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The effects of chemicals on hepatic heme biosynthesis
TIPS - March 1981
50 clinical trials and animal experiments in general, becau~ public opinion questions whether the~ are ~orth their effort in financial and ethical terms.
Conclusions
Want to change anytlring ?
valuable drugs, which will be given more bureaucratic regulation, which again hampers innovation. Once caught in this vicious circle, we will no longer be able to escape. Moreover, legislation will crack down on
My essay depicts only half the truth. Only the expected can be planned and written down. What can be planned is alread} anticipated and not innovative. For that reason, I have abstained from giving more than a frame into which the pharmacological sciences might develop, it is up to us, by observation and deliberation, to extrapolate the trends of our pharmacological activities into the, at present, unimaginable future. If a few of us discover this Brave New World and settle there, it will help the whole scientific community. We should all remember an address given by Max Planek to the Deutsche Gcscllschaft dcr Naturforscher und ,~,rzte in ! 910. He pointed to the absolute priority of the search for truth. independent of the results expected or
obtained, and concluded. "leh glctu~.., q,.'~c Worte ~o recht inl S i n c un~t:rcr (Jescllschaft ~u~pr,:chcn ,-t, durfcn, dcr
cs z~ b e ~ m d e r c m Rub~,c anrechne, muss. dass sic sich nient,ds an cine yon vornhercin festgelegte v, is~,:nschaftlich¢ Marscbroute gebunden, ~mdcrn et~aig¢ dahin gehende ¥ersueht. stct ~, rail Entschiedenheit zuri~ckg¢~ :~en hat ('1 presume to express the vlev, t f f , t ) u r St)cietx. ghich has - to its particular h, ,ntmr - ne,,er arranged its pursuit ~Jfscienct as a prefixed schedule, but has al~-ays deei~ edly rejected such demands'). I hope that the ,.catch tt,r truth v, ill not be misdirected b~ m~, e',',a~.. Accordingly ! conclude '~ith ~',tern ~arning against anticipating the .M ~rschroute ol pharmacolog} a,, ,i fixed schedule. man
l)r ,fred l-.rn.~t th;h,'r,nar~tt is Prt~]*.s ~,r ~ll the" R.,t,,I¢: Bu(hh(um-lrt~ntut ,~] P&ttr~nmoh~¢. ('Illler~ll~ ,~ (ile~e'n. ~:1¢ (.; tie ha~ ~orKt'd ,~rl rht' ~..,the'.nt'4r~ and pharmacoh,g~ o] the plasma k,n. ,i ~x llt"rl, tl~lgl"ltl[ and hattertal tottn~. ~tlll t'l~ltOlht~t oil .It'ltr~l~/t(lr-
1
The effects of chemicals on hepatic heme biosynthesis Gerald S. Marks Department o f Pharmacology, Oueen'.~ University Kit gston, Ontario, Cana la K 7 L
To the classically trained pharmacologist the study of the effects of chemicals on heme biosynthesis might appear to be a somewhat esoteric subject. However in recent years with developing concern of pharmacologists in environmental matters. this subject has become of increasing inter"st. A study of the interaction between chemicals and the heme biosynthetic pathway provides an elegant means of probing the detailed effects of drugs at a cellular and molecular level. In this review 1 will attempt to outline some of the interactions between chemicais and the heme biosynthetic pathway which concern pharmacologists. The tbll~owing areas will be considered. (1) The pot-
'L~'6.
phyrias are a group of disorders in which inborn or acquired derangements of enzymes involved in protohemc biosynthesis result in an increased excretion of porphyrins, their precursors, or both, in the excreta. Several novel drug effects ha~e a hereditary basis. An example is the interaction of barbiturates with the hepatic heine biosynthetic pathway, resulting in hepatic porphyria. (2) The levels of cytochromc P~so a hcmoprotein which plays a critical role in oxidative drug metabolism, is elevated by drugs such as phenobarbital. (3) In 1956, a number of Turks consumed wheat treated with the fungicide, hexachlorobenzene, and developed a disorder in hepatic porphyrin metabolism. A variet.~
of pol~ halogemtted ,arotnati¢ h xdrocctrbon., share this action ~ith hex~ehlorobcnzenc. (4) Gristofulvin arid ?,.Y - dtethox~carbon,,I - 1.4 - dihydmpyridine - 2.4.6 - trimcthylp.widinc (DDC) inhibit an ¢n,,',mc of the berne biosynthetic p,ith~,t.~. (5) I.ead interacts ~,,ith ~,,eral cnz}.me~ of heine bk~rynthesis prt~ucing effects +',hich arc useful in as.,,essing biologiczd effect,; of le+ld.
