Tocopherol Metabolism Using Thermochemolysis:Chemical and Biological Properties of γ-Tocopherol,

γ-Carboxyethyl-hydroxychroman, and Their Quinones

Rakesh Sachdeva,† Beena Thomas,† Xinhe Wang,‡ Jiyan Ma,‡ Kenneth H. Jones,§Patrick G. Hatcher,† and David G. Cornwell*,‡

Departments of Chemistry and Molecular and Cellular Biochemistry, and Division of Anatomy,The Ohio State University, Columbus, Ohio, 43210

Received December 30, 2004

Identification and quantitative estimation of quinone metabolites of γ-tocopherol (γ-T) andits derivative γ-carboxyethyl hydroxychroman (γ-CEHC) are complicated by their functionsas arylating electrophiles. We hypothesize that their biological properties are expressed througharylating quinone electrophile addition (Michael reaction) with thiol nucleophiles in cells andtissues. Glutathione (GSH) reacted with γ-tocopheryl quinone (γ-TQ) to form the hydroquinoneadduct, which was identified by electrospray time-of-flight MS (ESI-TOF-MS). Tetramethyl-ammonium hydroxide (TMAH) thermochemolysis reduced and methylated quinones and cleavedand methylated thioether adducts. These relatively nonpolar derivatives were readily separatedby GC and identified by MS fragmentation patterns. γ-CEHC was synthesized and oxidized toa product identified as the quinone lactone (γ-CEHC-QL). TMAH methylated both γ-CEHC-QL and its GSH adduct without opening the lactone ring, and these products were separatedby GC and identified by MS fragmentation patterns. γ-CEHC-QL reacted with both the cysteinylenzyme papain and fetal bovine serum, and TMAH thermochemolysis showed that each productmixture contained unreacted precursor and thioether adduct. Cytotoxicities of phenolicprecursors, γ-T and γ-CEHC, and their quinones, γ-TQ and γ-CEHC-QL, respectively, werecompared in COS1, NT2, 3T3, and N2a cell lines. Phenolic precursor γ-T had a small effectonly with NT2 and 3T3 cells while γ-CEHC had no effect in any cell line. Arylating quinoneswere highly cytotoxic in all cell lines with γ-TQ showing a significantly greater cytotoxicitythan γ-CEHC-QL. These data are consistent with our arylating electrophile hypothesis as anexplanation for some biological activities of Ts through their quinone metabolites.

Introduction

Quinones represent a highly interesting family ofbiologically active compounds provided in the diet andsynthesized in the cell. Their diverse biological activitiesare explained either by the generation of reactive oxygenspecies (ROS)1 through redox cycling or by the formationof Michael adducts with thiol nucleophiles (1-4). Toco-pheryl quinones (TQs) are all redox cycling compounds,but only the partially methylated quinones, arylatingelectrophiles, can form adducts with nucleophiles (5-12)and are therefore ideally suited for separating redoxcycling properties from adduct forming properties inexplaining biological effects.

Tocopherols (Ts) and tocotrienols, constituents of natu-ral vitamin E, have long been a topic of interest for theirantioxidant properties. However, the chemical and bio-logical actions of Ts, tocotrienols, and their derivatives

in living tissues are much more complex than actions thatmay be attributed to their redox properties alone (13).In 1956, a major metabolite of R-T, the carboxyethyl-hydroxychroman derivative R-CEHC, was identified asa product of phytyl side chain oxidation (14), and in alater study, a similar metabolite, δ-CEHC, was identifiedas the product of phytyl side chain oxidation of δ-T (15).Recently, an analogous phytyl side chain oxidationproduct was identified as a major metabolite of γ-T, andthis compound, 2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hy-droxychroman (γ-CEHC), is the subject of many currentstudies (16-24). γ-CEHC, like its precursor γ-T, showsinteresting and as yet unexplained biological propertieswith some cell lines in tissue culture, including its abilityto function as a natriuretic factor (25, 26) and cyclooxy-genase (COX II) inhibitor (27, 28). R-T and its metaboliteR-CEHC do not have these biological properties (25-28).Similarly, γ-T, γ-CEHC, and δ-T diminish proliferationin some but not all cell lines in tissue culture while R-Tand R-CEHC have either a much smaller effect or noeffect whatsoever (29-35). Chemical structures of Ts andtheir metabolites that will be discussed in this paperappear in Figure 1.

