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Poland A. and E, Clover. 1973. Studies on the mechanism of toxicity of the chlorinated dlbcnzo-p-Uloxine. Environmental Health Perspec tives 5: 245-231. HONS t00431 Studies on the Mechanism of Toxicity of the Chlorinated Dibenzo-p-dioxins* by Alan Poland1 and Edward Glover1 Introduction Concern about the potential health has* rds resulting from environmental pollution by the chlorinated dibenzo-p-dioxins and dibenzofu runs arise* from our recognition of the extraordinary potancy of these com pounds aa toxin* and teratogena and thalr Inadvertent dispersion In the environment as contaminants erf chlorinated phenolic prod ucts. Questions concerning the extant of en vironment contamination and those concern ing the mechanism of toxicity produced by these compounds are at present unanswer able. Several papers in this symposium hav* presented the historical background which led to our current understanding and concern about this problem: (1) the "chick-edema" outbreaks caused by "toxic feta" in poultry feed and the eventual isolation and identi fication of a hexachJorinated dibenzo-p-dioxin; (2) the occurrence of acne among work ers in several 2,4,5-T factories and recogni tion of 2^,7,8-tetrachlorodibenxo-p-dioxin (TCDD) aa the etloiofic agent; (8) the NCIcommissioned study on the potential taratogenicity, carcinogenicity and mutagenicity of 2,4,6-T;(4) and the widespread use of Agent Supported by: NIH Spedsi Postdoctoral Followship, S POSES 4StM; Ford Motor Compony Grant for Toxicology; and NIH Cantar Grant for Toxicology Rtwareh and Training, 2P11-GM 16I90-06AT. tDcpartmont of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, Now York 14642. Orange as a defoliant in Viet Nam and Cam bodia. The starting point of our atudiea was a re port by fileiberg and colleagues (I) that of 29 workers in a 2,4,5-T factory, all of whom had industrially acquired acne, 11 had por phyrinuria and aeverai bad overt clinical porphyria cutanea tarda. TCDD has been shown to be the causative agent of the acne; however, the cause of the porphyria was un certain. Porphyria cutanea tarda is an ac quired defect of hepatic porphyrin metabo lism characterised by an overproduction of porphyrins by the liver, increased urinary ex cretion of porphyrins, mechanical fragility and photosensitivity of the skin (blistering in areas exposed to sunlight), hyperpigmen tation, and hiruaitiam. We restudled the fac tory 5 years later and found no evidence of porphyria in the employees (t). The fact that this syndrome abated following meas ures to reduce the formation TCDD and mini mise employee exposure to this contaminant, suggested that TCDD might have been the causative agent of the industrial outbreak originally reported by Bleiberg et a], (1). Methods and Materials The halogenated dibenso-p-dloxins and dlbensofurans and analyses of their purity were generously provided by Dr. A. Pohland, Food and Drug Administration, Washington, D. C. and Mr. George Lynn, Dow Chemical Company, Midland, Michigan. In addition, Drs. J. Wade and A* Kq||0e synthesized September 1973 koms 200632 and analysed a number of dibenzo-p-dioxins which were tested and reported elsewhere in this symposium. Animals Our experiments were performed in chick embryos which were routinely 15--20 days of age. The various dibenzo-p-dioxins were dissolved in p-dioxane, and 26 *1 of the solu tion was injected into the eg? through a smalt hole punched in the air sac. Hale Sprague-Dawley rats, 70-100 g, were uaed in some experiments. Analysis S-Aminolevulinic acid synthetase acti vity was measured as previously reported (3). Aryl hydrocarbon hydroxylase was as sayed essentially by the method of Glelen, Goujon, and Nebert (4). One unit of hydroxy lase activity ia defined as that amount of enzyme catalyzing the formation per minute at 87*C of hydroxylated product causing fluorescence equivalent to that of 1 pmole of 3-hydroxy bento [a] pyrene. The assay was performed on the 10,000(7 supernatant, and results are expressed as units per milligram wet weight of liver. Result* A variety of xenobiotlcs product experi` mental hepatic porphyria and all have in com* mon the ability to induce the Initial and rata limiting enzyme in the heme biosynthesis pathway, i-aminolevullnic acid synthetase (ALA synthetase). To teet whether TCDD was in fact porphyrigenic, we administered the compound dissolved in 2Spl p-dloxane to chick embryos. The embryos were sacrificed 48 hr later, and the ALA synthetase activity asssyed in their livers. As seen in Figure 1, TCDD produced a dose-related increase in enzyme activity. As little as 4.66 x 10 '* mole egg (1.6 ng) produced a doubling enzyme activity and the highest level tested 1.56 x 10'* mole/egg (0.5 nff) produced a 85-fold induction. TCDD is more potent than any other inducer of ALA synthetase yet report ed by at least three orders of magnitude, and, unlike most other porphyrigenic chemicals, induction is very prolonged, moat likely a 