Biosynthetic path~ a~ Protoheme. a cornpk",, of iron ~lrtd protoporph}rin is the pro~,thetic group t+f hemoglobin, m}oglobin, extL_x:hrome',. pcroxidasc, calala~,e and tr.xptophan p.xrrol++~,e. The initiztl step in the biosvnthe,,i+ of protohcme is the condensation ila mitochoudria, by tile enz.~mc ,6-dnlmolevulinic acid (.-\LAJ ',~.nthetJsc. of succin}l-CoA ~ith gl.xcine It* fo;nl AI A (Fig. I ). AI.A rno~e, out of the r!qitt~hondnon into the c.~topl,i,m+ The monop3rrolc. porphobilinogen, fornlcd b} tht ct,ndcnsalion of t~o molecules of ALA is the precursor of the tetrap.xrrok, urot~wph~rinogtn Ill. The latter is decarbox}lated to coproporphyrinogen ii1 which enters the mitochondrion to serve as the precursor of protopt~rphvrinogen IX. Protoporph}rinogen IX is oxidized b~. the t | I-.2~t~f "4~rth.|io~l.llld ~h,fflcdl~.=. PTf.~ [ ~ l
T I P S - M a r c h 1981
6O
enzyrrne protoporphyrinogen oxidase to protol~rphyrin. Insertion of iron into protoporphyrin by the enzyme fcrrochelatase yields pr'~toheme. Tbe only porphyrin that serve~¢as an intermediate in the pathway is protoporphyrin IX. l~he remaining intermediates are the reduced hexahydroporphyrins, viz. porphyrinogens. Unde~ norm;d circumstances the pathway is carefully contrt)lled and porphyrins, other than protoporphyrin IX, are not formed. Howo ever when th~ regulatory process is dis.turbed other i!s~rphyrins accumulate (Fig. I ). An important mechanism for control of the pathway is fi:.~dback repression exerted by protoheme on A I A synthetase at a post-tran~riptional site. Pha~macogenetics= The key to the cla.~sification of por~ phyrias was provided by studies which showed that in some ca~es porphyrins accumulated in the bone marrow while in others porphyrins accumulated in the liver. The disorders of ix~rphyrin metalx~lism have therefore been classified as crythropoietie and hepatic porphyria. The hepatic ~rphyrias have in turn been cla.,,sifted into several sub groups. Acute intermittent porphyria, porphyria variegata and coprop*~rphyria are inherited disorders, and in people with Ihe inherited trait, sm~ll amounts of a variety of drugs can precipitate acute attacks. The genetic defects in the hepatic porphyrizs are represented by diminished activity of (me of the enzymes of the protoheme biosynthetic pathway, Since protoheme is involved in the feedback regulation of ALA-synthetase a block in one of the steps in heme biosynthesis would result in a secondary induction of ALAsynthetase. Thus a demonstrated decrease in uroporphyrinogen l-synthetase activib' in acute intermittent porphyria provides an explanation for the pattern of porphyrin ano porphyrin-precurg~r excretion and for the elevated ALA-synlhetase activity. Similarly a decrease in activity of coproSuccinyl_
CoA]
.--,
porphyrinogen oxidase might explain the findings in coproporphyria. Finally, diminished activity of ferrochelatase or protopol phyfinogen oxidase might explain the finditags in variegate porphyria. The hi:gh sensitivity of patients with inherited hepatic porphyria to drugs such as phenobarbital is explained as follows: inducers of hepatic cytochrome Pu, such as phenobarbital induce increased levels of A L A synthetase in order to provide additional he,he for synthesis of this hemoprorein. Porphyrin accumulation does not occur in normal humans because ALA is converted efficiently to berne, In the presence of an inherited partial block of one of the enzymes of the heine pathway, higher than normal levels of ALA-synthetase will result, leading to the accumulation and excretion of porphyrins and/or porphyrin procurers. When considering drugs fi~r use in the porphyric patient one should therefore select a drug which is unable to induce increased levels of cytochrome !~o Moreover when testing drugs for safe us,.: in patients with hereditary hepatic porph.vria the normal animal is not an appropriate model. Rather pa~ial blocks in the hepatic heine biosynthetic pathway should first be produced by appropriate chemical me.ms in order to imitate the patient with the hereditary disease. PorphyHas due to hexachlorobenzene a~.~d other polycyclic aromatic hydrocarbons:' A variety of polyhalogenated arom.:tic hydrocarbons (PAH) share the potential of hexachh)robenzene for hepatotoxicity and porphyria production in a variety of species. When PAH are administered to several varieties of mammals or birds, a massive over-production of uroporphyrin and heptacarboxylic porphyrin occurs after approximately one week. These porphyfins accumulate in the liver and are excreted in the urine. The excreti,.m of porphyrins is due to the fact that the activity of the enzyme uroporphyrinogen decarboxylase (UROG-D)
8-Ami~olevulinic
-,
falls progressively in rz=t liver as hexachlorobenze ne-porphyria develops. Chronic hepatic porphyrias hegin with accumulation of uro- and hepta-carboxylic acid porphyrins in the liver followed by a gradually increasing excretion of porphyrins in the urine. In humans exposed to polybrominated biphenyls and to 2,3,7,8 tetrachlorodibenzo - p- dioxin there was no significant change in urinary total porphytins. However, the urinary pattern was significantly different from normal. Thus while urinary, total porphyrin values will not indicate exposure to porphyrinogenic PAH the urinary porphyrin pattern may be useful as an indicator of such exposure 4. Porphyrias due to ailylisopropylacetamide (AIA), 3,$ - diethoxycarbonyl 1,4- dihydro 2,4,6 - trimethylpvHdine (DDC) and gHseofulvin B Both AIA and DDC have been shown to increase an induction specific RNA for ALA-synthetase or some other protein that activates this enzyme. In the rat AIA has been shown to destroy the heine moiety of cytochrome P,uo and the allyl group appears to be essential for this process. Both DDC and griseofulvin inhibit the insertion of iron into protoporphyrin catalyzed by the enz3,me ferrochelatase in rat and mouse liver. Thus AIA, DDC and griseofulvin, by dit/erent mechanisms lower the heine level in rat liver. It is therefore thought that two major mechanisms exist for the induction of hepatic ALAsynthetase. (1) A direct action on the nucleus to increase the amount of an inductionspecific RNA for ALA-synthetase. (2) An action to deplete a "regulatory heme pool', (a) by producing a partial block in heme biosynthesis, or (b) by causing destruction of the heme, or (c) by increasing the synthesis of apocy*.ochrome P4~ with increasing utilization of heine for synthesis of cytochrome P4so. Some drugs which induce hepatic ALA-
Porphobilinogen ---, Uroporphyrinogen III
Glycine
J
acid (ALA)
--,
Uroporphyrin III
(PBGt Coproporphyrinogenlll
Protoporphyrinogen III
Fe Protoheme ~-- Pre,toporphyrin IX Fig. I. The Iwme hic~symhelicpathway.