Although both R-T and γ-T are metabolized to theirpara quinones, R-TQ and γ-TQ, respectively, the amountof γ-TQ relative to its precursor γ-T found in living tissuesis much lower than the amounts of R-TQ relative to R-T

* To whom correspondence should be addressed. Tel: 614-292-7411.E-mail: cornwell.1@osu.edu.

† Department of Chemistry.‡ Department of Molecular and Cellular Biochemistry.§ Division of Anatomy.1 Abbreviations: T, tocopherols; TQ, tocopheryl quinones; γ-CEHC,

2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman; QL, quinonelactone; ROS, reactive oxygen species; GSH, glutathione; TMAH,tetramethylammonium hydroxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TIC, total ion chromatogram; SIC,selective ion chromatogram; AIC, analytical ion chromatogram.

1018 Chem. Res. Toxicol. 2005, 18, 1018-1025

10.1021/tx0496441 CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 05/13/2005

found in tissues, even in animals when the diet issupplemented with γ-T (36-39). Also, both R-T and γ-Tare metabolized to their CEHC derivatives, but in thisinstance, the amount of γ-CEHC relative to γ-T is muchlarger than the amount of R-CEHC relative to R-T (16-24). R-CEHC is further oxidized to a quinone lactone(QL), R-CEHC-QL, which has been found in urine (14)and tissues (20, 21). Interestingly, however, γ-CEHC-QLhas not been reported in tissues even though moreγ-CEHC than R-CEHC occurs in tissues (16-24). Thus,R-TQ and R-CEHC-QL are found in tissues while γ-TQis found in lower concentrations relative to its parent γ-Tand γ-CEHC-QL has not been identified. Because ary-lating quinone electrophiles have the tendency to formMichael adducts with thiol nucleophiles, we believe thatadduct formation may explain the low recovery of γ-TQand the absence of γ-CEHC-QL reported for biologicalmaterials. Standard analytical procedures based solelyon HPLC for lipophilic Ts or their quinones (38, 39) willnot separate and quantitate these adducts.

We have proposed that differences in chemical andbiological properties between quinones in the R-T seriesand quinones in the γ-T and δ-T series can be explainedby differences in their ability to serve either as arylating(γ-T and δ-T series) or nonarylating (R-T series) quinoneelectrophiles. A number of observations from our labo-ratories (5-11) including preliminary studies involvingtetramethylammonium hydroxide (TMAH) thermochemol-ysis (12) support this hypothesis. Arylating quinoneelectrophiles are highly cytotoxic agents that stimulateapoptosis resulting in cell death, but nonarylating quino-ne electrophiles such as R-TQ do not show this effect withcell lines and incubation conditions used in our studies.Arylating quinone electrophiles form Michael adductswith the nucleophilic thiol group in glutathione (GSH),and the purified glutathion-S-yl adducts are not cytotoxicin tissue cultures (7, 8). Furthermore, cytotoxicity witharylating electrophiles is enhanced when cultures aretreated with buthionine-[S,R]-sulfoximine to deplete in-tracellular GSH and cytotoxicity is diminished whencultures were treated with N-acetyl cysteine to increaseintracellular thiols.

Biological data summarized above are consistent withbut do not prove unambiguously, the formation of Michaeladducts in tissues and in cell cultures (5-11). Prelimi-nary studies from our laboratory suggested that thestrong methylating base TMAH under thermolytic condi-tions cleaved and methylated the thioether and aromatic

hydroxyl groups. Methylated derivatives were then sepa-rated by GC, analyzed by MS, and identified by charac-teristic fragmentation patterns (12, 40).

