Document pe3Zm50Y6DKBjaoEnq2Gqvpvd

PLAINTIFF'S EXHIBIT METABOLISM OF TOXIC SUBSTANCES is V. Per',:: J- R. Teevtyn Williams METABOLISM OF TOXIC SUBSTANCES DENNIS V. PARKE Ph.D. F.R.I.C. Dtparfment ofBiochemistry University ofSurrey R. TECWYN WILLIAMS F.R.S. Ph.D. D.Sc. Department ofBiochemistry St Mary's Hospital Medical School, London 1 Two-phase process of metabolism 2 Sites of metabolism 3 Excretion ami the nature of metabolites 4 Two-phase metabolism and toxicity a Phase I reactions and toxicity 0 Phase II reactions and toxicity 3 Factors affecting the metabolism of foreign compounds a Species b Strain: genetic factors c Age 1 Sex e Disease / Environment References The majority of chemicals normally regarded as foreign to the body are metabolized and transformed into other substances, irrespective of whether the foreign chemical is toxic or innocu ous. In the case of toxic compounds, metabolism can play an importict role in reducing or increasing the toxic effects, for a compound may produce signs of poisoning either because it is toxic per se or because it is converted in the body into a toxic substance. The extent to which a toxic substance can exert its deleterious effects may depend therefore on how effective the body is in metabolizing iL If a compound is toxic per se and is transformed in the body to a less toxic or non-toxic metabolite, then the more rapidly it is metabolized the less will be the toxic effect of a given dose. On the other hand, if it is metabolized to a more toxic agent, then the more rapidly it is metabolized the greater will bs its toxic effect. There are, however, some compounds which do notundergo metabolic changein the body and obviously metabolism is not involved in their toxicity. 1. Two-Phase Process of Metabolism The metabolism of a toxic compound is an enzymic process. The metabolism of foreign compounds appears to occur in two phases. The reactions of the first phase include those which may be classified as oxidations, reductions and hydrolyses, whilst those of the second phase, often referred to as conjuga tions. are reactions classified as syntheses. During phase I reactions, the compound may acquire reactive groupings such z> OH, NH:, COOH and SH through which it can undergo synthetic phase II reactions. If the foreign compound already possesses any of these reactive groupings, it could undergo phase II reactions directly without a phase I change. Some foreign compounds, Iioacv.t, rr.a- be metabolized almost entirely by phase I reactions, since* tii~ products, because of Iheir particular chemical or physical properties, do not undergo conjugation. Benzene is an example of a foreign compound winch undergoes a two-phase metabolism. During the fust phase of i: s metabolism, it is oxidized to phenol, and in the second phase i he phenol so produced undergoes a synthetic reaction to form the conjugates, phenyl glucuronide and phenyl hydrogen sulphate. If phenol itself is administered, it can undergo the phase i[ reactiojcdlrt ctly. C.,ajidc is a toxic ior. which aLo c_x is.de. ^ conjugation directly to form the relatively non-toxic thio cyanate. Ethanol is an example of a compound which appears to be metabolized mainly by pliase I reactions. Its mam metabolite is carbon dioxide. Acetaldehyde and acetic acid are intermediates in the oxidation of ethanol and, although acetic acid contains a carboxyl group which is potentially capable of undergoing conjugation, this does not occur-- presumably because acetic acid is rapidly metabolized to carbon dioxide through acctyl-cocnzyme A. 2. Sites of Metabolism The metabolism of toxic substances occurs principally in the liver aod in those organs and tissues which facilitate entry to. or exit from, the body--namely the intestines, skin, kidneys and lungs. Most foreign compounds are normally ingested via the gastrointestinal tract, and alter absorption are transported by the portal vein to the liver, where they tend to accumulate and undergo metabolism. It would thus appear that the enzymes which metabolize foreign compounds are strategically located at sites where they are best able to protect the body from the adverse effects of these compounds, and. may have developed as a defence mechanism against the.naturally occurring toxic chemicals of the diet and environment. In the liver, many foreign compounds.are. metabolized by enzymes present in the endoplasmic reticulum of the hepatic cells. The major class of these enzymes is the microsomal mixed-function oxidases which metabolizeforeign compounds, steroids andlipids by aprocessofhydroxylationoroxygenation, a phase I reaction (Parke, 1968). These enzymesrequire reduced nicotinamide-adenine dinudectide phosphate fNADPH) and molecular oxygen, and contain a hactnoproieyn,_ cytochrome P-4iid. wtuch is ihHiBTfe<mv~arrhon monoxide. The action of these enzymes is to introduce an atom of oxygen_ into tne torcigsmolecule, wiuctfresults mJheKydroxyiauo n c f* aromatic and aOcycGc rings and alkyl sicjicfaains. the retro* f ofalkyl groupslrom ethers and secondary and tertiary amu..?. itMwiMimn of ammes~~and the oxidatioa of sulphides to sulphoxides and sulpboncs. Many other enzyrreT concerned"uTlheTnetzBolism ol foreign compounds are also located in the hepatic endoplasmic reticulum, as, for example, the glucuronyl transferases, enzymes that catalyse.taesynthasU of glucilronic-aadcoajugates, a phase II reaction. 