---, Coproporphyrin Ill
T I P S - Man'h 1 ~.81
synthetase do ~a~by the first while othersdo so by the second mechanism. The most efficacious act by both mechanisms. It has been known for many years that a green pigment is formed in rat liver following the administration of an A IA analogue, sedormid. In the rat. phenobarbitalinduced hepatic cytochrome P.a, is selectively destroyed by AIAL In the process of inactivation, protoporphyrin IX, the ironfree prosthetic group of cytochromc P~so is transformed into an abnormal green porphyrin, whit h is covalently bound to AIA as a 1 : 1 porphyrin-AlA adduct. The green porphyrin is therefore formed when cytochrome P~s~ is destroyed by selfcatalyzed addition of AIA to its berne prosthetic group. The destruction of the heine moiety, of P~o might act as a signal for induction of ALA-synthetase. Griseofulvin and DDC, when injected into rodents, depress the level of ferrochelatase~. This is followed by the induction of ALA-synthetase and accumulation of porphyrins. Neither drug exerts any inhibitory effect on ferrochelatase in vitro. The reason for the activity of DDC and griseofulvin in vivo but notin vitro has been clarified. Thus when DDC and griseofulvin are injected into rodents these chemicals lead to the accumulation, apparently from heme breakdown, of green porphyrin-fike pigments which exert inhibitory activity on ferrochelatase~. The inhibitory green pigments closely resemble N-monomethylporphyrins and it is suggested that they bind to the active centre of ferrochelatase. The presence of a substituent at one of the nitrogen atoms would interfere with the normal incorporation of iron and thereby explain its ability to inhibit the enzyme. It is of interest to speculate how the green porphyrin arises and two possibilities have been suggested, viz. either a small fragment of DDC or griseofulvin or an endogenous reactive species of small molecular weight reacts with one of the nitrogen atoms of the porphyrin ring. While the green porphyrin isolated from AIA-treated rodents is a covalent 1:1 adduct of protoporphyrin and AIA s, the green porphyrin isolated from DDCtreated rodents does not contain DDC as a constituent. Moreover, the green porphyrio derived from AIA-treated rodents does not inhibit ferrochelatase. The lack of inhibitory activity of the latter compound has been attributed to the large size of the AIA-derived substituent on one of the nitrogen atoms of the porphyrin, resulting in steric hindrance to binding of the green porphyrin to the active centre of the enzyme.
hi Lead toxicity s
been
l'he t~o en.,'}mcs of the heine bit)s~nthctic palh~a~ most su~:cptiblc to inhibition by lead arc AI.A-deh',drata~ (AI_A-D) and fem)chelatase, l.cad in believed to excel its inhibitory effect b~ interacting with sulthydryl groups of these enzymes. The increa%" in AI.A in the urine of lead-poisoned individuals is thought t,~ be caused by inhibition of ALA-D in bone marrow red cells, liver and kidney. The increased excretion o | urinaD coproporphyrin in lead pmsoned individuak ,night be due Io inhibition of copropo[phyrinogen oxidase, the enzyme which converts copro- to protoporph} rinogen. Inhibition of termchelatase pro~ ide~ an explanation for increased erythroc}te pmtotx~rphyrin in el'Ionic lead poisoning Since circulating erythrocytcs are des old t,~ mitochondria, cJevation of cD'throc~t,: protoporphyrin is thought to be produce.I by the action of lead on bone marrow er~throblasts which contain mitochondria an t synthesize heine. Lead is preferentiali¢ deposited in bone marrow and it~ conce tratton is high at this site. Thus it is not s, prising that bone marro~ erythroblasts a e vulnerable to the toxic effects of lead. ,, n increase in erythrocyte protoporph.vrin a d an inhibition of erythrocyte ALA-D s apparent at relatively Io~ lead concenu lions. These two measurements are ther fore valuable for earl,,' detection of ex D ure to lead.
