Isoketals form cytotoxic phosphatidylethanolamine adducts in cells

Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are γ-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC50 2.2 μM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease. Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are γ-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC50 2.2 μM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease. Levuglandins and their regio- and stereo-isomers (isolevuglandins or isoketals) are highly reactive γ-ketoaldehydes (IsoK) formed by nonenzymatic rearrangement of prostaglandin H2 and their free radical generated counterparts (H2-isoprostanes) (1Salomon R.G. Miller D.B. Zagorski M.G. Coughlin D.J. Solvent induced fragmentation of prostaglandin endoperoxides. New aldehyde products from PGH2 and novel intramolecular 1,2-hydride shift during endoperoxide fragmentation in aqueous solution.J. Am. Chem. Soc. 1984; 106: 6049-6060Crossref Scopus (90) Google Scholar, 2Salomon R.G. Miller D.B. Levuglandins: isolation, characterization, and total synthesis of new secoprostanoid products from prostaglandin endoperoxides.Adv. Prostaglandin Thromboxane Leukot. Res. 1985; 15: 323-326PubMed Google Scholar, 3Salomon R.G. Sha W. Brame C. Kaur K. Subbanagounder G. O'Neil J. Hoff H.F. Roberts II, L.J. 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One feature of IsoKs that drew immediate interest was their extreme reactivity toward the lysyl residues of proteins, with reaction rates several orders of magnitude faster than the α, β-unsaturated aldehyde 4-hydroxynonenal, perhaps the most widely studied lipid peroxidation product (5Brame C.J. Salomon R.G. Morrow J.D. Roberts II, L.J. Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts.J. Biol. Chem. 1999; 274: 13139-13146Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 6Iyer R.S. Ghosh S. Salomon R.G. Levuglandin E2 crosslinks proteins.Prostaglandins. 1989; 37: 471-480Crossref PubMed Scopus (77) Google Scholar). This increased reactivity derives from the facile formation of stable pyrrole adducts on primary amines. In the presence of oxygen, these pyrrole adducts convert over time into highly stable lactam adducts, as well as hydroxylactam adducts (5Brame C.J. Salomon R.G. Morrow J.D. Roberts II, L.J. Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts.J. Biol. Chem. 1999; 274: 13139-13146Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7DiFranco E. Subbanagounder G. Kim S. Murthi K. Taneda S. Monnier V.M. Salomon R.G. Formation and stability of pyrrole adducts in the reaction of levuglandin E2 with proteins.Chem. Res. Toxicol. 1995; 8: 61-67Crossref PubMed Scopus (34) Google Scholar). Increased concentrations of IsoK protein adducts have been observed in vivo during a wide variety of conditions associated with inflammation and oxidative stress, including atherosclerosis, end stage kidney disease, myocardial infarction, Alzheimer's Disease, glaucoma, hyperoxia, allergic inflammation, and experimental sepsis (8Salomon R.G. Batyreva E. Kaur K. Sprecher D.L. Schreiber M.J. Crabb J.W. Penn M.S. DiCorletoe A.M. Hazen S.L. Podrez E.A. Isolevuglandin-protein adducts in humans: products of free radical-induced lipid oxidation through the isoprostane pathway.Biochim. Biophys. Acta. 2000; 1485: 225-235Crossref PubMed Scopus (71) Google Scholar, 9Fukuda K. Davies S.S. Nakajima T. Ong B.H. Kupershmidt S. Fessel J. Amarnath V. Anderson M.E. Boyden P.A. Viswanathan P.C. et al.Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels.Circ. Res. 2005; 97: 1262-1269Crossref PubMed Scopus (111) Google Scholar, 10Zagol-Ikapitte I. 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Med. 2006; 40: 1210-1219Crossref PubMed Scopus (61) Google Scholar, 14Poliakov E. Brennan M.L. Macpherson J. Zhang R. Sha W. Narine L. Salomon R.G. Hazen S.L. Isolevuglandins, a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo.FASEB J. 2003; 17: 2209-2220Crossref PubMed Scopus (46) Google Scholar). Whether IsoK adducts contribute to pathogenesis of these diseases or are simply byproducts of the disease remains an active area of investigation. Evidence supporting a potential role in pathogenesis comes from treatment of cultured cells with exogenous IsoK, which induces a variety of responses relevant to disease, including increased macrophage uptake of LDL, activation of platelet aggregation via p38-MAP kinase, inhibition of sodium and potassium channels, inhibition of proteasome function, and cytotoxicity (9Fukuda K. Davies S.S. Nakajima T. Ong B.H. Kupershmidt S. Fessel J. Amarnath V. Anderson M.E. Boyden P.A. Viswanathan P.C. et al.Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels.Circ. Res. 2005; 97: 1262-1269Crossref PubMed Scopus (111) Google Scholar, 15Hoppe G. Subbanagounder G. O'Neil J. Salomon R.G. Hoff H.F. Macrophage recognition of LDL modified by levuglandin E2, an oxidation product of arachidonic acid.Biochim. Biophys. Acta. 1997; 1344: 1-5Crossref PubMed Scopus (39) Google Scholar, 16Bernoud-Hubac N. Alam D.A. Lefils J. Davies S.S. Amarnath V. Guichardant M. Roberts Ii L.J. Lagarde M. Low concentrations of reactive [gamma]-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation.Biochim. Biophys. Acta. 2009; 1791: 307-313Crossref PubMed Scopus (21) Google Scholar, 17Brame C.J. Boutaud O. Davies S.S. Yang T. Oates J.A. Roden D. Roberts II, L.J. Modification of proteins by isoketal-containing oxidized phospholipids.J. Biol. Chem. 2004; 279: 13447-13451Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 18Davies S.S. Amarnath V. Montine K.S. Bernoud-Hubac N. Boutaud O. Montine T.J. Roberts II, L.J. Effects of reactive gamma-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function.FASEB J. 2002; 16: 715-717Crossref PubMed Scopus (93) Google Scholar, 19Davies S.S. Modulation of protein function by isoketals and levuglandins.Subcell. Biochem. 2008; 49: 49-70Crossref PubMed Scopus (11) Google Scholar). The mechanism by which IsoK induces these effects has been presumed to be its adduction to cellular proteins, because of the high concentration of lysyl residues in cells. However, these lysyl residues are not the only potential target of IsoK present in cells. In vitro, IsoK can form adducts with a variety of other primary amines including the amino­phospholipid phosphatidylethanolamine (PE) (20Amarnath V. Amarnath K. Amarnath K. Davies S. Roberts II, L.J. Pyridoxamine: an extremely potent scavenger of 1,4-dicarbonyls.Chem. Res. Toxicol. 2004; 17: 410-415Crossref PubMed Scopus (83) Google Scholar, 21Bernoud-Hubac N. Fay L.B. Armarnath V. Guichardant M. Bacot S. Davies S.S. Roberts II, L.J. Lagarde M. Covalent of isoketals to Radic. Biol. Med. 2004; 37: PubMed Scopus Google Scholar, S.S. P.A. Amarnath V. I. Boutaud O. Oates J.A. Roberts II, L.J. gamma-ketoaldehydes and 2006; PubMed Scopus (54) Google Scholar). In the reaction of γ-ketoaldehydes to form pyrrole adducts with the of the is faster than with (20Amarnath V. Amarnath K. Amarnath K. Davies S. Roberts II, L.J. Pyridoxamine: an extremely potent scavenger of 1,4-dicarbonyls.Chem. Res. Toxicol. 2004; 17: 410-415Crossref PubMed Scopus (83) Google Scholar). Oxidation of PE or of high density in produces by reactivity with suggesting the of PE by pyrrole products these conditions R. of in oxidized and Biochem. 2004; PubMed Scopus Google Scholar). the biological effects of PE have not been of the of PE by IsoK is to the of PE as the PE to an and PE modified by IsoK also in a to which via in and via and in Zhang D. Zhang C. J. M. et a induced by 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, M. N. N. of on macrophage through inhibition of and Biochem. 2009; PubMed Scopus Google Scholar). We therefore to the extent to which IsoK adducts to PE with proteins and whether IsoK-PE mediate from was from to the and the of PE in product is and from Streptomyces chromofuscus phospholipase was from and was from kidney cells from cells from human umbilical a from cell was from was from other from (IsoK) was from in as V. Amarnath K. Masterson T. Davies S. Roberts L.J. synthesis of of Levuglandin 2005; Scopus Google Scholar) and a of IsoK in by of synthesis of in was oxidized with to The was by and with to The was into IsoK as V. Amarnath K. Masterson T. Davies S. Roberts L.J. synthesis of