~Metabolism.of Xenobiotics ° Suggested Reading: : 7 . + Hodgson E, Goldstein JA. Metabolism of Toxicants Phase | Reaciions and Pharmacogenetics. In: Hodgson.E ‘Smart R:C. INTRODUCTION TG _ BIOGHEMICAL’ TOXICOLOGY 3# ed. Chapter 5. pp.67-113 (2001) + Rose, RL, Hodgson E. Adaptation Jo" “Toxicants, Chemical and Environmental Factors Affecting Metabolism of Xenabioties In: Hodgson EvSmart RC. TaTRODUCTION TO BIOCHEMICAL TOXIGOLOGY ‘rd:ed, Chapter. 8 . pp. 463-198 (2001) “Ipuengerich FP. Cytochromes PASO; Drugs, and Diseases. Molecular Interventions 3; 194-204 (2003), 2 : Optional Reading: . T ghimadaT..Xenobiotié-Metabolizing Enzyrnes involved in Activation arid aeration, of Carcinogenic Polycyesic Aromatic Hycros bons. Drug Metab. Pharmacokinet 24: 257-276 (2008) ‘| | | | ——————1. Xenobiotic Metabolism: Definition Xenobiotic = “foreign ‘Synonyms: BSiotransformation; Drug Metabolism Not: Detoxication Reactions 2, Phase ! and Phase ll Reactioris: Classification Phase | Reactions: Oxidation, Reduction, Hydrolysis. Introduce one of following groups into the intial compound: -OH, -COOH,-NH2-0" “SH Phase Il.Reactions: Conjugal) - Introcuces e highly nycophiic group to prémote exertion Examples: Phase I: Bencene (oxidation}> epoxide intermediate {rearrangement?>;phienot with pKa=t0 (el onlzed at pit7-4) Phase Il: tienot -{gucuronidaton)}> pany! acureide with pitae3.¢ (very watr soluble ‘greater than 99% lonized at pH 7.4 * 3. Phase | Oxidations (at least In part) by microsomal system 4.4. Aromatic Hydroxylation (via epoxide) : Sewertyedtten |X venteoaewet eae) Mat Nighi’ Benzene (aromatic hydroxylation)" ‘epoxide. Then either: i wc of 2 possible reactions: ~ Ce i (a) epoxide -(nonenzymatic redrangeriant}> phenol * oS [aebibyden aval {b) epoxids (epoxide, hytirolyase)> 1,2-dinydro-t,2-diol*‘+ Phase | Oxidations (at least in-part) by microsonal system 3:1. Aromatic Hydroxylation (via epoxide) NIH Shift $ intramolecular migirati telacular mation of substivent group at the site of oxi that riovés fo an aciacentagpdetion, nnn Sou ot este ondation WR Group Is Flot readily ionizable CH3, -OCHS, -phen\i, -hato,-niira), then 40 65% migration of D = IER Group is readily iofizable (-OH, -NH2); then 0-6% migrationaf Groups capable of migration: D or deuterium, 34 oF titlum, chloro, methyt => g a ® DY) as aonide. a Ss grin 3. Phase | Oxidations (at feast In-part) by microsomal system 3.4. Aromat ¢ Hydroxylation (via epoxide) Other substrates subject to'Aromatic Hydroxylation (via epoxidation): Bromobenzene ~> bromobenzene epoxide, . ‘“Chlérobenzene > chisrobenzene epoxide -> para-nyaroxyanline 3.3. Aliphatic Hydroxylation ipropyiebenzene. > pheriysCH2-CH2GH20D) maga hydroxylation. « and. phenyl: CHaCHORSCHS “omega minus 1 hydroxylation and: phenjXGHOPDCH2-CH3. alpha hydroxylation ‘The produits from Omega and Giega-iminus 1 hydroxylations'afe ah oe metabolites. a eens aimed metabolized either by aliphatic hydroxylation or aromatic . if substrate can be hydroxylation, aliphatic hydroxylation s always predominant| 3.4, N-Doalkylation (Oxidation of the Alkyl Group) “R-NH-CHS (oxidatin of the C) > R-NH2'+ HCHO (maihy! group Is oxksized to formalaéhyde) RANH-C(RQ)’ (oxidation ofthe carbon}-> RNH2. + ‘ketone (e.9., acetoie) Also O-Dealkyiaiion and $-Dealkylaién (always oxidation