Ic~,cl',o f fi:rrochchlta,,,c Ihen rills or m~ee. ll is titles important to select a sale drug [ol use m patient,, 'a.ilh hereditary hepatic poephi, fla. Fxpericnce indicates that te~t~ in the chick ¢mbr.~o appear lo ha',c prcdicti,,c ,,:due. If a drug induces I~wph} nn accumulali~ln in chick embr',o li~r cell culture and in the 17-day-olc chick cnlbr~(~ :rod is ,.~t a l~tency similar t~ that of secobarbital tt r, probable that it will have the capaot~ 1~ induce an attack ol porph}ria in patient,, ~',ith the inherited trait.
Differences in response to porphyrin-inducing drugs in different species s A variety of species and test s~stem~, have been used to study the effects of chemicals on heine biosynthesis and the following order of sensitivity re~ealed: chick embryo liver cells in culture -~ 17day-old chick embryo -> chickens - mammals. The remarkable sensitivity of chick embryo liver cells in culture can be attributed, at least :n part, to the unique pharmacokinelics of the system; unlike the intact animal, the cell culture cannot terminate the action of a drug b.~ redistribution to other organs or by excretion. Moreover. sufficient drug may be added to the cell culture to effectively ovcr~hehn the capacity of the liver cells to mctaboli:e and inactivate the drug. Since protoheme is involved in the feedback regulation of Al_A-synlhctase a low level of one of the enzymes of the heme biosynthetic pathway would make the pathway more susceptible to disturbance. It is therefore of interest that the 17-da:old chick embryo and the chicken have
fimnd
Io
ha~.c
considerabl~
!o~cr
Acknowledgement I his qud} was supported b~ the Medical Research ( o u n c d of ('anada. Reading lisl I ldll. (~ | t I lq-%) m tlandhook t l tpcr~.r,'n;a; Pharm,a.logx '..ol 44 I[)t:X|.:ll~l-. t .t;1J Aldr]dgc. \ t % . ed',L pp I ~.:"; %p~ln~v:-~,..'."l.~z. B e d i m ttciddb..'r¢ and N~:~ "l ork 2 M a t ~ c l l . D J ,;nd %lc'.~.'r. L A i I~%'1 in ~landbook o f at.;per'mcntal Pharmat,J,,~'~ X,~I 44 [DcMdIIcI'~, F. aPd Alrldgc ~, % Ld'..I pp "e4 25-!,. ~,prin+~..r %urI,LL'. Bt:rhrl. Hc+ddb,~r,= .rod Nc~ Y : q k Elder. ( L It. ( t ~ ' S ) m HLmdho.k ,,t ~ ;p¢,rI,~u',u,,: Pharmacol(,g) 'k'ol 44 t l k M a t t c ~ - . F .rod A l d r i d g c . '~V. N . cd~L pp I r," 2HII. %prm~c~ t ' c r l a g . Bcrtin. HcMt lbt-rg and Nov. '~,,rk 4 I}o-~. %!. (l~"~'J in (ht'rrltca[ ['t,rpl:~,rlt~ t'r I,l,i': (Stnk. J J f X,~, X . : m d K c c m a n / | l . c d - } pp I I - 2 ~ [ I,.o.vzr N o n h - H o l l . m d . ~.m~u:rd.m~ 5 D¢'~,tdllCl~. I- I 1'4".~) in Hatldt)¢,o& ,,tl tpr'rt~rzt'rll,i,~ I)harmdtoJt~ ~ol -I,A (l'k'%'i,t!lc~, l- .lilt] -Mdridgc. I,~, N . cds). pp. 1 2 o . 1 5 5 . Spn~wc; \ crlag. Bcrhn. Hc~del~,r~ .rod Nt..v. "fork h Orris" l ) c M,mtcl!an~,. P R . ".1~,,,. B \ and " l , , q
-
132 - l ~ De \ l , l l l C l s + |" a n d 1, ~iI~% \
"\ I | "451 ~/ell t,t /l¢',r/ j
8 ",a~s.t. S ( I t i " h ) m Handbo,,k o t t; tt, l~ "lr~tg.i! Pllartna, M,,qx ~,o1 44 t I ' k " q a t t c l , k .rod & l d n d g e . ~t N . cd'.] pp ~,;3 3 " 1 . Sprmgcr ~, c d a g . Bcrhn. H c i d c l ~ ' r ~ .rod Nee, ~ ork '4 Mark... ( i S I lq-'SI m H,mdhook or t tpt'rlztl¢ ¢11,a/ Pht~rtrzdt I'h'k'~ ~. ol 44 I [ ) c M . I t t c l . t and -\ktlldgt'. ~. N . cd~.), p p 2 1 t l - 2 3 " . hpll~lgi'r ~,crlag. g c r h n , tlc~dclbcr o ,rod Nov, ~ o~k
t ;er,dd S. ihtrk.~ obtained D. Phd. m orgumt the,m~lrv at OLk~rd University tn 1~54. A]ier re~e~rch and Lit tldt't?llt" clppomtrtlenf.~ an Sf ~uth .4tilt a, lilt" (-'.-g .4 . lht" U K and ('ana,hJ. he bee,row Pr,,fe,w,r and He~;tt ,,f the Dep(~ranem o1 Phatvnatx)logy ;at QueetF~ ( ni~er~IlV In IOO0.