In this paper, we present new studies investigating theMichael addition reaction using the arylating quinoneelectrophiles γ-TQ and γ-CEHC-QL with nucleophilicthiol groups in GSH, the cysteinyl protease papain (41),and fetal bovine serum (FBS) (42). We first validatedadduct formation by electrospray time-of-flight MS (ESI-TOF-MS) and identified adducts as their methylatedproducts made amenable to GC-MS by TMAH thermo-chemolysis. We then synthesized γ-CEHC and its QLoxidation product (Scheme 1) and studied their adductformation with thiol nucleophiles by TMAH thermo-chemolysis. Finally, a comparison of the cytotoxicities ofγ-T, γ-CEHC, and their respective quinones was carriedout in four different cell lines from different species.These cell lines have not been used previously in ourstudies on the cytotoxicity of arylating quinone electro-philes (6-11).

Materials and Methods

Materials. γ-T was a generous gift from Tama BiochemicalCo., Ltd. (Tokyo, Japan). Materials from specific vendors wereas follows: FBS (ATCC), RPMI 1640 (Gibco), MEM, DMEM,OPTI MEM (Invitrogen), papain from papaya latex and otherchemicals (Sigma-Aldrich). A mouse fibroblast line, 3T3, amonkey kidney epithelial cell line, COS1, and a mouse neuro-blastoma line, N2a, were purchased from ATCC. A humanteratocarcinoma cell line, NT2, was a gift from Dr. Virginia Lee(University of Pennsylvania, Philadelphia, PA). 1H and 13C NMRexperiments were carried out on a Bruker Avance 400 NMRspectrometer. Chemical shifts are reported in δ values (ppm)downfield from tetramethylsilane (TMS). IR spectra wereacquired as a thin film on a Perkin-Elmer 16PC IR spectropho-tometer.

Synthesis of γ-TQ (9). γ-TQ was synthesized from theparent T, γ-T, by FeCl3 oxidation, purified, and characterizedas previously described (7, 8).

Synthesis of Vinylbutyrolactone (13). To a solution ofethyl levulinate (32 g, 0.2 mol) in THF (20 mL) was addeddropwise a solution of vinylmagnesium bromide (200 mL, 0.22mol) (42) in THF. The reaction mixture was stirred for 30 minafter the complete addition of vinylmagnesium bromide, thentreated with 20% aqueous KHSO4, and extracted with ether.The combined ether extract was washed with water and brine,dried over sodium sulfate, and concentrated in vacuo. Vacuum

Figure 1. Structural representation of Ts and their metabo-lites.

Scheme 1. Synthesis of γ-CEHC and Its QL

Thermochemolysis of Arylating Tocopheryl Quinones Chem. Res. Toxicol., Vol. 18, No. 6, 2005 1019

distillation yielded vinylbutyrolactone as a clear colorless oil.Yield, 15.5 g (50%). IR (cm-1): 1770 (lactone CdO stretch). 1HNMR (400 MHz, CDCl3): δ 5.90 (dd, J ) 17.2, 10.8 Hz, 1H,olefinic H), 5.29 (dd, J ) 17.2, 0.43 Hz, 1H, olefinic H), 5.15(dd, J ) 10.8, 0.43 Hz, 1H, olefinic H), 2.57 (dd, Jgem ) 8.2 Hz,J ) 2.2 Hz, 1H, -CO-HCH), 2.55 (d, Jgem ) 8.2 Hz, 1H, -CO-HCH), 2.20 (m, 1H, CH3-C-HCH-), 2.0 (m, 1H, CH3-C-HCH-), 1.51 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 177.0 (CdO), 140.4 (olefinic), 114.1 (olefinic), 85.8 (C-O), 34.2 (CO-CH2),29.1, 26.7.