'CyracHTbme P^t50andseveralotKerettzyrrestiia) metabolite toxic compounds are found also in the kidneys. _C}iochrc'" P-450 is also present in the tissues of the gasYrointestical tree:, where the enzyme, benzpyrene hydroxylast^whick detoxiccuv. WBZuWneanfl other orailv ingested poiycyclic hydrocarbons, is also found (Wittenberg, Lcong Jc Strand, 1962). 3're-' plasma also contains a number of phase I enzymes, jcciud.r; the esterases which catalyse the fiydrbrysis of esters, such c> ,*>25' 6 * 921 u 'J I \ V er K ()5rt.) Sr n:^:. ' Mci'ABCUSM OP TOXIC SUL'jI ANCC3 Dennis l'. Turks T. Teeny:: Wiki: -or^; r.rJ lr. the nuiabolismof nr.piv.kn- ' < t:.- -...'.'Ohiia fewrd in the liver erv farther ;o J.I-diiiydroxyruiphthal-.-ne and 1,2-nnphtho- i.-i tivsu.;* of the eye. Tl'.is results in an increase in ^-i-r.dleacIstoretinaicleseneratioaandlensopacityCvan . & I'i.-ii. 1537). amines, sulphonamides and hydrazides are by acetylation of the amino sroup, a phase II . . ,.'licit occurs in the reticulo-cadcuhelial celts of the . r >')!-'-n. lung) and gastrointestinal mucosa (Govier, 1965). x ' !,i.v.h foreign and endogenous, may also undergo iiCi'.ioit, a conjugation reaction catalysed by a non- eiiic A-methyl transferase which is highly active in rabbit- ; ti'sc: (Axelrod, 1962). j'o-ci'jn compounds may also unaergo metabolism in the Mviu'.-: Anal tract by the aaion of the gut microflora, v :cv of 'hee reactions are reductive in nature, as in the `.TiroIr.ion of protocatechuic and hcmoprotocatechuic . (Dacrs & Williams, 1968) and in the reductive scission of " v f0'j<l dyes to toxic amine metabolites. Hydrolyses may .-.j occur: for example, cycasin, the (2-glucoside of methyl- methanol and the toxic constituent of cycad nuts, is - rolysedintoits hepatocarcinogenicaglycone by glucosidases ,:.c intestinal micro-organisms. The microflora of the gut y. -ive rise to the formation of a number of toxic compounds, ..1: as amines, indoles, and aromatic hydrocarbons (Scheline, . /cS). 3. Excretion and the Nature of Metabolites The metabolic transformation of a toxic compound in the vdy can result in the excretion in the urine of (i) the original . -ipoucc; (ii) the products of oxidation, reduction or hydro- -is (phase I products); and (iii) conjugates (phase II products), i a proportion in which these three types of products are ;d depends on several factors, including the chemical arc of the foreign compound, the dose, route of administra- n. species, strain, sex, diet and other environmental factors. W-ruoeHsm, polarity and excretion in the urine. When a - compound is metabolized in the body, it is converted j more-polar compounds. Phase I products of metabolism : -.uaiiy more polar than the parent compound, and phase rrcf ictTare usually aa<uc compounds, such as glucuronic .1 cor.:, rates, ethereal sulphates, anti glvane conjugates. veare -.'.ater solubieanamgniyiQMZKdatthepHof the blood tend to BeTeattilj exuetetTby the Kidney, ine metabolic ' \vss tHus converts liptd^ioluble'foreijinsjrtipoands, which * 2 to be reabsorbed by the kidney tubules, into water-soluble icr prod cets which arc readily excreted; detoxication of such ~:poucds may be a consequence of these processes. Thijjp^y r'.i-a v. by many of the compounds that do not undergo -ooiiTTr^n^ormanons in tne poqv arc olten strongly .-"--nr l- j-htyjniar -- -<cr:::jr. in the bile. Many toxic chemicals arc excreted in ' Me, mostly as glucuronides and other conjugates, and : cr their decomposition products are finally voided in the v Excretion by this route appears to be dependent on '/- c: the compound excreted for, when the molecular i is > biliaryexcrction may be the major route ofelimt- m of compound from the body. Conjugates excreted in - 'rri> be hydrolysed by the action of intestinal microflora *r e original toxic substances or may be decomposed to : new -.oxic substances. Polycyclic aromatic amines are metabolizedihe liver to o-hyclrozy-.mines v ivch arc excreted as glucurrcvd: corjttyr.tcs in the t:!:. 