced VSMC proliferation in cells isolated from female compared to male rats, further studies into the potential interplay between DAPM, the estrogen receptor, and COX-2 seem warranted.

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Section: Discussionsupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Role of COX-2 in the Bioactivation of Methylenedianiline and in Its Proliferative Effects in Vascular Smooth Muscle Cells

et al. 2011

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“…The driving force for this process is likely the stability of the products: aniline can either remain neutral or further decompose to 77 Da as shown in Figure 2e,f, and the 106 Da fragment can further rearrange to a tropylium-like ion of the same mass. 7,39 This mechanism describes the metastable behavior of these ions; therefore, detection of o -NH 2 protonated 2,4′-MDA or 2,2′-MDA is minimal. As a result, the primary conformation observed for the 2,2′-MDA [M + H] + ion (199 Da) is composed of ring protonated species.…”

Section: Results and Discussionmentioning

confidence: 99%

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies: I. Electrospray Spectra of 2-Ring Isomers

et al. 2014

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Purified methylenedianiline (MDA) regioisomers were structurally characterized and differentiated using tandem mass spectrometry (MS/MS), ion mobility-mass spectrometry (IM-MS), and IM-MS/MS in conjunction with computational methods. It was determined that protonation sites on the isomers can vary depending on the position of amino groups, and the resulting protonation sites play a role in the gas-phase stability of the isomer. We also observed differences in the relative distributions of protonated conformations depending on experimental conditions and instrumentation, which is consistent with previous studies on aniline in the gas phase. This work demonstrates the utility of a multifaceted approach for the study of isobaric species and elucidates why previous MDA studies may have been unable to detect and/or differentiate certain isomers. Such analysis may prove useful in the characterization of larger MDA multimeric species, industrial MDA mixtures, and methylene diphenyl diisocyanate (MDI) mixtures used in polyurethane synthesis.

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“…Acetylated metabolites were reported to be found in urine, blood and vascular smooth muscle cells. [10,11] However, their presence cannot explain the toxicity of DAPM. Other reactive and toxic metabolites are expected in bile, the likely route of exposure of bile duct cells to the proximate toxicant.…”

Section: Introductionmentioning

confidence: 99%

Liquid chromatography–electrospray tandem mass spectrometry investigations of fragmentation pathways of biliary 4,4′-methylenedianiline conjugates produced in rats

2008

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Abstract4, 4′-methylenedianiline (DAPM) is the main building block for production of 4,4′-methylenediphenyldiisocyanate that has been widely used in the manufacturing of polyurethane materials including medical devices. Although it was revealed that damage to biliary epithelial cells of the liver and common bile duct occurred upon acute exposure to DAPM, the exact mechanism of DAPM toxicity is not fully understood. Both phase I and II biotransformations of DAPM, some of which generate reactive intermediates, are characterized in detail by liquid chromatography electrospray tandem mass spectrometry. The two most prominent metabolites found in rat bile (M2 and M7) implicated glutathione, glucuronic acid and glycine conjugations (phase II) following hydroxylation, and N-oxidation (phase I). Their decomposition pathways, as evidenced by MS n experiments, have been elucidated in detail.

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Identification of metabolites of 4,4′‐methylenedianiline in vascular smooth muscle cells by liquid chromatography‐electrospray tandem mass spectrometry

Cited by 11 publications

References 19 publications

Role of COX-2 in the Bioactivation of Methylenedianiline and in Its Proliferative Effects in Vascular Smooth Muscle Cells

Role of COX-2 in the Bioactivation of Methylenedianiline and in Its Proliferative Effects in Vascular Smooth Muscle Cells

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies: I. Electrospray Spectra of 2-Ring Isomers

Liquid chromatography–electrospray tandem mass spectrometry investigations of fragmentation pathways of biliary 4,4′-methylenedianiline conjugates produced in rats

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