50 clinical trials and animal experiments in general, becau~ public opinion questions whether the~ are ~orth their effort in financial and ethical terms.
Conclusions
Want to change anytlring ?
valuable drugs, which will be given more bureaucratic regulation, which again hampers innovation. Once caught in this vicious circle, we will no longer be able to escape. Moreover, legislation will crack down on
My essay depicts only half the truth. Only the expected can be planned and written down. What can be planned is alread} anticipated and not innovative. For that reason, I have abstained from giving more than a frame into which the pharmacological sciences might develop, it is up to us, by observation and deliberation, to extrapolate the trends of our pharmacological activities into the, at present, unimaginable future. If a few of us discover this Brave New World and settle there, it will help the whole scientific community. We should all remember an address given by Max Planek to the Deutsche Gcscllschaft dcr Naturforscher und ,~,rzte in ! 910. He pointed to the absolute priority of the search for truth. independent of the results expected or
obtained, and concluded. "leh glctu~.., q,.'~c Worte ~o recht inl S i n c un~t:rcr (Jescllschaft ~u~pr,:chcn ,-t, durfcn, dcr
cs z~ b e ~ m d e r c m Rub~,c anrechne, muss. dass sic sich nient,ds an cine yon vornhercin festgelegte v, is~,:nschaftlich¢ Marscbroute gebunden, ~mdcrn et~aig¢ dahin gehende ¥ersueht. stct ~, rail Entschiedenheit zuri~ckg¢~ :~en hat ('1 presume to express the vlev, t f f , t ) u r St)cietx. ghich has - to its particular h, ,ntmr - ne,,er arranged its pursuit ~Jfscienct as a prefixed schedule, but has al~-ays deei~ edly rejected such demands'). I hope that the ,.catch tt,r truth v, ill not be misdirected b~ m~, e',',a~.. Accordingly ! conclude '~ith ~',tern ~arning against anticipating the .M ~rschroute ol pharmacolog} a,, ,i fixed schedule. man
l)r ,fred l-.rn.~t th;h,'r,nar~tt is Prt~]*.s ~,r ~ll the" R.,t,,I¢: Bu(hh(um-lrt~ntut ,~] P&ttr~nmoh~¢. ('Illler~ll~ ,~ (ile~e'n. ~:1¢ (.; tie ha~ ~orKt'd ,~rl rht' ~..,the'.nt'4r~ and pharmacoh,g~ o] the plasma k,n. ,i ~x llt"rl, tl~lgl"ltl[ and hattertal tottn~. ~tlll t'l~ltOlht~t oil .It'ltr~l~/t(lr-
1
The effects of chemicals on hepatic heme biosynthesis Gerald S. Marks Department o f Pharmacology, Oueen'.~ University Kit gston, Ontario, Cana la K 7 L
To the classically trained pharmacologist the study of the effects of chemicals on heme biosynthesis might appear to be a somewhat esoteric subject. However in recent years with developing concern of pharmacologists in environmental matters. this subject has become of increasing inter"st. A study of the interaction between chemicals and the heme biosynthetic pathway provides an elegant means of probing the detailed effects of drugs at a cellular and molecular level. In this review 1 will attempt to outline some of the interactions between chemicais and the heme biosynthetic pathway which concern pharmacologists. The tbll~owing areas will be considered. (1) The pot-
'L~'6.
phyrias are a group of disorders in which inborn or acquired derangements of enzymes involved in protohemc biosynthesis result in an increased excretion of porphyrins, their precursors, or both, in the excreta. Several novel drug effects ha~e a hereditary basis. An example is the interaction of barbiturates with the hepatic heine biosynthetic pathway, resulting in hepatic porphyria. (2) The levels of cytochromc P~so a hcmoprotein which plays a critical role in oxidative drug metabolism, is elevated by drugs such as phenobarbital. (3) In 1956, a number of Turks consumed wheat treated with the fungicide, hexachlorobenzene, and developed a disorder in hepatic porphyrin metabolism. A variet.~
of pol~ halogemtted ,arotnati¢ h xdrocctrbon., share this action ~ith hex~ehlorobcnzenc. (4) Gristofulvin arid ?,.Y - dtethox~carbon,,I - 1.4 - dihydmpyridine - 2.4.6 - trimcthylp.widinc (DDC) inhibit an ¢n,,',mc of the berne biosynthetic p,ith~,t.~. (5) I.ead interacts ~,,ith ~,,eral cnz}.me~ of heine bk~rynthesis prt~ucing effects +',hich arc useful in as.,,essing biologiczd effect,; of le+ld.