Synthesis of γ-CEHC (6). In a modification of a publishedprocedure (43), BF3-etherate (20 mmol) was added dropwise toa solution of 2,4-dimethylhydroquinone (1.38 g, 10 mmol) in drydioxane (20 mL) and maintained at a constant refluxingtemperature of 110 ( 1 °C under a nitrogen atmosphere.Vinylbutyrolactone 13 (1.89 g, 15 mmol) was dissolved in drydioxane (10 mL) and added dropwise to the refluxing solution.When the dropwise addition was complete, the solution wascooled to room temperature, diluted with water, and extractedwith ether. The combined organic layer was washed with brine,dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The residue was purified on a silica gelcolumn using gradient elution (0-35% v/v ethyl acetate inhexane) to obtain γ-CEHC as a viscous oil (yield: 0.8 g, 37%).The oil was triturated with ether and hexane to obtain a whitesolid that was identified as γ-CEHC (yield after crystalliza-tion: 0.4 g, 15%). IR (cm-1): 3500 (broad, COOH), 1710(carboxylic acid CdO stretch). 1H NMR (400 MHz, MeOH): δ6.32 (s, 1H, aromatic H), 2.58-2.52 (m, 2H, CH2), 2.33-2.29(m, 2H, CH2), 2.07 (s, 3H, CH3), 2.05 (s, 3H, CH3), 2.00-1.94(m, 1H), 1.90-1.70 (m, 3H), 1.22 (s, 3H, CH3). 13C NMR (100MHz, MeOH): δ 176.6 (CdO), 147.7, 144.6, 125.1, 122.1, 117.8,111.9, 74.2, 34.7, 31.6, 28.4, 22.7, 22.0, 10.9 (CH3), 10.8 (CH3).MS (m/z): 264 (M+), 151 (base peak).

Synthesis of γ-CEHC-QL (12). An aqueous solution of FeCl3(307 mg, 1.14 mmol) was added to a solution of γ-CEHC (100mg, 0.38 mmol) dissolved in ether (10 mL). The biphasic reactionmixture was stirred at ambient temperature and monitored byTLC (hexane:ethyl acetate, 80/20, v/v) for complete conversionof γ-CEHC to its quinone γ-CEHC-QL. The ether layer wasseparated, and the aqueous layer was extracted with ether. Thecombined ether extracts were washed with water and brine, andconcentrated in vacuo. The residue was purified by elution froma silica gel column with hexane:ethyl acetate (95/5 v/v) andconcentrated to obtain a product as a yellow viscous liquid(yield: 0.032 g, 32%). IR (cm-1): 1770 (lactone CdO stretch),1648 (quinone CdO stretch). 1H NMR (400 MHz, MeOH): δ 6.5(s, 1H, CHdCH-CO), 2.66-2.60 (m, 2H, CH2), 2.55-2.49 (m,2H, CH2), 2.21-2.13 (m, 1H, CH2), 2.05 (m, 1H, CH2, overlapwith CH3), 2.02 (s, 3H, CH3), 2.01 (s, 3H, CH3), 1.85 (dd, 2H,CH2), 1.45 (s, 3H, CH3). 13C NMR (100 MHz, MeOH): δ 187.6(CdC-CdO), 187.6 (CdC-CdO), 176.6 (O-CdO), 148.2, 141.4,141.1, 132.8 (-CHd), 86.2, 39.41, 33.2, 29.2, 25.8 (CH3), 24.4,12.6 (CH3), 12.3 (CH3). MS (m/z): 262 (M+), 99 (base peak).

Synthesis of Thiol Adducts of Arylating Quinones. Theglutathionyl adducts of γ-TQ and γ-CEHC-QL were preparedby taking equimolar solutions of GSH and quinone in a biphasicether/water system and shaking on a Rugged Rotator (Glas-Col, Terre Haute, IN) for 48 h. The organic layer was separatedand washed with water and concentrated (5, 8, 9). The reactionsof γ-CEHC-QL with papain and FBS were carried out in aMeOH:water mixture. The reaction mixtures were stirred onthe Rugged Rotator for 48 h, concentrated to remove methanol,and freeze-dried to remove water.

ESI-TOF-MS of Adducts. ESI-TOF-MS was performed ona Micromass LCT in the direct infusion mode. The instrumentwas operated at a capillary voltage of 3000 V, and the conevoltage was varied between 35 and 70 V to obtain the bestsignal. The desolvation temperature was set at 100 °C usingN2 as the desolvation gas. Adducts were dissolved in appropriatesolvent systems for analyses.