ofthe carbon): 3.5 N-Oxidation (Oxidation of N) ‘Arématio priniary arid segondary amines yield aromatic hydroxylamine Products very. reactive and toxic *‘Aliphatié tertiary amiries yield N-Oxide’ Am wen -(CH3)3N > (GH3)QN=O "1 .” a ; pe Aromatic fertiaty aimiries > in oride 4 pnine "Paap ‘Two pathways leading to N-oxidatidr: . _ _ a 7 i + Microsomal FAD-containing monooxygenase. ES Not inhibited by CO + Cytochrome P450 puigionp sagan Inhibited by CO ‘ ; 23.6. S-Oxidation come P60 are active Both EMO and oytect a sulfone sulfides, sulfoxide (major product) thioetners nes es 5 - é 1" é os si 2 BH Hethioear 4. Non-Microsomal Oxidationis i 4.4. Alcohol Oxidation . Aliphatic‘alconol > Aljehyde > Carborylié Acid 7 ADIt = alcohol dehydrogenase ALDH = aldehyde dehycrogenase Fete ch etaboom: pimary seals > soconday scan >>> teria aleohals (iter nat tnetabolized by alcohol dehycrogensse). ° ; ETHANOL METABOLISMi p Bre cnt | Bininot pSommn atragneseg BOR Low i ihe ay ene 7 same { el ina atte i { «+ [9 alcool acyaripansas hay | Cl, wT i Nethani "| RS foildchye OS (ferme al | (prompt neg. | Spor ie, a " | elas J ch els orn Stan toni = eae shin yom pti a procs sed ForsldteSdropsaeto yl farted ‘tows Tomas dare tooceone “teint hiiion ih orogmss —- ; Pipseistyepetsimmenptn : {ieskonsepyca ies i * Fahslene Gipcot, HOKCU:CH-O (entire deers) nial petablism by Alebo! Dergerateto Oberst, Evert metab 9 Oral Ad = “Foxtety: Denosion of alin amlaeexst ts bles ofRhiney 2nd sal Blo veh ‘ibe bain aa) ° ee “Theiupyt inhibin of cabo hopes Fegan hime it tart 5, Phase i Reactions: , in mammalian tissues where oxygen Favored by anaerobic cdtiditions. Do occti concentrations low, "°° + + oxidized substrate + water soluble vampounds are ‘pettet substrates for gluble substrates”Cytochio; oa -Mechirome' 4s 08 tani : obistie Tretabengreaniy Of smiarfémopfotein, arf eticalyimporiant 7 Se ‘Ne tiumen.getiome ange, es 57480 proline: (i ! ‘Sinohedin meiatondrces . (Gusngerich, 2003), | Rormally finda te boay enOHete cecal (chemin euch as dagen 40 pepe ince z i Atl volved the metabotipm of sols (ncidng te ais) ‘that oxidize fat-soluble vitamine; and ° 7 S invelved in the itetabotisin of fatty atids ad eicosanoids: ~ Substrates are essentially unknown forthe fomaining 16 ofthe 57. 4. Cytochrome P450: Terminal Oxidase in’ Xenobiotic Metabolism ‘Substratas: “"The cytochrome P450'géne superfamily encod tnanjisezyinee] that are unusual in the variety of ciemical reactions catalyzed and the ruinber of substrates (metabolized): ‘The. [sibstrates} include physiologically important substances,such as steroids, eleosanoids, fatty acids iid hydroperoxides, retinoids, and-othor lipid metaboltes, and xenobiotics such ‘as-drugs, alcohols; procarciriogens, antioxidants, organic solvents, anesthetics, dyes, [and] pesticides" . : Allimited number.of the P450 isazyines, e.g. these P4508 vihich rhetabolize steroids, are moderately specific in the nature of the substrates metabolized. However, “many of the 460s, [especially those in the hepatic ondoplasmic reticulum, catalyze a... large number of ‘chemical reactions with ar’ almost unlinited number of biologically accurring and xenobiotic compounds. In the latter category are synthetic environmental chemicals, now estimated at about 250,000, most of which