Biosynthetic path~ a~ Protoheme. a cornpk",, of iron ~lrtd protoporph}rin is the pro~,thetic group t+f hemoglobin, m}oglobin, extL_x:hrome',. pcroxidasc, calala~,e and tr.xptophan p.xrrol++~,e. The initiztl step in the biosvnthe,,i+ of protohcme is the condensation ila mitochoudria, by tile enz.~mc ,6-dnlmolevulinic acid (.-\LAJ ',~.nthetJsc. of succin}l-CoA ~ith gl.xcine It* fo;nl AI A (Fig. I ). AI.A rno~e, out of the r!qitt~hondnon into the c.~topl,i,m+ The monop3rrolc. porphobilinogen, fornlcd b} tht ct,ndcnsalion of t~o molecules of ALA is the precursor of the tetrap.xrrok, urot~wph~rinogtn Ill. The latter is decarbox}lated to coproporphyrinogen ii1 which enters the mitochondrion to serve as the precursor of protopt~rphvrinogen IX. Protoporph}rinogen IX is oxidized b~. the t | I-.2~t~f "4~rth.|io~l.llld ~h,fflcdl~.=. PTf.~ [ ~ l
T I P S - M a r c h 1981
6O
enzyrrne protoporphyrinogen oxidase to protol~rphyrin. Insertion of iron into protoporphyrin by the enzyme fcrrochelatase yields pr'~toheme. Tbe only porphyrin that serve~¢as an intermediate in the pathway is protoporphyrin IX. l~he remaining intermediates are the reduced hexahydroporphyrins, viz. porphyrinogens. Unde~ norm;d circumstances the pathway is carefully contrt)lled and porphyrins, other than protoporphyrin IX, are not formed. Howo ever when th~ regulatory process is dis.turbed other i!s~rphyrins accumulate (Fig. I ). An important mechanism for control of the pathway is fi:.~dback repression exerted by protoheme on A I A synthetase at a post-tran~riptional site. Pha~macogenetics= The key to the cla.~sification of por~ phyrias was provided by studies which showed that in some ca~es porphyrins accumulated in the bone marrow while in others porphyrins accumulated in the liver. The disorders of ix~rphyrin metalx~lism have therefore been classified as crythropoietie and hepatic porphyria. The hepatic ~rphyrias have in turn been cla.,,sifted into several sub groups. Acute intermittent porphyria, porphyria variegata and coprop*~rphyria are inherited disorders, and in people with Ihe inherited trait, sm~ll amounts of a variety of drugs can precipitate acute attacks. The genetic defects in the hepatic porphyrizs are represented by diminished activity of (me of the enzymes of the protoheme biosynthetic pathway, Since protoheme is involved in the feedback regulation of ALA-synthetase a block in one of the steps in heme biosynthesis would result in a secondary induction of ALAsynthetase. Thus a demonstrated decrease in uroporphyrinogen l-synthetase activib' in acute intermittent porphyria provides an explanation for the pattern of porphyrin ano porphyrin-precurg~r excretion and for the elevated ALA-synlhetase activity. Similarly a decrease in activity of coproSuccinyl_
CoA]
.--,
porphyrinogen oxidase might explain the findings in coproporphyria. Finally, diminished activity of ferrochelatase or protopol phyfinogen oxidase might explain the finditags in variegate porphyria. The hi:gh sensitivity of patients with inherited hepatic porphyria to drugs such as phenobarbital is explained as follows: inducers of hepatic cytochrome Pu, such as phenobarbital induce increased levels of A L A synthetase in order to provide additional he,he for synthesis of this hemoprorein. Porphyrin accumulation does not occur in normal humans because ALA is converted efficiently to berne, In the presence of an inherited partial block of one of the enzymes of the heine pathway, higher than normal levels of ALA-synthetase will result, leading to the accumulation and excretion of porphyrins and/or porphyrin procurers. When considering drugs fi~r use in the porphyric patient one should therefore select a drug which is unable to induce increased levels of cytochrome !~o Moreover when testing drugs for safe us,.: in patients with hereditary hepatic porph.vria the normal animal is not an appropriate model. Rather pa~ial blocks in the hepatic heine biosynthetic pathway should first be produced by appropriate chemical me.ms in order to imitate the patient with the hereditary disease. PorphyHas due to hexachlorobenzene a~.~d other polycyclic aromatic hydrocarbons:' A variety of polyhalogenated arom.:tic hydrocarbons (PAH) share the potential of hexachh)robenzene for hepatotoxicity and porphyria production in a variety of species. When PAH are administered to several varieties of mammals or birds, a massive over-production of uroporphyrin and heptacarboxylic porphyrin occurs after approximately one week. These porphyfins accumulate in the liver and are excreted in the urine. The excreti,.m of porphyrins is due to the fact that the activity of the enzyme uroporphyrinogen decarboxylase (UROG-D)
8-Ami~olevulinic
-,
falls progressively in rz=t liver as hexachlorobenze ne-porphyria develops. Chronic hepatic porphyrias hegin with accumulation of uro- and hepta-carboxylic acid porphyrins in the liver followed by a gradually increasing excretion of porphyrins in the urine. In humans exposed to polybrominated biphenyls and to 2,3,7,8 tetrachlorodibenzo - p- dioxin there was no significant change in urinary total porphytins. However, the urinary pattern was significantly different from normal. Thus while urinary, total porphyrin values will not indicate exposure to porphyrinogenic PAH the urinary porphyrin pattern may be useful as an indicator of such exposure 4. Porphyrias due to ailylisopropylacetamide (AIA), 3,$ - diethoxycarbonyl 1,4- dihydro 2,4,6 - trimethylpvHdine (DDC) and gHseofulvin B Both AIA and DDC have been shown to increase an induction specific RNA for ALA-synthetase or some other protein that activates this enzyme. In the rat AIA has been shown to destroy the heine moiety of cytochrome P,uo and the allyl group appears to be essential for this process. Both DDC and griseofulvin inhibit the insertion of iron into protoporphyrin catalyzed by the enz3,me ferrochelatase in rat and mouse liver. Thus AIA, DDC and griseofulvin, by dit/erent mechanisms lower the heine level in rat liver. It is therefore thought that two major mechanisms exist for the induction of hepatic ALAsynthetase. (1) A direct action on the nucleus to increase the amount of an inductionspecific RNA for ALA-synthetase. (2) An action to deplete a "regulatory heme pool', (a) by producing a partial block in heme biosynthesis, or (b) by causing destruction of the heme, or (c) by increasing the synthesis of apocy*.ochrome P4~ with increasing utilization of heine for synthesis of cytochrome P4so. Some drugs which induce hepatic ALA-
Porphobilinogen ---, Uroporphyrinogen III
Glycine
J
acid (ALA)
--,
Uroporphyrin III
(PBGt Coproporphyrinogenlll
Protoporphyrinogen III
Fe Protoheme ~-- Pre,toporphyrin IX Fig. I. The Iwme hic~symhelicpathway.