1!;l.oI>s:j of :!:cs: glucuronides in the liberates the free o-hyiroxyamincs which are evreinou-ihe and could be the caiii,. of intesti:;.! tumours. Tlu antibiotic chloramphenicol, which is poitrocenic in rats, is c.xcrctcd in the bile as chloramphenicol jlucuronidc and then undergoes hydrolysis and decomposition in the gut to form amines. These arsines, which appear to be the goitro genic ngents, are subsequently reabsorbed from the gut (Williams, Millbuin & Smith, 1965). Excretion by other routes. Toxic substances and their metabolites may also be excreted from the body by other routes. Many volatile compounds such as aromatic hydro carbons are excreted unchanged in the expired air, ns are volatile metabolites such acarbon dioxide. Nicotine and other basic compounds are secreted in the gastric juice into the stomach, and afiatoxin, pesticides and many drugs may be secreted into milk. 4. Two-Phase Metabolism and Toxicity From the point of view of toxicity, pliase I and phase II products may differ. Products of phase I metabolism may be less toxic, more toxic, or possess a different type of toxicity from their precursors. In contrast, the majority of phase II products are either non-toxic or considerably less toxic than the parent compound, or its phase I metabolites should they be formed. However, foreign compounds may also be concerned in certain synthetic reactions that produce toxic products or toxic consequences. These reactions are known as lethal syn theses and should be distinguished from the phase II or con jugation reactions. Lethal syntheses may come about as the re sult of a foreign compound or its phase I metabolite mimicking a natural intermediary metabolite, as, for example, fluoroacetate which mimics acetate, enters the tricarbo.xyHr-.-f:ri .cycle and finally blocks this pathway of metabolisms. Alternatively, (.euaiii important-macromolccules may become slightly modified by the aaion of a foreign compound, for example, dimcthylnitrosamine is converted by an oxidative process to an active methylating agent which reacts with nucleic acids. These nucleic acids may be important in the development of cancer (Craddock & Magee, 1966). a. Phase I Reactions and Toxicity As a result of phase I reactions, an initially toxic compound may be converted into less toxic substances or an initially nontoxic compoundmay be converted into more-toxic metabolites. A substance may also be convened into several metabolites, some ofwhich may be more toxic and others less toxic than the original compound. Tetraethyltin is not itself biologically active but it shows the same toxic effects towards the central nervous system as does triethyltin, except that the symptoms arc delayed in onset This is apparently because of the hepaiic de-ethylation of tetraethyltin to triethyltin which then exerts its toxic effects upon the animal (Crcmer, 1953). Methanol causes blindpgss ioaun,and this is the. result ofia axiSafcr.loiormaiocEyde which then disturbs oxidative phosphorylation in tKereuna (Mm Jt looper, lSr&l). The insecticide paracluon is inactive until it has beenconvertedinthe tissues into paraoxon which is a powerful cholinesterase inhibitor (Gage, 1953). Again, the neurological effects of tri-o-cresyl phosphate (TOCP) ire da: in part to its oxidation and conversion to a toxic cyclic phosphorus compound (Eto, Casida & Eto, 1562). 257 3 r METABOLISM OT TOXIC SU 'STANCES Demus V. Parke <?. II. Teev.y:i ll'kk.u.is i ..' -O'uL t'niyjO.:!:.-! showsiomL.';-:.iso:n:nnimals u!-. i n i' i'.i"'' by Eat net when it i - i~j-Cicd. In this c. (-. i: .uvc.w- tl-.it the glycoside is hydro!: v.cd, probably by f'l.-.-.'jiJ.iwi ` t^c Eut flora, to m-mdcloniir.le which then b.-.-.As down to give the highly toxic ev.-nide ion. jivinipL'i of the reduction ortoxnTty'and pharmacological ;w'of substances undergoing metabolism by phase 1 rej.'iions arc numerous. The carcinogen benzpyrene is o* d to be a proximate carcinogen and undergoes [ tvT.n by aromatic l.yJroxylation to give 3-hydroxy- ; -_--v :_-nc and 6-hydroxybenzpyrene, which are not cci'vceric. Many barbiturates are similarly detoxicated and ir'ictiwiteJ by oxidation of their side chains, whether these be a'.rb o.ic. as in pentobarbital (5-ethyl-5-(l'-methylbutytybar- l :.:rie acid), or aromatic, as in phenobarbital (5-ethyl-5ry.rn>(barbituric acid). Tyramine, a toxic hypertensive amine pic.e'r.t in cheese, wine and certain other food-stuflfs, is de- uwieaied by the enzyme monoamine oxidase and undergoes oxidative deamination to give p-hydroxyphenylacetic acid. 1 lx erranophosphorus insecticide, malathion, an inhibitor of cr.:>me cholinesterase, is metabolized in mammals by rxe-aiysis of the ethyl ester bonds .to give the relatively noatov; malathion diacid; and the chlorinated hydrocarbon i. '. ,c;;cide dicophane (DDT; l,l,l-trichioro-2,2-<ii-(4-chloro- ~V.; ny P<thane) is similar!y detoxicatedin mammalsbymetabolic d. lwdrochlorination to DDA (2,2-bis-0>-chlorophenyl)acetic uN). b. Phan II Reactions and Toxicity The conjugation of toxic compounds or their metabolites, II reactions, almost invariably results in reduction of : Hydrazine, a chemical used as a rocket propellant, - ;h is probably toxic by virtue of its reducing properties -rl its tendency to form bydrazides with physiologically :. c r.'