of Levuglandin 2005; Scopus Google Scholar). HEK293 cells on to in and in in concentration was added to the cells and To protein adducts from adducts, of was and the lower phase by The protein the the aqueous and chloroform phase was also and To measure the of adducts, HEK293 cells treated with and after the the cells by and in The was with and the The was by of IsoK-PE was by IsoK-PE was as N. Fay L.B. Armarnath V. Guichardant M. Bacot S. Davies S.S. Roberts II, L.J. Lagarde M. Covalent of isoketals to Radic. Biol. Med. 2004; 37: PubMed Scopus Google Scholar). In PE in was and in of a phase of IsoK in was added to the and the was and The chloroform phase was in and PE in a phase of in of the phase IsoK-PE in was The reaction of with PE was with in a as the reaction of IsoK with using in of the was added to the To was added to IsoK in and was in a using was in some in of was extracted with and the product by of pyrrole the reaction was treated with and the measure by of IsoK-PE reaction was added to with of and only also The in and of was added to the and of was added to the chloroform in and LC/MS/MS of the products of the reaction IsoK and the PE into the in ion and and induced product ion was on the using in the was was on the PE with to and a the ion LC/MS/MS of was using a a of Solvent a to Solvent in and Solvent to the The was to the in reaction and PE and of the products of the reaction IsoK and or IsoK and into the in ion and and product ion was on the and using in the was LC/MS/MS of was using a a of Solvent with with a to Solvent with and Solvent to the The was to the in reaction and IsoK was with human kidney in of well The treatment was to the cell and IsoK in the treatment extracted with and by ion LC/MS/MS by To measure IsoK adducts in cells, was added to the cells after the cell and cell and to of was added to to the and the lower chloroform was to a new The protein the was also to a of of as the the chloroform was and in and The was was added to the and The extracted with The chloroform was and in LC/MS/MS of adducts as adduct after was on the protein as after of the umbilical vein endothelial cells in well with and to to The was and the cells with and treated with was in as HEK293 cells, that was added to cells, to and the cells and and HUVEC in into well with and to to The was and the cells with PE treated with IsoK, or was as and in To the concentration of IsoK-PE induced HUVEC the concentration of IsoK, or treated PE from the of IsoK or added to treated PE the total PE concentration and was of treatment and was than the treated PE or PE added to of to with their treated PE and added to well an the with and the extent of by the as was to the only treated cells. To the of IsoK-PE that the cells during the to that of IsoK-PE formed IsoK was with cells, of HUVEC cells treated with IsoK-PE the and IsoK-PE after as IsoK treated HUVEC cells. To significant cellular of IsoKs, a radiolabeled of a IsoK, was with HEK293 cells and the adduct by HEK293 cells cellular because of their and of We that only of the radiolabel associated with the protein 40% of the radiolabel associated with the chloroform lower phase that (Fig. The radiolabel associated with the aqueous phase potentially adducts with and other amines. When was from treated cells, of the radiolabel was associated with The of radiolabel into the lower chloroform is with the formation of of adducts with as is that the radiolabel in the lower chloroform also IsoK or we the formation of adducts in treated cells. The of PE adduct formed in cells on the of PE and the reaction of PE other primary amines. The with faster than the reaction with (20Amarnath V. Amarnath K. Amarnath K. Davies S. Roberts II, L.J. Pyridoxamine: an extremely potent scavenger of 1,4-dicarbonyls.Chem. Res. Toxicol. 