---, Coproporphyrin Ill
T I P S - Man'h 1 ~.81
synthetase do ~a~by the first while othersdo so by the second mechanism. The most efficacious act by both mechanisms. It has been known for many years that a green pigment is formed in rat liver following the administration of an A IA analogue, sedormid. In the rat. phenobarbitalinduced hepatic cytochrome P.a, is selectively destroyed by AIAL In the process of inactivation, protoporphyrin IX, the ironfree prosthetic group of cytochromc P~so is transformed into an abnormal green porphyrin, whit h is covalently bound to AIA as a 1 : 1 porphyrin-AlA adduct. The green porphyrin is therefore formed when cytochrome P~s~ is destroyed by selfcatalyzed addition of AIA to its berne prosthetic group. The destruction of the heine moiety, of P~o might act as a signal for induction of ALA-synthetase. Griseofulvin and DDC, when injected into rodents, depress the level of ferrochelatase~. This is followed by the induction of ALA-synthetase and accumulation of porphyrins. Neither drug exerts any inhibitory effect on ferrochelatase in vitro. The reason for the activity of DDC and griseofulvin in vivo but notin vitro has been clarified. Thus when DDC and griseofulvin are injected into rodents these chemicals lead to the accumulation, apparently from heme breakdown, of green porphyrin-fike pigments which exert inhibitory activity on ferrochelatase~. The inhibitory green pigments closely resemble N-monomethylporphyrins and it is suggested that they bind to the active centre of ferrochelatase. The presence of a substituent at one of the nitrogen atoms would interfere with the normal incorporation of iron and thereby explain its ability to inhibit the enzyme. It is of interest to speculate how the green porphyrin arises and two possibilities have been suggested, viz. either a small fragment of DDC or griseofulvin or an endogenous reactive species of small molecular weight reacts with one of the nitrogen atoms of the porphyrin ring. While the green porphyrin isolated from AIA-treated rodents is a covalent 1:1 adduct of protoporphyrin and AIA s, the green porphyrin isolated from DDCtreated rodents does not contain DDC as a constituent. Moreover, the green porphyrio derived from AIA-treated rodents does not inhibit ferrochelatase. The lack of inhibitory activity of the latter compound has been attributed to the large size of the AIA-derived substituent on one of the nitrogen atoms of the porphyrin, resulting in steric hindrance to binding of the green porphyrin to the active centre of the enzyme.
hi Lead toxicity s
been
l'he t~o en.,'}mcs of the heine bit)s~nthctic palh~a~ most su~:cptiblc to inhibition by lead arc AI.A-deh',drata~ (AI_A-D) and fem)chelatase, l.cad in believed to excel its inhibitory effect b~ interacting with sulthydryl groups of these enzymes. The increa%" in AI.A in the urine of lead-poisoned individuals is thought t,~ be caused by inhibition of ALA-D in bone marrow red cells, liver and kidney. The increased excretion o | urinaD coproporphyrin in lead pmsoned individuak ,night be due Io inhibition of copropo[phyrinogen oxidase, the enzyme which converts copro- to protoporph} rinogen. Inhibition of termchelatase pro~ ide~ an explanation for increased erythroc}te pmtotx~rphyrin in el'Ionic lead poisoning Since circulating erythrocytcs are des old t,~ mitochondria, cJevation of cD'throc~t,: protoporphyrin is thought to be produce.I by the action of lead on bone marrow er~throblasts which contain mitochondria an t synthesize heine. Lead is preferentiali¢ deposited in bone marrow and it~ conce tratton is high at this site. Thus it is not s, prising that bone marro~ erythroblasts a e vulnerable to the toxic effects of lead. ,, n increase in erythrocyte protoporph.vrin a d an inhibition of erythrocyte ALA-D s apparent at relatively Io~ lead concenu lions. These two measurements are ther fore valuable for earl,,' detection of ex D ure to lead.