.ial carbonyl compounds such as pyridoxal, is detoxicated u:c:..lation to diacetylhydrazine. The corrosive poison, ; Vcnel. is detoxicated bv conjugation"with vulnhuric nr ~*~..u.*Llmc acids; and the narcotic drug, morphine, is similarly j. T.tiva-ed by formation of the 3-glucuronide which is A-r.i.u in thtbile. Salicylic acid, an anurheumatic drug that c".:oup!e oxidative phosphorylation, is deactivated by . with glycine to form salicyluric acid. The toxic x. : :rv., chlorobenzene, is detoxicated by a similar conjugation . h t'cuthionc which, after further metabolism, is excreted ji iht acetylcysteine derivative, p-chloropbenylraercapturic * Hmally, the conjugation of cyanide km by the enzyme w.iphate sulphurtraosferase gives the non-toxic thio... c. S. lac tors Affecting the Metabolism of Foreign Compounds Ti.c metabolism of a foreign compound is controlled by and any factor which affects these enzymes also ' 4 metabolism of the compound and consequently its 'ay. Several Actors are now known which affect this * 'u--these being species, strain (genetic factors), age, si r, stress, diet, chronic administration, and the r; `finn of other foreign compounds. The nature of the ' ' r r 1 ,!,ay also be important, since they may play a role in ' Soli .m of toxic compounds ingested orally cr of '.ccretsd in the bile. These organisms may vary to species. Ofeven within a giveospecies, dependant * ' ' t!i.: and environment. a. Sprcirs The safety of drugs and other chemicals used by men ii assessed largely by tests on laboratory animals, but the acti-. :: v and toxicity of these chemicals are not always the m animals as they arc in man. One reason for these c'iTcrerc;, is that the metabolism of a compound in laboratory animals often differs from that in man both qualitatively and quantira- tively. Species differences in the metabolism of foreign com pounds (see Williams, 1967a,-1967b; )Smith,_196S) dsp-erc mainly upon variations'in the enzymes which control phase I and phase II reactions. These variations in enzyme activity can be due to differences in the following: (i) the absolute amount or the specific activity of an enzyme; (ii) the amour:; or nature ofnatural inhibitors ofan enzyme; (iii) the activity of an enzyme reversing the reaction; or (iv) the activities of enzymic reactions competing for the same substrate. Examples ofspecies differences in toxicity which appear to be related to differences in the metabolism of a compound are as follows. The carcinogenic activity of the 2-acctamidofluorene (I; R - H) is believed to be related to the conversion of the compound in vivo to W-hydrcxy-2-acctaraidofluorcne (1; R -- OH). The dog, cat. rabbit, rat, mouse nrf hamster are R 1 susceptible to this carcinogen and they all convert it in part to the W-hydroxy compound. The steppe lemming and guinea-pig are not susceptible and do not A'-hydroxyiate the carcinogen to any appreciableexteat. Man and monkey also Af-hydroxylate the compound, but it is not known whether they develop cancerfrom acetamidofluorene, although by inference from the evidence of other species they should (Weisburger, Grantham, Vanhorn, Steigbigel, Rail St Weisburger, 1964). The development of certain rodesticidss has been based on species differences in toxicity, a case in point being x-naphthylthiourea (ANTU) which is highly toxic to rats but not to domestic animals. A relatedcompound, pheoylthiourea, shows similar species differences in toxicity to ANTU (for rats the LDjo of pheoylthiourea is 3 mg./kg. body-wt.; for rabbits, 40 mg./kg.; for guinea-pigs, 250 mg./Vg.; and for hens, about 1 g./kg.). There is evidence to suggest that in this case, also, toxicity is related to metabolism (B. Bringioe, R. L. Smith and P. T. Williams, unpublished data, 1964; see Williams, 1963). PhenyIthiourea produces several urinary metabolites, include; pbenylcyanamide, and it has been postulated that its toxicity is due to the highly toxic hydrogen sulphide which is liberated in this reaction (II): ^ ^NH-CS-NH, ------- + oPhenylihrourei HS NH-CN Phenyleyantmide II - Other mttibot<ce:. 921001 RowVerK OSH I Sr. r:. mciaijolis OF TOAIC StJK.Sl ANLFS Di'nnii lI'eu <T; 1:. Tccxy. c<> ,'l *.! '.1 I'.sici:y i-. eirxytui: of HjS is . i. f...; iha: L. . to tic l-:.-ti.,l-l-pli'.nyl- t (I.O,-, 2Cj mg./fcg. botiy-ut. in r:.:s) protects rats - -.ins'. a1 ;;hr.! do .c of pb/rnylihinurea, p.-ob.zl'.y by competing for the enzyme releasing H-S. Methylphenyithiourea would picl-jb-.