% # , _ Chem. Res. Toxicol. 1991, 4, 330-334 _.,_ 0 i_jl)d Hydroxyl RadicalMediatedDemethylenationof , (Methylenedioxy)phenylCompounds * \, Yoshito Kumagai,t Lena Y. Lin, Debra A. Schmitz, and Arthur K. Cho* M_/t _ _//A ! Department of Pharmacology, UCLA School o[ Medicine, Center [or the Health Sciences, Los Angeles, California 90024-1735 Received December 10, 1990 The oxidative demethylenation reactions of (methylendioxy)phenyl compounds (MDPs), (methylenedioxy)benzene (MDB), (methylenedioxy)amphetamine (MDA), and (methylenedi- oxy)methamphetamine (MDMA), were evaluated by using two hydroxyl radical generating systems, the autoxidation of ascorbate in the presence of iron-EDTA and the iron-catalyzed Haber-Welss reaction conducted by xanthine/xanthine oxidase with iron-EDTA. Reaction products generated when MDB, MDA, and MDMA were incubated with the ascorbate or xanthine oxidase system were catechol, dihydroxyamphetamine (DHA), and dihydroxymethnmphetemine (DHMA), respectively. The reaction required the presence of either ascorbic acid or xanthine oxidase. Levels of each catechol increased in proportion to ferric ion concentration and were suppressed by desferrio-nmine B methanesuLfonate (desferal). Catalase (CAT) inhibited the oxidation by the ascorbate system whereas superoxide dismutase (SOD) had little effect. The ,_; · _ wasadditinoton oinfitiahydterogd enby phyerodxirodgeen to ptheeroxidereactionalonmie, xtusurgge estimstingulatetdhat thheydrogoxidation,en peroxibutdetheacretsactaiosna _: _ '. precursor of hydroxyl radical. SOD and CAT suppressed the demethylenation reactions in the · xanthine oxidase system. Hydroxyl radical scavenging agents such as ethanol, benzoate, DMSO, _,', _ _ and thiourea effectively inhibited the oxidation by both systems. Urea, which has little effect on hydroxyl radical, was without any effect. Theose .results indicated that hydroxyl radical can ._ Introduction Experimental Procedures "'_ ' Although xenobiotics are generally oxidativeiy metabo- Chemicals. MDB and formaldehyde were obtained from lized by mixed-function oxidases (1), interaction of some Aldrich Chemical Co. Inc. (Milwaukee, WI). As the MDB drugs with hydroxyl radical has also been studied since it rained ,mn!! amounts of catechol, the catechol wasremoved with iSa common byproduct produced in biological systems and 1 N NaOH. MDA and MDMA were obtained from the Research i Technology Branch of The National Institute on Drug AboM ,._ hsextremely reactive (2). For example, it has been found that the dehydrogenation of ethanol to acetaldehyde (3), (Rockville, MD). DHA was obtained from Merck Sharp ,mci Dohme Laboratories (West Point, PA). DHMA wassynthesized demethylation of DMSO 1 to produce formaldehyde (4), accordingto published procedures (I1). Catechol, ferric chloric_ _ tdieonnitroofsatianionline oftoN-nitp-amroisnophenodimetholylam(ine6), and (5gen), heydroxyration ]ao- f dEi,mDTAutas, ase c(SODorbic),accat,id,1, ,xan,e (OthiAne,T), exanthatnholin,ebeoxinzodaseic a,cid,supdeimethytroxide ., ethylene from methionai (7) can proceed by hydroxyl sulfoxide (DMSO), and urea were obtained from SigmaChemicg radical mediated pathways. Co. (St. Louis, MO). Desferal was obtained from CIBA Lib_ ' ' . (Methylenedioxy)amphetamine (MDA) and (methy- ratories (Horshnm, Sussex, U.K.). All other chemicals used were lenodioxy)methamphetamine (MDMA) are serotonergic of the highest grade available. neurotozins that cause a long-term depletion of 5- IaeulMtion. The ascorbatesystemconsisted of MDPs (MDB, hydroxytryptamine (5-HT) content in the brain (8, 9). It 2 mM; MDA and MDMA, 1 mM), 10_M ferric chloride, 20 _M EDTA, and 30 mM pota_ium ph_phate buffer, pH 7.4, in a final has been suggested that the neurotoxic effect is due to volume of 1.0 mL. The reaction was initiated by the addition metabolites (10). Recently, we have found in studies with of ascorbate (final concentration 1 mM). The zanthine oxidaM rat liver microsomes that MDMA is demethylenated to system consisted of the MDPs described above, 0.5mM xanthine, DHMA by cytochrome P-450 and that the catechol me- 10 _M ferric chloride, 20 ,M EDTA, and 30 mM potassium tebulite was further oxidized to a quinone or semiquinone phosphate buffer, pH 7.4, in a final volume of 1.0 mL. The which reacts with sulfhydryl functions (11). The reaction reactionwasinitiated by addition of 0.025unit of xanthine oxida_ has been reported for brain homogenates also (12), but as Both reactionswere carriedout at 37°C for 10rain and termlnAted the levels of cytochrome P-450 in brain are low, the pos- by the addition of 0.5 mi, of 7.5% perchloric acid containing 30 t sibility that demethylenation may occur by another mM thiourea (the ascorbic acid system mediated reaction could not be completely stopped by perchloric or trichloroacetic acid). pathway was considered. The purpose of the present study The reaction mixtures were centrifuged at 13500gfor 5 rain, and was to determine whether hydroxyl radical could promote the supernatants were analyzed by h/gh-performance liquid chemical cleavage