are potential P450 substrates if not inducers or inhibitors of Tthe.individual [P450s]™ ca flchromes P450. . jely on the-sequence similarty among, P4908. individual P4505" and does 8.4. Nomenclature of Cyto ~The nomenclature syste is based $2 pot ingicate the propertios oF function o pasos are named using the 00k oS tamily number, 2 Veter re ating the {product of] he 460 in farnlly 2s omenciatuce'system,[}, the pytacheorns “rrabie numeral designating romatie.numeral repre epee is the eytoonvome P subfamily. sn the current n symioci CYP... followed by an denoting the subfamiy, and another 2 individual gene’. Thus CYPZEt [nol subfamily E. and gene product 1 in t ces numerous ofzymes, of which mare than 150 nave $0 iy from abdul 10% 10 over 90 ‘Sequence identity.” tity 2re | ~The P450 gene superfamily enc far bes characterized. These va the current nomenclature sctieme? ‘ {e] "Those PASO picsie rmal soures with 407 oF sequence ide! (0 vr ee Pre faml, aedosignaled by an Avabie mame ” [b) "Those with greater than 65% Identiy are then included In the,eer"e, subfeimily,, 25 dosignated by a capital letter.” Pet {c} *Thie individual genes (and gene products) Names of'the géhes:are written in talics;¢.g., OYPIAT- 5 signed aumbers" ) are then ariveriy ‘p450 Gene Superfamily, Family, Subfamily, and gene dgsignation (Wobert et al. 1987} Family 4 (polycyelié arom ‘compouind-inducible) on only one subfamily s 5 Al : a2 . Family 3 (steroitndice) Facilly 2 : onlyone gubfarily 2a subfaonly a ‘ = SAL 2a we ‘2B subfamily (phénobartatétinductle) - Family 4 (peroxisome pfalferator-insutble) - on 28h cniyone suber 2¢ subfamily’ ~aily'17 (sterol 17ealpha-hyaroxyiase) 261 a (enone subfamily? foe aly 24 (toroid 27-nysrog!a86) 303. ‘only ono subfamily ea Family 44 (ero. 11-betasiydroniose) oes. ee “only one subfaily - ace. ‘amily 2 (crotesterl side-chain clesv202) oa : (ryone subfamily. aa amity $1(plant P&8D) 1 gene Or a a tprharyate F480) an iy (gene; CIA posterity” . only one subfamily (genes IAT) 201 : 202 26 subfamily (ethanotind 248.2: Cytochrome P450 Intracellular Localization (2) Cellar: microsorns. ‘Techni: The endoplasmic rtieulury (thé intracelusr sto of Cytochy lsrpted into mall aac, The'emal eaca are callested by vacant pallet refered to as mlerosomes. ‘The microsomes donot occur #3 (b) Outer nuclear metnbrane Lot, (0) Micctcntiin nose testes oars orion) tt exsnata o> ise yr Sens 3 rome P450) fagavon 8 @ geno el veiation e © Heap |: aagnono08 Boomer terme jurium teét for mutagenesis : eee oa gone compas or tab reat of te em te taing to OSIS MNS importance of "S-9" ‘Amos Salmonelt typhi Salmonella test for mutago "55.9" contains both microsomes jens 2.3. Tissue Distibition'ef Cytochronie PASO, Liver so. Kdney, hun, Sraltintesine >> heaitymiscle, praln. 0 tamu 2 : Iva Aniline Hydraxyiation, Hexubathital ne N-demethylation, and’NADPH-Dehylroensaé rome Po4S0 (a Varioud Rat Tienes (10) iatetbution of Re styeeexplation, Artnopy MMetieiles arst Argus at Cytoeh ‘aniliga |, Mexobarbital Ariinopyrine."" NADEH- Amount ‘riasue hydroxylation raxplation A-damethylation « detydrogerase of P-180 Ta ete hive {ine * 7} tov'aat 400 #8 3007s. L9DeS Kidney © =" $42 oe Tasos aes owes orig ings sae Cath eg et Swell Miestiie 7 Fae BT aat aka Muscle EL o. her vee Sirele ° ° A veit Oxidation anism of Cytoctirome P-450 Dependent . 