2004; 17: 410-415Crossref PubMed Scopus (83) Google Scholar). We the reaction of and PE using methods to that we a to the of the In the reaction of with PE was which was faster than the reaction of with that we To the IsoK-PE adduct and an to these adducts, we a of PE and of and PE (Fig. with a IsoK, (Fig. and the products (Fig. by ion of IsoK the of and and to the formation of new and The shift of these novel from the PE is with formation of adducts and reaction we not formation of oxidized pyrrole adducts or after in to with IsoK protein adducts, because the the PE reaction to to a greater extent than aqueous protein However, the PE product was and to several we observed a with the formation of a lactam adduct The of product and (Fig. The product and are with the (Fig. the product is with a The product ion is with the of the from and the product ion is with the of the the fragmentation of the product formed by other PE modified by IsoK can be we PE to an LC/MS/MS to measure the of IsoK-PE and Modification of the the of from significant of PE from PE (Fig. of IsoK-PE LC/MS/MS of of ion from LC/MS/MS of IsoK-PE PE and PE IsoK, induced IsoK, reaction We treated HEK293 cells with or IsoK and the formation of IsoK-PE using of the ion of IsoK-PE or and their product or In HEK293 cells treated with the IsoK-PE (Fig. In of IsoK to HEK293 in significant the a of potential IsoK-PE (Fig. The of these IsoK-PE The of the PE with PE in cells treated with IsoK was potential in PE and IsoK-PE on these these results that IsoK potentially modified of the PE modified by IsoK in cells can be by high concentrations of IsoK to significant that not be cell lower concentrations of of the total IsoK-PE formed in cells after of the IsoK-PE and their potentially than PE present in cells, of IsoK-PE by we to an that the cellular of IsoK-PE in a One potential to is to or the modified and the To be quantified by stable we IsoK with an of or and the products by ion The most product formed by the reaction of IsoK with was a with an pyrrole adduct (Fig. products and are with formation of the and hydroxylactam The of the ion a product ion with with and (Fig. The of the ion product and The of the ion product and The of the ion product and with an adduct product was not studied because with IsoK protein adducts that the is an that rapidly to the pyrrole adduct. The reaction of IsoK with generated products and and the of these products from the of their counterparts We these to a stable LC/MS/MS these the hydroxylactam adduct with a time of the lactam product with a time of and the pyrrole adduct with a time of (Fig. When the products of the reaction with IsoK or IsoK with using the and hydroxylactam adduct only present in the reaction IsoK and of the IsoK with reaction that the pyrrole product the most after the was that of the lactam adduct. pyrrole and lactam in of adducts. ion the and hydroxylactam adducts in reaction IsoK or IsoK and IsoK, reaction The phospholipase from S. chromofuscus is to PE modified with suggesting that be to IsoK-PE as We therefore whether IsoK-PE be to treatment with was added as an to with IsoK-PE or IsoK-PE and with S. chromofuscus only not significant (Fig. the pyrrole and lactam adducts. The with IsoK-PE only results (Fig. In the with S. chromofuscus treated IsoK-PE with pyrrole and lactam adducts (Fig. These results that S. chromofuscus IsoK-PE to the IsoK-PE lactam the hydrolysis product significant of the lactam suggesting that during the with and the pyrrole adduct to the lactam adduct. We to the total of IsoK-PE formed in cells treated with IsoK and to the total of adduct. HEK293 cells treated with IsoK the treatment and IsoK, and the levels of PE and protein adduct in the cells. than of the added IsoK was present in the after of the IsoK suggesting that the of IsoK with cellular amines. of protein and PE adducts in HEK293 cells increased as the concentration of IsoK in the treatment increased (Fig. these the of PE and protein adducts and of the IsoK, with the greater reactivity of To whether IsoK-PE adducts also formed in cell relevant to disease, we the extent of IsoK-PE formation in human umbilical vein endothelial cells treated with of HUVEC with IsoK also in the formation of IsoK-PE adducts and protein adducts (Fig. and the of PE adduct formed was greater than that of protein adduct. These results indicate that PE is a significant target of IsoK in cells, the that IsoK-PE mediate some of the effects of IsoK on cells. To test whether