TMAH Thermochemolysis. In a typical procedure (12, 40),about 1 mg of the test sample was placed in a Pyrex glassampule and 200 µL of TMAH (25% in methanol) was added.The mixture was shaken and evaporated to dryness under astream of N2. The tube was flame sealed under vacuum andplaced in an oven maintained at 250 °C for 30 min, cooled,opened, and extracted with ethyl acetate. Combined extractswere passed through a glass wool filter and concentrated to avolume of about 100 µL under a slow stream of N2. Off-linecapillary GC/MS analysis was performed on a Hewlett-Packard6890 GC fitted with a 7683 autosampler using a Zebron-5 (5%phenyl, 95% methylsiloxane) capillary column (30 m × 0.25 mm× 0.25 µM). The GC was interfaced to a Pegasus II time-of-flight mass spectrometer from Leco Corporation (St. Joseph, MI).

Cell Lines and Culture. COS1 and 3T3 cells were main-tained in DMEM supplemented with 10% FBS, 100 U/mLpenicillin, and 100 mg/mL streptomycin. NT2 cells were main-tained in OPTI MEM, supplemented with 5% FBS, penicillin,and streptomycin. N2a cells were maintained in MEM supple-mented with 10% FBS, penicillin, and streptomycin. Cells weregrown as monolayer cultures in a 95% air/5% CO2 water-saturated atmosphere at 37 °C.

Cytotoxicity. COS1, 3T3, NT2, and N2a cells were seededin 24 well plates at a density that yielded 95-100% confluencywhen cells were harvested. After overnight culture, completemedium was refreshed and duplicate cultures treated with0-200 µM γ-T, γ-TQ, γ-CEHC, or γ-CEHC-QL dissolved inethanol (0.5% final volume) and grown for 24 h. Viability (7-9)was then measured by the bioreduction of 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a coloredformazan product (44), which was dissolved in 500 µL of2-propanol, centrifuged, and measured at 570 nm with a MR600 microplate reader. Assays included duplicate readings foreach sample. Ts and their quinones did not reduce MTT in theabsence of viable cells.

Results

TMAH Thermochemolysis of γ-TQ and its GSHAdduct. The pure reactant γ-TQ was subjected to TMAHthermochemolysis, and the peak obtained at 16.2 min onthe GC chromatogram (see the Supporting Information)shows a molecular ion of m/z 462 corresponding to thedimethoxy derivative 15 of the parent quinone. TheMichael addition reaction between quinones and thiolnucleophiles yields hydroquinone adducts, which aresometimes oxidized by excess precursor quinone as isobserved with menadione and GSH (5; Thomas, B., et

Scheme 2. TMAH Thermochemolysis Products ofγ-TQ and Its GSH Adduct

1020 Chem. Res. Toxicol., Vol. 18, No. 6, 2005 Sachdeva et al.

al. Manuscript in preparation) and sometimes remainsin the hydroquinone form as is the case for γ-TQ or δ-TQand GSH (7, 8). In the current study, when γ-TQ reactedwith GSH, the quinone was the limiting reagent in thebiphasic ether/water system used for adduct formationand the hydroquinone was the expected product (Scheme2). The formation of hydroquinone adduct was confirmedby ESI-TOF-MS (Figure 2), which identified the adductfrom the m/z values of 740.7 for (M + 1)+ and 738.2 for(M - H)- in the positive and negative ion modes,respectively.

TMAH thermochemolysis of the product mixture ob-tained from the reaction of γ-TQ with GSH gave a fairlycomplex total ion chromatogram (TIC) (Figure 3) withtwo broad peaks at Rt ) 16.7 and 19.0 min, whichcorrespond to products 15 and 17. Identification of theTMAH thermolysis products 15 and 17 clearly indicatesthe presence of unreacted γ-TQ and the adduct 16 in theproduct mixture. Product 17 is the dimethoxy derivativeof the quinone moiety and the cleavage/methylation ofthe -S-alkyl moiety from precursor adduct 16.

TMAH Thermochemolysis of γ-CEHC, γ-CEHC-QL, and Its GSH Adduct. The products of TMAHthermochemolysis of γ-CEHC and its quinone (Scheme3) both have the same molecular ion m/z 292 but differentGC retention times (Figures 4 and 5). The peak at Rt )11.8 min on GC (Figure 4) was assigned the structure18 while the peak at Rt ) 12.3 min on GC (Figure 5) was

assigned the structure 19. The base peak with m/z 165in the mass spectrum of 18 further suggests a hydroxy-chroman structure for 18. The base peak with m/z 99 inthe mass spectrum of 19 strongly suggests that thelactone group is intact and electron impact leads to thebreakage of the bond between the lactone and the alkylchain forming the stable ion 22 (45).