9, Mecha ‘eid jee 0 i a 9. Mechanism of Cytochrome P-450 Dependent Oxidation step 4: Association of the sutistrate witti oxidized (ar fémig) eytochrome P450 Fy 2 F450 ———=——"> PA 50-8 (Fe 3) ol e?,e forte @FISO- step 2: The-niéxt Step is @ one-electron tiansfer from NADPH to yield a complex between reduced (or ferrous) cytochrome P450 and the substrate. rie reaction is catalyzed by NADPH-cytochrome P450 reductase, : Jets fie 3 P450-85~__» FS : (Fe>) fos WADPH (Fez) ~~. a _ reduced, (vid evens P450-substrefeNote tate rte substrate complex Oevoceseeets Step 4: Te teriary coniploxun i Tiary complex ‘un the oxygenato lex ungerdoes a secnd bne-lscron rei roenated reduced cytochrome Pas0-qusstate complet ne oon ot P4spzs AMile teder ¢ peeps ‘ : X _S : (fey AD os “he donor at hes ; ; Fnac oetein ni sian mea iADPHsorecone PA wisn: ~ peodoninort | {@)NAOH cyterhvome BS reducace vert “hater apna to be nparantin ritrsomal eet t atyie. 9. Mechanism of Cytochrome P-450 Dependent Oxidation +| stop 5: There'is a! decompiostion.of the oxygenaléd cytochrome P450.vith the Téledse of water , : -Heo a P4S0-S Ae: pq50cS . (Fe? Soe 6 NG . oxene na eharge) Step 6; The final step is the release of the:hyidroxylated substrate and the oxidized | cytochrome’ P450: . ‘The oxidized cytochrome’ car’ then récyéle by binding to another. molecule of ‘substrate: a 8 : PASE So “4 SH (Fe3)-.°8 es Ds oKene. bjdeniy lat Sub siede - i redyafe.yMonooxygenase 18. FAD-Contaning Maniooxygénase, oF Flavin-Containina Mo Cotayee manoonygenaton of nuceophiie. NP, $8 ‘Substrates: tatary and secondary amines tarlary amines ald Ne ‘ ‘Sura, ioetners, hs, tNocarbamatesarganophosphorus compound dations, Many of ese sunstates ae subject o bol P50 meiabolsm and ERO meiabotsin: N.S. P O sesuturations Feap Bicbininal Toereieey 11, Inhibitors of Cytochrome P450 Omeprazole and déeazepam =». ss. 7 7 ” Both substrates are metabolized by crezcis The competition for: the same-P450 decreases the Slearance of. diazepai and prolongs its plasma half life . Occurrence of this méchdnism; many examples ive fe bi itor Is not CYP2D6 metabolizes dextromethorphan : Quinidine‘binds to and inhibits‘CYP2D6 And inhibits the metabolism of dextromethorphan - Occurrence. of this: mechanism:-few or rare = ‘most jon! Metabolism-dependent (probably the best term) Suicide inactivation. °_ - a450;or ant a hat dociaan Peete an ase activity f Cytochrome PASO ig, a Soren intra. One nhs ray anor oorere - (0) Mechanism-based inhibitor deni ls n-based: in 7 8, : ibltor definition, . >, Cf Rectnmcane ‘aholte by cytochrome P50; reac , =i hw cr ee peat mee And thereby inhibits the Cytochrome Paso, (1) sie sosa, rae (02) Metiyieneconyphonyt compounds (example: piperonyl butoxide) pov (3) Metyrépone Initial, short-term effect, inhibiion-- °°. 