| Furan-containing compounds refer to a class of compounds containing furan ring(s),which are abundant in fruits,herbs,foods,and beverages.Many furan-containing compounds have been reported to be toxic and/or carcinogenic.A number of furans have also been documented as mechanism-based inactivators of cytochromes P450.The mechanism by which furan-containing compounds induce cytotoxicity,carcinogenesis,and P450 enzyme inactivation lies on cytochromes P450-mediated metabolic activation of the furans to furanoepoxides or/and the corresponding cis-enediones(cis-enedials or ?-ketoenals).The cis-enediones(cis-enedials or ?-ketoenals)are also suggested to be generated by rearrangement of the furanoepoxides.The electrophilic metabolites generated in situ react with nucleophiles of biomolecules,possibly triggering the development of cytotoxicity and genotoxicity as well as enzyme inactivation.The risks for intake of toxic furans have been rising,due to the rapid growth of globe-wide consumption of medical remedies,dietary supplements,and "natural" foods.However,detection of the reactive metabolites of furans generated in situ is a challenge,because of the high reactivities of the electrophilic species.In addition,covalent modification of proteins by reactive metabolites is suggested to be associated with the toxicities and enzyme inactivations of harmful furans.The studies performed are summarized as below.1.A selective and sensitive platform for function-based screening of potentially harmful furans was developed.In this study,2,5-dimethylfuran(DMF)was used as a model furan compound.To improve the selectivity of the analytical platform,4-bromobenzylamine(BBA)was employed as the trapping agent of reactive metabolites,in place of N-acetyl lysine(NAL)applied in the traditional detection method.DMF was incubated with rat liver microsomes supplemented with glutathione(GSH)and BBA as trapping agents,to produce a GSH/BBA-derived pyrrole.The weak C-N bond of BBA would be cleaved to produce product ions(PIs)of m/z 169 and 171 in mass spectrometry.Therefore,this would allow us to spontaneously monitor four characteristic fragments,including neutral loss(NL)scan of 129 Da and PI scans of m/z 272,169,and 171,to detect the GSH/BBA-derived pyrroles.NL of 129 Da and PI of m/z 272 come from the fragmentation of GSH portion,and PIs of m/z 169 and 171 originate from the fragmentation of the BBA moiety.Additionally,the ratio in intensities of the detected PI scans of m/z 169 and 171 should around be 1:1.Moreover,because of the inorganic element,bromine,which the resulting pyrroles contain,the results indicate that the pyrroles resulting from the reactive metabolites,cis-enediones(cis-enedials or y-ketoenals)can also be detected by inductively couple plasma mass spectrometry(ICP-MS).The GSH/BBA pyrrole derived from DMF was successfully detected by the four-channel monitoring system.Additional six furan-containing compounds were tested by the same approach,and similar observation was obtained.Crude extracts obtained from traditional Chinese medicinal herbs,including Dioscorea bulbifera L.and Lindera aggregata(Sims)Kosterm,were analyzed by the approach.Due to the extraordinarily high selectivity and sensitivity,the platform shows its great capability to analyze complicated mixture samples,such as fruit and herb extracts.2.In Lindera aggregata(Sims)Kosterm,two furan-containing compounds,i.e.linderane and linderalactone,were detected by our developed platform.Linderane(LDR)caused a time-and concentration-dependent inactivation of CYP2C9.In addition,the inactivation of CYP2C9 was NADPH-dependent and irreversible.More than 50%of CYP2C9 activity was lost after its incubation with LDR at the concentration of 10 mM for 15 min at 30?.The maximal rate constant for inactivation(kinact)was found to be 0.0419 min-1,and the concentration required for half-maximal inactivation(KI)was 1.26 ?M,respectively.GSH,catalase,and superoxide dismutase(SOD)failed to protect CYP2C9 against LDR-induced inactivation.Diclofenac,a substrate of CYP2C9,prevented the enzyme from inactivation produced by LDR.The estimated partition ratio of the inactivation was approximately 227.Two reactive intermediates,including furanoepoxide and?-ketoenal,might be responsible for the observed enzyme inactivation.The formation of the intermediates was verified by chemical synthesis.Multiple P450 enzymes,including CYPs 1A2,2B6,2C9,2C19,2D6,3A4,and 3A5,were found to be involved in the metabolic activation of LDR.Taken together,LDR was characterized as a mechanism-based inactivator of CYP2C9.3.Many furan-containing compounds are known to be toxic.Metabolic activation of toxic furans to reactive metabolites is generally considered as the initial step towards the processes of their toxicities.Sequential modification of key proteins by the electrophilic reactive intermediates is suggested to be an important mechanism of the toxic actions.In the present study,we developed a novel and simple analytical platform to detect protein modification resulting from metabolic activation of model compound 2,5-dimethylfuran(DMF).BBA and 4-bromobenzylmercaptan(BBM)were employed to trap protein adductions at cysteine and lysine residues,respectively.The resulting protein samples were proteolytically digested by chymotrypsin and Pronase E,followed by LC-MS/MS analysis.Modifications of cysteine and lysine residues of proteins were observed in microsomes and animals after exposure to DMF.In conclusion,the approach established has been proven highly selective and reliable.Not only does this advance allow us to detect the protein adductions arising from metabolic activation of potentially harmful furan-containing compounds,but also to define the structural identities of amino acid residues modified.The protein adductions were found to correlate with the hepatotoxicity of DMF.4.Diosbulbin B(DIOB),a harmful furanoid found in traditional Chinese medicine Dioscorea bulbifera L.,was reported to be hepatotoxic to mice.We proposed that covalent modification of key proteins by the electrophilic reactive intermediates of DIOB is an important mechanism of toxic actions.The present study systematically investigated the protein modification resulting from metabolic activation of DIOB by our developed analytical platform.BBA and BBM were employed to trap protein adductions at cysteine and lysine residues,respectively.Reactive metabolites of DIOB were found to modify both cysteine and lysine residues,and the amino acid adducts further reacted with lysine/cysteine residues to form crosslinks.The protein adductions were time-and concentration-(in vitro)and dose-dependent(in vivo).To probe the role of CYP3A in DIOB-induced protein adductions,DIOB was incubated with mouse liver microsomes prepared from mice pretreated with ketoconazole(P450 3A inhibitor),dexamethasone(P450 3A inducer),or vehicle.The observed protein adductions were found to correlate with the enzyme activities of CYP3A.Pretreatment with buthionine sulfoximine increased the protein adductions in mice given DIOB.The results of the study were consistent with the observed toxicity and metabolic activation of DIOB obtained in our laboratory.In summary,we established a bromine-tagged platform to monitor the formation of reactive metabolites of furan-containing compounds.This approach,lying on the natural isotope abundance of bromine,has been approved to be selective,sensitive,and particularly suitable for analysis of complicated systems,such as extracts of herbal medicines.By this technique,we succeeded in identifying DIOB and 8-epidiosbulbin E acetate from Dioscorea bulbifera L.,as well as LDR and linderalactone from Lindera aggregata(Sims)Kosterm.DIOB and 8-epidiosbulbin E acetate are known hepatotoxins,and we found LDR was a mechanism-based inactivator of CYP2C9.Using similar method,we identified and quantified protein adductions resulting from metabolic activation of DMF and DIOB.The technique we developed was a breakthrough which facilitates mechanistic study of toxicities of furanoid-containing traditional Chinese medicines. |
PDF Full Text Request