of (methylenedioxy)phenyl compounds chromatography-electrochemical detection (HPLC-ECD). (MDPs) using (methylenedioxy)benzene (MDB), MDA, Determination of Metabolites. The catechol products were and MDMA as substrates (Figure 1). Two hydroxyl analytic1 by HPLC-ECD using a Biophase ODS (250 x 4.6 mm radical generating systems were used,the autoxidation of i.d., 5 mn particlesize;Bieanalytical Systems, Inc.,West Lafayette, ucorbate in the presence of iron and EDTA (4, 13) and the-iron-catalyzed Haber-Weiss reaction in which super- oxide is oxidized to oxygen and H202 reduced to hydroxyl (methylened_Abbreviationsioxy)ben: MzI)ene;Ps, (methMDAy,lencl(methylenediozy)ampioxy)phenycompoul n_ds;hetMDB,amiae; radical (12, I4). MDMA,(methylensd/ozy)meth-mphetamine;DHA,dihydrozyamphet_ ' rural.e; DHMA, dihydroxymethamphetamine; dederal, dederrio,_mine B methanmulfonate;SOD,superoxidedismutaoe;CAT,catalaoeDM; SO, t Merck postdoctoral fellow (1990-1991). dimethylsulfoxide;ECD,electrochemicaldetection. 0893-228x/91/2704'0330502.50/0 C 1991 American Chemical Society Oxidationsi MDPs by Hydroxyl Radical Chem. Res. Toxicol., Vol. 4, No. 3, 1991 331 ] o_NH2 O.,_/%./-._,jNHCH3 MDB MDA MDMA _ 8o Figure 1. Structures of (methylenedioxy)benzene (MDB), (methylen.edioxy)amphetamine (MI)A), and (methylenedioxy)- mettlamphetamine (MDMA). A B e C .. ._ 0.45 0.55 0.65 0.75 0.85 *: = Electrode potential (V) Figure 4. Electrochemical response curves of eatechol and a reaction product generated during metabolism of MDB by the I ascorbate system°: (la) authentic catechol; (m)reaction product. Two Hydroxyl Radical Generating Systems" conditions catechol DHA DHMA _, ___.g,[ l__ _' _ _ asTrecorblbatee I. syDstomemethylenation profoducMDBt . formMOatiA.on, andnmoMDMl/10 A minby complete 5.78 4- 0.57 9.11 4- 0.29 11.51 4- 0.48 -ascorbate 0 0 0 Figure 2. High-performance liquid chromatograms of MDB (A), -Fe s*- EDTA 0.53 4- 0.23 0.65 4. 0.14 0.49 4. 0.11 MDA(B), and MDMA (C) oxidation products obtained from the -Fe s - EDTA + 0 0 0 incubationmixture in the ascorbate system. The chromatographic desferal (100 _M) conditions are described under Experimental Procedures. +Fe8+- desferal6 0.34 4- 0.03 0 0 xanthine oxidase system A _ *' _ complete 4.33 4- 0.04 4.48 4-0.17 3.21 4-0.69 '_Go -- '* -xanthine ozidase 0 0 0 (1 f_ -Fe s+ - EDTA 0.06 4- 0.02 0.55 4- 0.06 0.65 4- 0.01 /_ d-Feesfa+era-l E(ID00TA,M+) 0 0 0 +Fes+- desferalb 0 0.27 _- 0.02 0.04 4. 0.01 from con- aEach MDP concentration was 1 mM, and ail incubations were ch Procedures. Each value is the mean 4- SD of two to six determi- nations, bIncubation was carried out in the presence of 100 _M 'p am desferal instead of EDTA. tesis_ loride coelution with authentic compounds. These reaction wiArozid,_use _ ; p_riedoductsout wundereernotthe condidetectedtions deswhecrribed_ theundinecrubaExperimtion ewasntal nethy '_ _ carried out in the absence of each substrate. The elec- emica trochemical responses due to authentic catechol and the Labs Figure 3. High-performance liquid chromatogr,ms of MDB (A), product formed from MDB by the ascorbate system were i weM MDA (B), and MDMA (C) oxidation products obtained from the compared as the oxidation potential of the detector was incubation mixture in the xanthine oxidase system. The chro- varied over the range 0.5-0.8 V. As shown in Figure 4, the MDB matographic conditions are described under Experimental Pro- K)#M cedures, product peak, (expressed as fraction of its height at 0.8 V) fi_ varied in exactly the same way,as the catechol standard. titio_ IN) column and a glassy carbon working electrode (LC-4, Bioa- When the comparison between authentic DHA or DHMA ddaM nalytical Systems, Inc.) set at 0.7 V (vs Ag/AgCI reference and the appropriate product was carried out, similar results thine, electrode). The mobile phase consisted of 0.1 M citrate, pH 3.5, were also obtained (data not shown). The identical Jsium contsining 1 mM octyl sodium sulfate/aeetonitrile/methanol (&Iff chromatographic and electrochemical behavior of the c_me_ v/v) at a flow rate of 0.7 mL/min. To confh'mcatechol production proposed reaction products with authentic standards ina"_ from MDB, 0.1 M citrate buffer, pH 4.0/acetonitrile (4:1v/v) was supports the conclusion that the MDPs are de- also employed as a solvent. The peak height of each metabolite methylenated to the corresponding catechols. insso was momtored by a Hewlett Packard 3390A recording integrator. The role of the components contained in the ascorbate _zould Formaldehyde formation from DMSO was determined by the and xanthine oxidase systems on the oxidation were ex- acid), method of Nash (15). stained, and the results are shown in Table I. No products were formed in the absence of ascorbate or xanthine ox- R®solt$ idase. The addition of desferal (100 _M), an iron chelator, Incubation of MDPs with the two hydroxyl radical markedly inhibited or blocked the oxidation. Although generating systems resulting in the formation of electro- small amounts of catechols were observed in the absence chemically active products.