Ic~,cl',o f fi:rrochchlta,,,c Ihen rills or m~ee. ll is titles important to select a sale drug [ol use m patient,, 'a.ilh hereditary hepatic poephi, fla. Fxpericnce indicates that te~t~ in the chick ¢mbr.~o appear lo ha',c prcdicti,,c ,,:due. If a drug induces I~wph} nn accumulali~ln in chick embr',o li~r cell culture and in the 17-day-olc chick cnlbr~(~ :rod is ,.~t a l~tency similar t~ that of secobarbital tt r, probable that it will have the capaot~ 1~ induce an attack ol porph}ria in patient,, ~',ith the inherited trait.
Differences in response to porphyrin-inducing drugs in different species s A variety of species and test s~stem~, have been used to study the effects of chemicals on heine biosynthesis and the following order of sensitivity re~ealed: chick embryo liver cells in culture -~ 17day-old chick embryo -> chickens - mammals. The remarkable sensitivity of chick embryo liver cells in culture can be attributed, at least :n part, to the unique pharmacokinelics of the system; unlike the intact animal, the cell culture cannot terminate the action of a drug b.~ redistribution to other organs or by excretion. Moreover. sufficient drug may be added to the cell culture to effectively ovcr~hehn the capacity of the liver cells to mctaboli:e and inactivate the drug. Since protoheme is involved in the feedback regulation of Al_A-synlhctase a low level of one of the enzymes of the heme biosynthetic pathway would make the pathway more susceptible to disturbance. It is therefore of interest that the 17-da:old chick embryo and the chicken have
fimnd
Io
ha~.c
considerabl~
!o~cr
Acknowledgement I his qud} was supported b~ the Medical Research ( o u n c d of ('anada. Reading lisl I ldll. (~ | t I lq-%) m tlandhook t l tpcr~.r,'n;a; Pharm,a.logx '..ol 44 I[)t:X|.:ll~l-. t .t;1J Aldr]dgc. \ t % . ed',L pp I ~.:"; %p~ln~v:-~,..'."l.~z. B e d i m ttciddb..'r¢ and N~:~ "l ork 2 M a t ~ c l l . D J ,;nd %lc'.~.'r. L A i I~%'1 in ~landbook o f at.;per'mcntal Pharmat,J,,~'~ X,~I 44 [DcMdIIcI'~, F. aPd Alrldgc ~, % Ld'..I pp "e4 25-!,. ~,prin+~..r %urI,LL'. Bt:rhrl. Hc+ddb,~r,= .rod Nc~ Y : q k Elder. ( L It. ( t ~ ' S ) m HLmdho.k ,,t ~ ;p¢,rI,~u',u,,: Pharmacol(,g) 'k'ol 44 t l k M a t t c ~ - . F .rod A l d r i d g c . '~V. N . cd~L pp I r," 2HII. %prm~c~ t ' c r l a g . Bcrtin. HcMt lbt-rg and Nov. '~,,rk 4 I}o-~. %!. (l~"~'J in (ht'rrltca[ ['t,rpl:~,rlt~ t'r I,l,i': (Stnk. J J f X,~, X . : m d K c c m a n / | l . c d - } pp I I - 2 ~ [ I,.o.vzr N o n h - H o l l . m d . ~.m~u:rd.m~ 5 D¢'~,tdllCl~. I- I 1'4".~) in Hatldt)¢,o& ,,tl tpr'rt~rzt'rll,i,~ I)harmdtoJt~ ~ol -I,A (l'k'%'i,t!lc~, l- .lilt] -Mdridgc. I,~, N . cds). pp. 1 2 o . 1 5 5 . Spn~wc; \ crlag. Bcrhn. Hc~del~,r~ .rod Nt..v. "fork h Orris" l ) c M,mtcl!an~,. P R . ".1~,,,. B \ and " l , , q
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8 ",a~s.t. S ( I t i " h ) m Handbo,,k o t t; tt, l~ "lr~tg.i! Pllartna, M,,qx ~,o1 44 t I ' k " q a t t c l , k .rod & l d n d g e . ~t N . cd'.] pp ~,;3 3 " 1 . Sprmgcr ~, c d a g . Bcrhn. H c i d c l ~ ' r ~ .rod Nee, ~ ork '4 Mark... ( i S I lq-'SI m H,mdhook or t tpt'rlztl¢ ¢11,a/ Pht~rtrzdt I'h'k'~ ~. ol 44 I [ ) c M . I t t c l . t and -\ktlldgt'. ~. N . cd~.), p p 2 1 t l - 2 3 " . hpll~lgi'r ~,crlag. g c r h n , tlc~dclbcr o ,rod Nov, ~ o~k
t ;er,dd S. ihtrk.~ obtained D. Phd. m orgumt the,m~lrv at OLk~rd University tn 1~54. A]ier re~e~rch and Lit tldt't?llt" clppomtrtlenf.~ an Sf ~uth .4tilt a, lilt" (-'.-g .4 . lht" U K and ('ana,hJ. he bee,row Pr,,fe,w,r and He~;tt ,,f the Dep(~ranem o1 Phatvnatx)logy ;at QueetF~ ( ni~er~IlV In IOO0.