- be metabolii-.u similarly to give metlur;thiol, a product less tc.tic than HjS. I'lienykhiourea is desulphurized : e r.bcur the same extent in the rat, rabbit and guinea-pis, JO-50% of the doss being excreted as sulphate in 24 hours, but i!;e guinea-pig excretes more neutral sulphur compounds than the rat, and excretion of metabolites is more rapid. Moreover, guinea-pigs are less susceptible to H;S than are rets, the approximate intrapedtoneally administered LDS0 being 2-3 mg./kg. body-wt. in rats and about 10 mg./kg. bocly-wt. in gumea-pigs. Species differences in the toxicity of pi.enylthiourea may therefore be related to species differences in the metabolism of the compound and in susceptibility to the toxic metabolite. The insecticide dimethoate is 70 times more toxic to the h?.v* I<a-' <' in ['. -.-'i ad erty: : v.many cclt-rs at.: C.. :ti\e:es ere;., such as tf:c murcic relaxant succir. ti.'.olir,: ('' e.';, 1963). A i'.'.zof this enzyme in susceptible i:..,.ia.:..;i can result in rcJ; ir..:ory failure following the admin ..u:.'.. ..." normal doses of succinyicholice. Wide individual in the detoxication of pheuazonc (aniipyritw), an an,!pyrc:.; metabolized by hydroxylation, has been also c'.'tervrd in ro:s and man (Vcscll & Page, 1963). The toxicity of foreign compounds may also be affected by genetic variations in the activity of enzymes that are con cerned only indirectly in the metabolism of these suostarces. For example, a number ofdrugs and other foreign compounds such as divicine and isoun.mil, pyrimidines that are present ia the broad bean, produced haemolytic anaemia in certain susceptiblcindiviJualswhohavebeenfo'jndtohaveabnormally low levels of glucose-6-phosphate dehydrogenase activity in their red blood cells (Magcr, Glaser, Razin, Izak, Bisn &. Noam, 1965). cockroach and 325 times more toxic to the housefly than to the mouse. Dimethoate is detoxicated in the tissues by enzymic hydrolysis which can occur both at the amide and the thiol ester bonds. Studies with in-vitro preparations from various species have shown that the in-vitro rate of hydrolysis is inversely related to the toxicity of the compound (Uchida, Diuterman & O'Brien, 1964): c. Age Newborn and foetal animals metabolite some toxic sub stances slowly (see Parke, 196S) because of the low activity of many of the respective enzymes which metabolize foreign compounds. The hepatic microsomal phase I enzymes, including cytochromeP-450, together with thephase II enzymes responsible for the formation of glu (CHjO)-;PS-S-CHj-CO-NH-CHj-------- (CH,O)iPSSCH,C0OH------- * (CH,0),PS SH. Dimethoate Dimethoate acid Dimethyl phosphorodithionate curonic acid, glycine and glutathione conjugates are all at a low level or absent at birth. The enzymes which b. Strain: Genetic Factors Differencesin the individual susceptibility to toxic substances, aad idiosyncrasies towards certain drugs and food-stuff's, are often the result of impaired detoxication of these compounds resulting from genetically determined variations in the enzymes concerned. Differences in phase I metabolism have been observed in the rate of metabolism in vitro of a number of foreign compounds indifferent strains ofrats and dogs (Siegert, Alsleben. Liebensctoutz St Remmer. 1964) and rabbits (Cram, catalyse the formation of sulphate znd acetyl conjugates are present, however, at normal levels m some species. In the newborn rat, glucuronyl transferase is already at adult level(Dutton, 1964). From birthomvards these enzymes develop rapidly, reaching adult levels in about 30 days in the rat and in about S weeks in man, aad it is possible that their activity is partly stimulated by foreign chemicals present in the diet. Ia consequence of these enzyme deficiencies the newborn animal is generally more susceptible to the toxic Jcchau <5c l-'outs, 1965). From Table I it may be seen that j- "':ons by os much as a factor of 15 may occur in the rates o' ---tiboliim of toxic compounds in livers of different strains ot rabbit. Furthermore, no correlation is apparent between ihs different metabolic reactions in the various strains. TABLE I. Differences in the rates of metabolism in vitro of toxic compounds in various strains of rabbit (Cram, Juchau A Fours, I9S5) Cottontail rabbits in general appear to metabolize toxic compounds the least effectively, yet have the highest activity among the different strains for the metabolism of 3,4-benzP.-Tcne. Similarly, Dutch rabbits have the highest activity for the metabolism for amphetamine and yet have the lowest Svfcnru* imoMiMI tiHBiuWlwi twg!wijwjM>uwfhtViwmMitginkmkir<h Oocdi Eniah New CoUtttMil * activity for the metabolism of3,4-benzpyrene. Individual differences in the ability to metabolize drugs may bzve considerable bearing on thenpentie efficacy and toxicity. As a consequence of this, many investigations into genetic 4;Terences in the metabolism of toxic substances have been concerned with drugs. Hydrazine derivatives, such as isoniazid Ciomcotinic acid hydratide), hydrallazine and phenelzine, and <-Lc sulphonamide, suiphadimidioe, all of which are metabolic-Mly deactivated by acetylation, exhibit polymorphism of the cr'yme involved, liver acetyltransferase, which divides human t.ejects into "slow" and "fast" deactivators (Evans, Hexobarbitxl Aniline 3.4-Benzpyrene Amidopyrine Amphetamine p-Nitrobenxoic acid Aliphatic hydroxylation Aromatic hydroxylation Aromatic hydroxylation Demethylation Oxidative deamination Reduction 140 3S 25 70 ISO 90 170 250 130 ICO 35 30 220 so w 80 70 120 20 70 50 15 10 50 '-35). As would be expected, the "slow" deactivators exrv'.'eceed greater therapeutic efficacy and greater toxic sider'Teets than the "fust" deactivators. Similar genetic variants The liver supernatant was orepired by homogenizing iivr in 2 vol. M5% KCI solution, followed by centrifuging it 9.0C0 g for 33 min. 91001 RcvvVerK 05H..' '>. 