γ-CEHC-QL was reacted with GSH as described, andthe reaction mixture was analyzed by TMAH thermo-chemolysis. Four peaks were obtained by GC (Figure 6)and identified by MS: lactone 19 at Rt ) 12.6 min withm/z 292 from the precursor, the S-methylated product21 at Rt ) 13.5 min with m/z 338, and two additionalpeaks at Rt ) 12.7 min (m/z 322) and Rt ) 13.6 min (m/z324). Suggested structures from the analyses of frag-mentation patterns for these peaks are presented inTable 1. The formation of the methoxylated product m/z322 has been observed in other instances (40; Thomas,B., et al. Manuscript in preparation) and may be due tonucleophilic attack of hydroxyl ion of TMAH followed bymethylation. We have not been able to assign all of thedaughter ions formed in the mass spectra, but it isimportant to note that all of the compounds fragment ina similar fashion and the daughter ions formed are aresult of loss of exactly the same neutral fragments foreach of the compounds, indicating that they are all partof a structurally related series of compounds. Also,multiple fragmentation modes are observed for eachcompound.

Figure 2. ESI-TOF-MS of the γ-TQ/GSH reaction in a) positiveand b) negative ion modes indicating the formation of ahydroquinone adduct.

Figure 3. TMAH GC/MS TIC of the product mixture of a γ-TQ/GSH reaction.

Scheme 3. TMAH Thermochemolysis Products ofγ-CEHC, γ-CEHC-QL, and Its Thiolated Adducts

Figure 4. TMAH GC/MS TIC of γ-CEHC.

Thermochemolysis of Arylating Tocopheryl Quinones Chem. Res. Toxicol., Vol. 18, No. 6, 2005 1021

TMAH Thermochemolysis of γ-CEHC-QL Adductswith Papain and FBS. γ-CEHC-QL was reacted withpapain as described above, and the reaction mixture wassubjected to TMAH thermochemolysis and analyzed byGC/MS. The TIC for the mixture is very complex with alarge number of products (Figure 7). The selective ionchromatogram (SIC) shows the four characteristic peaksobserved previously in case of the GSH adduct of the QL:Rt ) 12.3 min with m/z 292, Rt ) 12.4 min with m/z 322,Rt ) 13.2 min with m/z 338, Rt ) 13.3 min with m/z 324(Figure 6), the slight difference in retention times be-tween Figures 6 and 7 being due to changing columnlengths resulting from maintenance performed. Thepeaks at Rt ) 12.4 (m/z 322) and 13.3 (m/z 324) mincontain coeluting compounds whose identities could not

be determined. When γ-CEHC-QL was reacted with FBS,a chromatogram similar to that observed with the papainadduct was obtained (Figure 8). Thus, in both reactionmixtures, TMAH thermochemolysis was successfullyemployed to identify both the unreacted arylating quino-ne and its thiol adduct.

Cytotoxicity of Arylating Quinone Electrophilesand Their Parent Phenols. Cytotoxicities of four newcell lines, as indicated by decreasing cell viabilities,showed similarities with and distinct differences betweenphenolic precursors and their arylating quinones (Figure9). γ-T but not γ-CEHC was slightly cytotoxic in both 3T3and NT2 cell lines while neither precursor phenol wascytotoxic in COS1 or N2a cell lines. Unlike their phenolicprecursors, quinones were cytotoxic in the four cell linesbut differed in that cytotoxicity was first apparent at 5µM with γ-TQ and first apparent at 50 µM with γ-CEHC-QL. The data summarized in Figure 9 show that cyto-toxicity is a function of three independent variables,arylating quinone structure, agent concentration, and cellline, with arylating quinone structure overriding all otherindependent variables in establishing cytotoxicity.