2 . Longer term exposure, induced syntiesis of cytochrome PégO: - (€) ‘Inhibitors‘of heme synthesis glycine + sisceinyl CoA : _deltarapinctevaline acid aw, | oe PBG (porphobilinogén) 3 ‘ protéporphyrin 1X 7 le heme. Pb+ : . Inhibits ALA dehydratise and ferrdchelatase (= heme synthetase) Cobatt'co2+ Inhibits ALA synthetase, prevents normal association of heme to apocytachrome P450, tase, stimulates héme ox'ygenase (the énzyme Involved in heme Inhibits heme synthe degradation) i (d) Agents causing destruction of cytochrome P450 (eweon) g ; AIA = allylisopropylacetamide THON, (mechanism based Inhibitor, : Ch \ metabolite Is the epoxide that reacts hy | with-the hieme of cytochrome P450) ‘Allyksepropylacetamida: Cadmium ¢cd2+ (P4s9} Carbon tetrachloride (.ccI3 trichloromethyl free radical)Phase II: Conjugation Réactions * themical Parent compounds or their Phase I metabolites that contain fultable chem groups undergo conjugation’ reactions with endogenous substrat MINy excreted. Conjugates. In general, conjugates are polar molecules that are readily cana i ‘ezsamess | pyroscies, | I joc: sulfa ooo x 7 [a co | Aceyizamsieae HO, i Sotihare Bas i = [waste [Orphans | cutee aie feos! | Stamcname goes) igen” Rosa | i [Sees | : ~ Paesiea = hE Taare | Pieler vein oan beoeite “Yocmay PO, oo. Sid aoa —— ies tar eon ce | | elibeine Ltt |g, sectobnio jst | I L : i | ee a 15.A. Glucuronidation: [Saisie raasease — pac besa” Coie fai, F gluctresie “TUDE ~~ | OF id | ueuresasyt °} Coos Humstnse | xB i | Moverysomesy "| sit Pe Le5 Reactions ani Products: | ore 8 na. «ther. eee ee "| (@) O-alucurorides ethers pee nati wecse: |. ether, | (rom phenolicrhydroxyls and Pecans =. soecap: ”. getare” | aliphatic hydroxyls) | Ro eos (0) O-alucurorides esters | (rom carboxglic aélds, oooh) +. | (6) N-glucuronides, _ | @) S-glucuronldes (@) C*plucuronides CG Le Siow BS cere - —— "Qe on Opens pa eee! tod Enzymes?" , ‘ UnP glucurcnosy transferases (UGT) are products of a.multigene superfamily © 1 Rat lived UGTSs are members of two gerie families, UGT1 and UGT2. Each gene family consists of at least 4:distinct' ‘enzymes, “ Humane produée at Toast 6 distin UGT# gone products. . in the rat, UGT1 family'members are inducible by Ah-receptor ligands (TCDD, 3- methylcholanthrene); phenobaftital, and clofibrate Reaction Characteristic’: Glucufonidation: low-affinity, high-capacity catalysis, and provides for efficient substrate conjugation at high substrate concentrations. Reactions and Products: (page-3, Table 6.2) Note upper hydroxylamine (replaces H on OH) and lower hydroxylamine (replaces H on the N).1S. Sulfation wor oT iSomples | Vigne dations" Wanarese cae] Types at Pom } suneation | react ety ony | ___ ts Sitiste—" Phoraadnoa | Silfovanetsase “Nip ees | secu “eopjugation | Phosphosulfare “| (prinoarity: ‘i omni. I * J} eee ea nnnin | 7050) son: ae Enzymes t The sulfotransferases comprise & superfamily of enzymes, z L Major subfamilies of cytosolic sulfotransferases.are reffered “Suttetion oe to as SULT L SULT 2 and SULT 3, Reaction Characteristics: : e Sulfation often provides for high-affinity, tow capacity a H catalysis and provides for efficient substrate, conjugation at ree tow substrate condensations, 7 Sulfate is rate limiting component within cells. Total extent of Ts sulfation‘in increased by including sulfate (or cysteine or ‘methionine, which are degraded into Inorganic sulfate.)" Reactions and Products: Sulfate conjugation involves the trahsfer of stlfonate, (50,7), Not sulfate (SO,-) from PAPS to the xenobiotic. "(The commonly ised terms sulfate and sulfate conjugation ate“ used here, even though sulfonation and sulfonate are more appropriate descriptors.)"; (Klaassen, 2001,,p. 203]: AAG, map ban Conf ‘kote Peay Arma Aine