3 METABOLISM or TOXIC SUBSTANCES D:.mis V. Parke d R. Teexyn iVilXanu ;-.ud ."jiNgrt compounds. Trout : ;;uby of the i:w/.z'-j- in re. born rats of a r.umb:r of dregs, Ye.try, I: ' .! C. fir'.eh-.A". (lO'j.'i) showed that i.toit dregs r.'c 2-iO . r toxic to ay-old rut than to adult rats. `I..-.- M.tus >aiii: its lack of d;tn ticr.tirg ep-/ymcs would r to ba i.i a pauicularly vulnerableposition, since many c>i::i;!0'.i.ids Iiava been shown to cross the placental b i -r _ 'id enter the foetus. Moreover, tlw protection of the /a_::;r,al Li. :r may also become impaired, since the level of h.^atic cytochrome P-450 may be reduced in pregnancy and r. ."y of t'- j detoxicating enzymes appearto be inhibited by the high pl'.sma lc\els of progestogens that occur in pregnancy. Recent work has suggested that the human placenta, at least, has a slight ability to detoxicate foreign compounds, and this t- .. .her with tiie somewhat diminished capacity of the n-..rnal liver may afford reasonable protection to the foetus (M. Neale and D. V. Parke, unpublished data, 1969). d. Sex Certain foreign compounds show differential toxicity in male and female animals. Hurst (1953) gives a list of some 30 compounds that have been reported to be-more toxic to one sex t! n to the other. In this list human beings were mentioned or. .. -. vice, suggesting that women may be more susceptible than rr.cn to amidopyrine and benzene. When toxicity has been rc-.'-itcd to metabolism it does appear that rats show sex <f..ccs in toxicity more frequently than other species. T..-- Uf gives the LD0 values of five relatively toxic compounds in r^iii and female animals. Strychnine administered intracr ;_,;y is eguaiiy toxic to male and female rats but, when civ;- intmceritoneally or subcutaneously, it is more toxic to f.mairs. Kato, Chiesara <Sc Vassanelli (1962a, 1962b) have shov-r. that strychnine is more rapidly metabolized to less to vie metabolites by liver microsomal preparations from male rev. than from females and is consequently less toxic to the r.rf.v. Unlike strychnine, parathion is metabolized to a toxic rr. taboiite, namely paraoxon and this occurs more rapidly in C--a.c rat-liver preparations than in male (Davison, 1955). Parcthion is thus less toxic to the male (DtCois, Dou'.I, Salerno &. Coon, 1919). In mice, cats and cogs, there is tta scxtiiff.raucc in th; torticity of parathion and it would appear that the cotv.pt '.K.l i> metabolized equally a.ell by bothrsxci. Paraoxon, lite to;.ic mrtubolit: of pt.rathicn, show; no s.-x difference ir. toxicity in the rat. It is nvctaboMeed by. >tcr?.ses to less toxic products and there is probably no sex dilference ia the activity of these enzymes (III): NO,/~\o?(OSt)1 Entyms > >*KdC7Sinc Parathion NO, O II OP(OEt), Enz/mei art not sx4^ofi4ant Panoxon (toxic) oNO, O il OH -r (:0)^0H. p-Nitrophenol 111 Schradan, like parathion, is metabolized to a toxic substance but, unlike parathion, it is more toxic to male than to female rats, a consequence of its more rapid metabolism in male rat liver (Davison, 1955). Warfarin, the last compound in Table II, is less toxic to male than to female rats, and like strychnine is probably more rapidly metabolized in the male to less toxic metabolites. With these compounds, sex differences in toxicity are found only after puberty. It appears therefore that, as far as the rat is concerned, the metabolism of these compounds and hence their toxicity are under the influence of the sex hormones. Strychnine Parathion Paraaxon Schr.viin TA8LE II. Sex differences in toxicity and metabolism Rowta (mfjitu.>M* r --a) Mmate ^w1* sot ptitrmmTtfoltfitsiirffoccaiftlrliwmoifth Alta tf Owwiiaii 0.S7Rat Intravenous 0S7 2M 1.62 FRat Intraperitoneal 1.81 FRat Subcutaneous 4.01 j> Less toxic M>F 7 FRat IS 6 FRat 10Mouse J- sCat 12-20 JDot Intravenous Orel Intraperitoneal Intraperitoneal Intraperitoneal 4 9-10 3- S 12-20 More toxic F >M F >M 1.2Rat Intraperitoneal 3.5 3JRat Oral 1.2 ^ Leu toxic -- 7 27Ra: Intraperitoneal M More toxic M> F jRat Oral 323 58 F Leu toxic -- &Kato, Chiesara Vassanelli (1962a. 1962b) DuBois, DouU, Salerno & Coon (1949); Davison (1955) OuBois etel. (1949) Davison (I9SS) AHagan Radomskl (1533) -- means tha: no meuureme.-.u have been made *921001 RowVerK Sr. Kid. METABOLISM OI- TONIC SUBSTANCES Dennis V. Parke d P. Teevryn Williams e. Diicase 1; i; wellruw.