IsoK-PE induce effects as IsoK, we the to IsoK-PE to induce endothelial cell cell is a of and IsoKs are some of the most potent formed by lipid To test whether IsoK-PE induced endothelial we IsoK-PE as with PE treated with the or with the isoketal umbilical vein endothelial cells with IsoK, or treated PE and the in cell and treated PE significant on of the IsoK-PE concentration dependently the of HUVEC (LC50 2.2 (Fig. To the of IsoK-PE that the cells these conditions with the of IsoK-PE formed HUVEC treated with IsoK, we HUVEC with the treatment from the cells, and the IsoK-PE in the We that treatment with IsoK-PE in in the treated the of IsoK-PE formed IsoK to the These results are with the that IsoK induces cytotoxicity in by with PE to form a the formation of protein adducts by IsoK has been well in vivo and presumed to mediate the biological effects of IsoK, the extent of IsoK adduction to other relevant amines in vivo and their biological remains that other of PE adducts, are formed in greater in cells and may mediate some of the effects of et a IsoK modified PE in human as well as in the of treated W. L. S. R. Crabb J.W. Salomon R.G. in and of hydroxylactam Radic. Biol. Med. 2009; PubMed Scopus Google Scholar). the levels of PE adducts that in are than the levels of protein adduct than we have in plasma C.J. Boutaud O. Davies S.S. Yang T. Oates J.A. Roden D. Roberts II, L.J. Modification of proteins by isoketal-containing oxidized phospholipids.J. Biol. Chem. 2004; 279: 13447-13451Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The plasma of PE to protein adducts in these are to the of PE to protein adducts that we in cellular during the by et that IsoK formed adducts on Amarnath V. Oates J.A. Boutaud O. of adducts of and formed by reaction with 2009; PubMed Scopus Google Scholar) is with that IsoK also formed adduct to a extent than protein adducts. The that PE adducts are formed to a greater extent than protein adducts the that IsoK-PE is the active of some of the biological effects of that IsoK-PE is to induce cytotoxicity in HUVEC is with the that IsoK induces cytotoxicity in by IsoK-PE which induces cell in indicate that of by IsoK can the biological function of associated and also be by with IsoK-PE. Modification of the of PE by the and IsoK be to and and and protein potential of by IsoK-PE the cytotoxicity to HUVEC induced by IsoK-PE. IsoK-PE also as to that via in Zhang D. Zhang C. J. M. et a induced by 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) and via and in M. N. N. of on macrophage through inhibition of and Biochem. 2009; PubMed Scopus Google Scholar). The activation of a to cell by IsoK-PE be with the activation of p38-MAPK by IsoK in N. Alam D.A. Lefils J. Davies S.S. Amarnath V. Guichardant M. Roberts Ii L.J. Lagarde M. Low concentrations of reactive [gamma]-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation.Biochim. Biophys. Acta. 2009; 1791: 307-313Crossref PubMed Scopus (21) Google Scholar). lipid modified PE have been to several and as high and the that may well lipid modified PE One of is the of to their The that the from S. chromofuscus can IsoK-PE in to that active in other also be to IsoK-PE. interest in is the that in cells are J. K. T. N. characterization of a phospholipase and its Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and through and a role in Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google that be of interest to IsoK-PE is a of these and the of of these on endothelial cell or during oxidative stress and the greater of PE with protein adducts, is of interest to whether the effects of IsoK as which cells from induced cytotoxicity S.S. P.A. Amarnath V. I. Boutaud O. Oates J.A. Roberts II, L.J. gamma-ketoaldehydes and 2006; PubMed Scopus (54) Google be mediated in by the formation of IsoK-PE adducts. be to the of cytotoxicity and other cell are also In that IsoK-PE is of the most adducts of IsoK in cells a new potential of the effects of IsoK and methods to the extent of its formation in relevant The the of on of and induced human kidney human umbilical vein endothelial cell reaction phosphatidylethanolamine phospholipase