Discussion

Previous studies from our group are consistent withthe hypothesis that the biological properties of TQs areprincipally related to their role as adducting agents (5-12). The formation of Michael adducts with the TQs wasdeduced from our understanding of the TMAH thermo-chemolysis reaction, but adducts were not isolated andcharacterized prior to the thermolytic process. In thisstudy, we confirm the formation of Michael adducts byfirst isolating adducts and identifying them using ESI-

Figure 5. TMAH GC/MS TIC of γ-CEHC-QL.

Figure 6. TMAH GC/MS analytical ion chromatogram (AIC)of a product mixture of the γ-CEHC-QL/GSH reaction.

Table 1. Stable Lactone and Other Significant IonsObtained From Common Fragmentation Patterns in

TMAH Thermochemolysis of γ-CEHC-QL and Its ThiolAdducts

Figure 7. TMAH GC/MS TIC of a product mixture of theγ-CEHC-QL/papain reaction; inset, SIC showing four specificpeaks.

Figure 8. TMAH GC/MS TIC of a product mixture of theγ-CEHC-QL/FBS reaction; inset, SIC showing four specificpeaks.

1022 Chem. Res. Toxicol., Vol. 18, No. 6, 2005 Sachdeva et al.

TOF-MS. We then subjected γ-TQ to TMAH thermo-chemolysis and showed the reductive methylation of thequinone moiety to the dimethoxy derivative. The reactionof γ-TQ with GSH was performed in a biphasic ether-water system rather than methanol-water due to theextremely low solubility of γ-T in methanol. Interestingly,the choice of solvent system provided a reducing environ-ment (γ-TQ limiting reactant) for the adduct yielding thehydroquinone 20, which was identified by ESI-TOF-MS(Figure 2). TMAH thermochemolysis of the product

mixture gave the fully O- and S-methylated compound21 (m/z 508) identified in the initial and present studies.

Studies with γ-CEHC and γ-CEHC-QL further dem-onstrate the versatility of the TMAH thermochemolysisreaction in that products are readily separated by GCand identified by retention times and mass spectra.Although both compounds yielded tandem reduction/methylation derivatives, 18 and 19, respectively, withmolecular ion m/z 292, the products have differentretention times on GC and distinctly different MS

Figure 9. Cytotoxicities of arylating quinone electrophiles in COS1, N2a, 3T3, and NT2 cells. Arylating quinones were highlycytotoxic in four different cell cultures with γ-TQ > γ-CEHC-QL. Precursor phenols had limited cytotoxicity with γ-T showing asmall effect only in NT2 and 3T3 cells and γ-CEHC showing no effect in any cell line. Cytotoxicity varied inversely with viability,which was measured by the MTT assay (see the Materials and Methods) and reported as relative viability.

Thermochemolysis of Arylating Tocopheryl Quinones Chem. Res. Toxicol., Vol. 18, No. 6, 2005 1023

fragmentation patterns. The mass spectrum of the TMAHthermochemolysis product of γ-CEHC-QL is dominatedby the daughter ion m/z 99, which is characteristic of suchlactones (44). The TMAH thermochemolysis product 21of the thiol nucleophile adduct 20 is also characterizedby the presence of the lactone fragment, m/z 99 (Table1). Thus, the carboxyethyl hydroxychroman 6, its QL 12,and its thiol nucleophile adduct 20 were all readilyidentified. We contemplated that a similar identificationof the products would be feasible in an in vitro biologicalsystem using the three criteria described above. Indeed,when the reaction of γ-CEHC-QL was carried out withthe cysteinyl enzyme papain or with thiols in FBS, weobtained after TMAH thermochemolysis complex mix-tures as seen in their TICs on GC/MS (Figures 7 and 8).However, SICs showed that the product(s) obtained fromthe addition of -SH group(s) of the proteins to γ-CEHC-QL upon TMAH thermochemolysis resulted in the cleav-age of the S-alkyl bond in the adduct and subsequentmethylation to give 21. This was confirmed by the factthat the products did elute out at the same time andshowed the same fragmentation patterns as the GSHadduct of γ-CEHC-QL.