< that iiun::u> patient.-: and ,t: ..-..aU with liver arc ii'.r.w: than normally susceptible n toxic effects 0f r.inl forriga chemicals, and since the ll.ee is she major of metabolic detoxication this is not surf Patients miwi liscr diseases such as cirrhosis, obstructive jaundice and r.petii'S showed impaired ability to form e.Iucuronide and sulphate conjugates (Muting, 1953) and rabbits with experi- obstructive jaundice produced by ligation of the bile de.t showed impairment of the hepatic phase I enzymes that oxidatively metabolize drugs (McLucn & Fouts, 1961). The amount of hepatic cytochrome P-450 and the activities of t!<: microsomal enzymes of the liver are also diminished as the result of poisoning with the hepatotoxins carbon tetrachloride c,r riimethylnitroramine (Smuck'.sr, Arrhenius & Hulun, 1967). Liver damage may also change the pattern of metabolism, a nd [he carcinogen 2-acctamidofluorene is metabolized to the carcinogenic A-hycroxy-2-acetamido"uorene to a greater cctcot in rats with liver damage than in normal animals (Margreth, Lotlikar, Miller St Miller, 1964). /. Environment Stress. Adverse environmental conditions such as cold and rrise, which give rise to stress in animals, may result in an in crease in the activity of the hepatic microsomal enzymes that is .-.-..-.lilted through the pituitary-adrenal axis. Thehydroxylatioa ofacetaailide by rat-livermicrotomes is almost doubled by previous exposure of the animals to cold stress, and the meta bolism of 2-naph:hylamine to 2-amino-l-naphthol in mice is increased more than 50% by exposure to cold and more than 100% by cold plus noise (Dewhurst, 1963). As 2-amtno-loaphthol is possibly the proximate carcinogen of 2-naphthyls.-ur.e, the stress conditions may be considered, in this case, to r:,'.iit in an increase in toxicity. S'tt'iiion. For a discussion on nutritional aspects see the ; -; rr by McLean St McLean, p. 276 of this Bnlletin. /. :->ition and stimulation of metabolism by foreign chemiC3:;. The metabolism of a toxic compound by 'he hepatic nicrosomal enzymes may be initially inhabited by the presence of competing substrates, but subsequently stimulation of metabolism may result. Pre-treatment with drugs, steroids. Tied additives, toxic chemicals such as pesticides, polycyclic fydrocarbons, polycyclic amines, and normal constituents of ''sod of no nutritional value such as (3-ionooe and safrole (?'.;fce Se Rahman, 1969) can result in a twofold to tenfold se in the activity of hepatic enzymes that metabolize `rnzs and toxic substances (Cooney, 1967). This increase of enzyme activity differs according to the inducing chemical, but could be mediated through an increased rate of synthesis of enzyme protein, a decreased rate of turnover of enzyme, or an activation of enzyme possibly by changes of structure or conformation. The results of this phenomenon are also that chronic administration of a drug or toxic compound may nh;*.nee the activity ofthe enzymesthat catalyseits metabolism. 'Mu-r. metabolism results in a reduction in the toxicity of the compound, chronic administration will reduce the toxicity of a given dose. When metabolism first gives rise to a toxic ^t'ENCtS l.vd.(1462) J. Plrarmoc. exp. Vttr. 1J\ 23 -a, E. & Sim s. F. (1967) Btoehtm. J. 104,394 V H. (19A?) Pi:arrr.nt. Pto. 19. 317 ' M. Cv Magee, P. N. (1966) Bioeh.em. J. 100,724 metabolit!., which is subsequently duUv.ic;- Ly mcutboli.m, the effects o:t OYcr-ail toxicity aic conseqt:.-;:;'. . more complex. Ph.-nyr.tmidol, cn cntinauronr.I blocking ryi-.hibits :>.c mctaboiij.ni of ccum-rio anticoagulant drugs, such as di- coumntol.so uiat the usual therapeutic dose Lcco.r..-sc.vc.iw, t and spontaneous, haemorrhage may occur (Solomon C~ Schrogie, 1966). On the other hand, pre-trtatmen; v. ,':h phcnobarbital increases the metabolism of diaumarol, thus reducing its anticoagulant activity. The da-.Jopmcm c; tolerance to barbiturates and certain other drugs is similarly due to the stimulatory action of a drug on its out. metabolism (Cooney, 1967). The induction ofthese enzymes by p-e-rrer'ment i;h N.-.-.r-i compounds does not necessarily increase the activity of cli enzymes to the same extent. Certain metabolic pathways may be preferentially stimulated. The hepatocarcinogen, 2- acetanudofluorene, is metabolized by hydioxylation to give 5-hydroxy-, 7-hydroxy- and W-hydroxy-2-acetamidofluorene, the last compound being the proximate carcinogen. Con tinuous administration of 2-acetamidofiuorenc to rats results in a preferential induction of the A-hydroxylation. Pre- treatment with acetanilide, or 2-acetamidofiuorer.e plus acetanilide, docs not increase the extent of A'-hydroxylauon, and consequently prevents or delays carcinogenesis by 2- acetamidofluorene (Grantham, Mohan, Yamamoto, V.eis- burger St Weisburger, 1968; Yamamoto, Glass, Frenkel, Weisburger St Weisburger, 1966). 