Aisiafemnatens Silo « MPS ems” Sabo}OIes ADP ene Cae . . on, voscn + Gilugoronidation id Suita v-ufinly,high-ebpacity slp, ant provides foreffcfdtaubuine** high ackavata cohecntymions ae ay Sulfliign fen prowktes fr higfroTiny, low exputiy eatalysis and provides or eAficenn A _sbsate cxjugatie at ow subs epee Wh sbsnses wieght snetondto nd win fe. settee ng we ‘stato penton is sus ager cdg gucanaeg Jestuniats nig cncettens or aan. Pexcwag af lean need Sp, Silas. ycine. pe tative sale or abymstred. ,Queen cepnomemapraoanenennemninns Roja nelatenemente ee 7 Tama Re Typeset Saami cat las [sobs a ali : t Phesch, ibes, | Pye | cs) wu re Fi Scedismiies [Beams | Endogenous substrate: ‘s-Adendsyimethionine ae, - <== Réaction and Products: ; JB i | ii ‘Setnylation isa common'but generally minor pathway of Hencbiotic metabolism. Methylation phase tl reactions lh ‘general docrease the wator-slubiy of xenebolcs ~~ Sha macks funcional groups that might ethers be onjugated by ther phase Il enzymes. However, io ‘methyation reactions tat produce quaternary.” Ae we Ci. ‘ammoniumions of methylation that produces positively Charged sulfonium ions increasethe solubilty. eR a et a Te 3 tam pra eS‘reactions'oceurt two types of acetylation ivated conjugating intermediate, One involves a activated conjugating Interment ‘acetyl CoA, and the xenobiotic, The rea: referred to'as-acetylation.. The second type involves thie activation of the xenobiotic to an acyl CoA derivative,.which then feacts with all amino acid to form-an amine acid conjugate. Acetylation typically results in the ‘masking of the amine group with a non-ionizable acetyl moiety. As a result, the acetylated. derivatives are generally less water soluble than the parerit compound. Enzymes: ‘Two cytoplasmic N-acetyltransferases;-NATL-and, NAT2 have been identified'tn-humans..A third enzyme, NAT3; has been Identified In mice, 7 Bs 7 Importance to toxicity: Génetic polymorphisms in N-acetylaticn enzymies'have been identified in humans’and other species, The human population is segregated into slow acétylators and fast acetylators based on the rates of acetylatiol of the drug Isoniazid. The slow-acetylator pheriotype is the result Of polymorphisms in thé NAT2 gene. Siow acetylators are predisposed to toxicity of drugs that are inactivated by acetylation such as isoniazid and dapsone. This enzyme also acetylates aromatic aming dyes to which workers have been exposed industirially such a8 benzidine dyes, 4aminobiphenyl, and o-toluidine. Workers in the a¢rylamine dye industry who are slow acetylators have been shown to nave an Increased risk of bladder ‘cancer. The low activity ofNAT2 In the liver-of slow acetylators may make the aromatic amihes-fnore available for hydloxylation, The resulting hydroxylamines ther’ accumulate in the bladder where they are.acetylaled by. NATL.‘Masa 3 ics 6 rca sia tometli een Sains rin on : wo lassen elayred ty aston te a Sees een cmon sean Macnee | : stn ng meecenceaee:, | SE — heer gation il be covered ih deta “Protec systems" lectures Glutathione Con}16, Mewbotise of Caveinogens soa Compound =p Frosiimate Carvings se | “Ss Ultimate Curemogen buceaseons Motwoolsty sdinyatodiol polyeyelic byshoearbons een 7 sate oo Oo ra on ox oe Ts FIGURE 15° Exsinjles of DNA dormeaing crctuogens 2 (6 {OVO