Previous studies from our laboratories found no cyto-toxicity with either the nonarylating electrophile R-TQor its phenolic precursor R-T, high dose and incubation-time dependent cytotoxicity with the arylating electro-phile γ-TQ, no cytotoxicity with its phenolic precursorγ-T, and moderate cytotoxicity with the arylating quinoneprecursor δ-T (6-12). These studies also found littledifference between the arylating quinones δ-TQ and γ-TQexcept that δ-TQ was less stable in solution (6, 7). Thepresent study allowed us to compare structurally differ-ent T and CEHC precursors with their quinone metabo-lites in four new cell lines, and we found that γ-CEHCshowed no cytotoxicity in any cell line while γ-T showeda small and concentration-dependent cytotoxicity in 3T3and NT2 cell lines. The two quinone electrophiles, γ-TQand γ-CEHC-QL, were both highly cytotoxic but differedgreatly in the concentrations required to induce cytotox-icity, beginning at <5 µM with γ-TQ and >50 µM withγ-CEHC-QL. These data confirm our initial studies (7-10) and recent studies (30-35), which show that cyto-toxicities of γ-T and δ-T are dependent on structure, dose,incubation time, and cell line. Furthermore, formationof Michael adducts with thiols in FBS suggests that thetoxicity of the quinones may be underestimated in cellviability studies where FBS is present in the medium.

The data that we report here are consistent with ourhypothesis (6-12) that Michael adduct formation is theprimary explanation for many of the biological effects ofTs and their metabolites. Quinone cytotoxicity greatlyexceeds the phenolic precursor cytotoxicity that we foundin only two cell lines viz 3T3 and NT2. We suggest thatthe cytotoxicity in 3T3 and NT2 cells occurs because theoxidation of phenolic precursors is greater in these celllines. This hypothesis should be explored by measuringoxidation and adduct formation through TMAH thermo-chemolysis in systems where γ-T and γ-CEHC apparentlyfunction as natriuretic factors, COX II inhibitors, andcytotoxic agents (25-32) and where δ-T functions as asignificant cytotoxic agent (8, 30, 31).

The unexpected differences between the cytotoxicitiesof γ-TQ and γ-CEHC-QL do not appear to be related tothe cell line because γ-TQ cytotoxicities are similar in

the four cell lines and γ-CEHC-QL cytotoxicities aresimilar in the four cell lines. Differences between γ-TQand γ-CEHC-QL could be explained either by differencesin cellular uptake of the two quinones or by differencesin the reactivitiy of the two quinones toward detoxifyingthiols. For example, the effective concentration of theγ-CEHC-QL could be lowered by formation of thioladducts prior to uptake or intracellularly after uptakehas occurred. Further studies are needed to resolve theseissues.

The detoxification hypothesis due to GSH adductformation is consistent with biological data, which showthat diminished expression of metallothionein, a majorsource of intracellular thiols, is associated with enhancedcytotoxicity (46-48). It will be interesting to examinebiological systems deficient in metallothioneins by TMAHthermochemolysis for precursor quinones and their ad-ducts.

Conclusion

We have demonstrated that various arylating quinoneelectrophiles react with nucleophilic thiol groups ofpeptides and proteins in vitro to form Michael adducts.We have used TMAH thermochemolysis to identify GCamenable marker molecules that provide conclusive prooffor the formation of adducts. Thus, we have shown thatESI-TOF-MS and TMAH thermochemolysis become use-ful tools to identify thiolated addition products. Work isin progress for optimizing the assay procedure for quan-tification to identify precursor quinones and their addi-tion products in vivo. Furthermore, we believe that theTMAH thermochemolysis can become an important toolto study the metabolomics of the reactivity of Ts in cellsignaling and cell viability studies.

Acknowledgment. This study was supported in partby the National Science Foundation (CHE-0089147) andthe Ellison Medical Foundation. We thank the CampusChemical Instrumentation Center for providing accessto the ESI-TOF-MS instrumentation.

Supporting Information Available: TIC of TMAH GC/MS analysis of γ-TQ and γ-CEHC-GSH adduct. AIC of TMAHGC/MS analysis of γ-TQ-GSH adduct. This material is availablefree of charge via the Internet at http://pubs.acs.org.

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