7,I2-Dimethylbcnz[a> anthracene (DMBA), a carcinogen which also produces adrenal necrosis, is hydroxy!ated principally at the methyl groups. The 7-hydroxymethyl derivative is the active adrenocorticoly'ic agent, whereas the 17-hydroxymethyl derivative and ring- hydroxylated raeta .' es are probably true detoxication products. Pre-tree - -.t with 3-methylcholanthrene and certain other polyc . hydrocarbons protects against the DMBA-induced ac necrosis because of a preferential induction of the mi. - mal enzymes which shifts nydroxyla- tion of DMBA fror .- methyl groups to the aromatic ring. Phenobarbiial pre-t . -rent gives similar protection, but acts by a different indi;: sn mechanism involving the further hydroxylation of the /-hydroxymethyl derivative to 7,12-di- (hydroxymethyl)benz(fl]amhraccne and other products (Boy- land St Sims, 1967). Cytochrome P-450, an essential component of the hepatic microsomal enzymes, is inhibited by carbon monoxide, and an atmosphere containing only 2% carbon monoxide inhibits the in-vitro hydroxylation of anDine and oxidative demethyl- ation of amidopyrine in rats by about 50% (Kato, 1966). Inhibition of the hepatic microsomal enzymes is also produced by certain methyleoedioxyphenyl and methylenedioxynaphthyl compounds such as pipcronyl butoxide and sesamex (Metcalf, Fukuto, Wilkinson, Fahmy, El-Azix St Metcalf, 1966). These compounds are used as insecticide synergists since, by inhi biting the insect enzymes which deactivate insecticides, they potentiate toxicity and insecticidal activity. It is pertinent to speculate on the possible effect of the chemicals which row contaminate the environment on the metabolism of toxic compounds in man. Cram, R. L., Juchau. M. R. St Fouts, J. R. (1965) Froe. Sue. exp. Biol. Med. 115,872 Cremer. J. E. (1933) uioe.'iem. J. 68, 635 Dacre, J. C. Si Williams, R. T. (l96S)J.Pherm. Phartnae. 20, 610 **61 92VC j1 w.wVarK OJjfH i METABOLISM OL TOX.C SUBSTANCES Dennis V. Parke &. R. Teewyn V/illiasns I) ivi-'ot, A. N. O'i.A-1TUchvn.J. 61, '03 l;. {19A3j Lxperi.'ntta, 19, 646 n.:;\v;, M. P,, J.. Salerno, P. R. A Coon, 1. M. (1949) J.Pkt.anac. exp. I her. i!5, 79 Dutton, Ci. J. (1954) Proc. Eur. So:. Sir.dy Dm; Taxi:. 4,121 F.'.n, M . C.v Ja, J. E. A Eto, T. 096') i'-hr/jri. Pharmac. 11,337 Evans, L>. A. P. (1965) Ann. A'. 1'. Acad. Sci. 123.173 Olz'. C. (1953) Kiocb.em. J. 51. 426 Cr.i.i, V,'. C. (Ivi5)y. Pharmac. exp. Ther.150, 305 Cranfirjn, P. H., Mohan, L.. Yamamoto, R. S., Weisburser. E. K. i: Weisburger. J. H.095S) Toxic. c.ppl. Pharmac. 13,118 Hagan. E. C.. A Radomsfci, J. L. {1953) J. Am. pharm. Ass. 42,379 H.y: .iger., R. van A Pine, A. (1967) liiochem.J. 102, 842 Hutv., L. V.'. (1953) In: Walpole, A. L. & Spinks, A., cd. The evalua tion c.fdrug toxicity, p. 12. Churchill, London Karo, P*. (1956)/. Biochtm^ Tokyo, 59, 574 Ka.:o. R., Cr.icsara, E. A Vassanelli, P. (1962a) Biochtm. Pharmac. 11,913 Ka:c>, R.. Chitsara, E. A Vassanelii, P. (1962b) Jap. J. Pharmac. 12,26 Kim, M. M. A Cooper, J. R. (1961) Biochtm. Pharmac. 8, 207 McLutn, E. F. A Fouti, J. R_ (1961) /. Pharmac. exp. Ther. 131, 7 Magcr, J,, Glaser, G., Razin. A., Izak, G., Bien, S. A Noam, M. (1965) Biockem. biophys. Bits. Commiin. 20,235 Margreth. A, Lotlikar, P. D,, Miller, E. C. A Miller, J. A. (1964) Cancer Res. 24,920 Metcalf. R. L., Fukuto, T. R, Wilkinson. C.. Fahmy, M. H., El-Aziz, S. A. A Metcalf, E. R. (1966) /. agric. Fd Client. 14, 555 Muting, O. (1963) Germ. med. Mott. 8,193 Parke, f>. V. (1965) The biochemistry of foret;r. <. o-, Pcrgmnon Pres<, London Parke, D. V. A Rahxar., 11. (1969) Biack-tr.. J. 113,;i, Schtline, R. R. (1 CIO) Ada pharmac. tc :. 23,332 Siegert, M,, Alilebe.i, B,, Licoensahutz, W. ARcmme.-.II. " Kaunyr-Schniidebcrgs Arch. exp. Pr.tr.. Ph.srmak. 2 :7, 50j Smith, J. N. (1963) Ada. comp. Physio!. Biochcm. ?, 173 Smurklcr, E. A., Arrhenius, E. A Huliin, T. (1957) iir'c' .- - / 103,55 Solomon. H. M. A Schrogie, J. J. (1956) J. Pharmac. exp. . hrr 151, 660 Uchida, T,, Dautermaa. W. C. A O'lLien, R. D. (1934) J. c Fd Citcm. 12,43 Vcsell, E. S. A Tage. J. G. (1968) Science. K. Y. 101, 72 Watter.barg. L. W, Leons. J. L. A Strand. P. J. 11952) Crrs-- Fxs. 22,112C Weisburger, J. H,, Grantham. P. H., Vanhorn. E., Stcijbi :el N. II., Rail, D. P. A Weisburger, E. K. (1964) Cancer Kes. U, 475 Williams, R. T.(i965) Annis Biol. din. 23,7 Wiliams, R. T. (1967a) Fedn Proc. Fedn Am. Socs exp. Bio! 25. 1029 Williams, R.T. (1967b) In: Wotstenholme, G. E. W. A Porter, R., ed. Drag responses in man, p. 71. (Ciba Foundation Symposium.) Churchill, London Williams, R. T,, Millburn, P. A Smith, R. L. (1965) Ann. .V. Y Acad. Sci. 123,110 Yamamoto, R, S,, Glass, R. M.. FrankeJ, H.H,, Weisburger. E K. A Weisburger, J. li. (1968) Toxic, appi. Pharmac. 13,108 Yeary. R. A., Banish, R. A. A Finkelstein, M.(1966)/. Pediat. 69, 663 059i) ' 262 Br. nr. S "