24G Ficuu 1. Lorarithade Inn mutii mm fer the induction of ALA synthetase by TCDD. Chicken CCS of 17 days' ((station were injected with ZS *1 of solvent (control) or solvent containing vari* ous doaea of TCDD, end hepatic enzyme activity was assayed 41 hr later. The points represent the mean * standard error of three or four creeps of pooled liven. reflection of the long biological half-life of TCDD. We next acreened a aeries of 15 halogenated dibenzo-p-dioxins for their abil ity to induce ALA synthetase. As seen in Fig ure 2, all the isomers which ware inducers Ficuss t. Structure-activity relationships of the halofsnated dibeiuto-p-dioxins: Induction of ALA synthetase. Seventeen-day embryos wore Injected with the solvent (p-dionene) or solvent eontaiainf the dioxin tested and ensyme activity was assayed 43 hr later. Uninjected control values (s -- ), At not differ appreciably from solvent iajected controls (a at It). Kaeh bar for the tost groups represents the value for s eindfetHHip of three to Avs pooled livers. Environmental Haallfi Perspectives i t 1 : j ii t. j 4 \ I r1 had two common properties: (1) halogen atoms occupy at least three of the four lateral ring position! (2, 3, 7, and 8) and (2) there is at least one free, nonhalogenated carbon atom. Note that octachlorodib*nzo-p-dioxin is inactive. The compounds that were not inducers were tested at 200-400 times the molar concentration of TCDD that produced a significant response. To the extent that toxicologic data are available (5), all those dioxins which are at low doses lethal, teratogenic, or produce acne, also induce ALA synthetase, and those dioxins which are not potent toxins do not induce ALA synthetase. We have also test ed a limited series of dibenzofurans; the unsubatituted compound and 2,8-dichloro and octachloro derivatives all fail to induce, and a mixture of di- tri- and tetrachlorodibenzofurans is potent as an inducer of ALA synthetase. While the data are very limited, it appears the structure-activity relation ship is similar in the dibenzofuran aeries. As reported elsewhere in this symposium (6), TCDD does not induce ALA synthetase in several laboratory mammals. We have found it to be a poor inducer in the rat. This should not be interpreted to mean that the results obtained in the avian embryo have no relevance to man. For instance, many sex steroids appear to play a role in precipitating acute intermittent prophyria and porphyria cutanea tarda in man; how ever, while induction of ALA synthetase by these compounds can be shown in the chick embryo, it does not occur in the rat There is an empiric relationship observed by numerous investigator* that many com pounds which induce ALA synthetase also induct microsomal mixed-function oxygen ase activity in the liver (also called the "drug metabolising enzymes"). Two points are of note about this correlation: not all drugs in duce both enzyme activities; also, the rela tionship many have a theoretical baata, in that heme is the prosthetic group of the ter minal component of microsomal oxygenase, cytochrome P-450. The high concentration of cytochrome P-450 and rapid turnover relative to all other hepatic hemoproteins. accounts for a large fraction of the total hem* synthesized in the control liver. Some investigators have suggested that coordinate induction of ALA synthetaae, the rate- lim iting step in heme synthesis, and cytochrome P-450 and microsomal oxygenate activity may have a basis in providing the extra heme necessary lor the new cyto chrome P-4B0. DwffRinwi not very satisfy ing teleologic explgligrijifc there is a sizable literature reporting fhinancommitant induc tion of ALA synthetaae and microsomal ox ygenase activity. We next studied the effect of TCDD on microsomal oxygenase. As a measure of this enzyme complex we choose to investigate aryl hydrocarbon hydroxylase activity, because aromatic hydroxylation ia induced primarily by aromatic compounds, which in our view chemically resemble TCDD. As seen in Figure 3, TCDD produce* a dose- related induction of aryl hydrocarbon hydro xylase in chick embryo liver. At the lowest dose tested, 1.55 x 10*" mole/egg (0.5 ng) there is a nearly twofold increase in enzyme activity, and maximal induction is produced o-u TC00 (notnAes) Ficus* 3. Logarithmic doae-rtaponae curve for the induction of ary) hydrocarbon hydroxylase. Eighteen-day ambryaa war* injected with TCDD diaaolved in p-dloxane or p-dtoxane alone (control), and hepatic anxyma aetMty was aaaayed *4 hr latrr. Each point rapnaaSSa the mean s atandard arror of four groupa f psafad Uvera. September 1073 247 HONS 2H06S4 of TCDD at a dose of 3.11 x 10'1* mole/kg In Figure 6 we have plotted the reauita of 11' produces nearly a fivefold induction in rat of the time course of induction produced by liver enzyme activity (Fig. 5). Maximal in 3-methylcholanthrene (20 mg/kg in corn 0- ! duction of ary) hydrocarbon hydroxylase was oil), TCDD (10 pg/kg in p-dioxine) and ;d n produced by a dose of 3.11 x 10'* mole/kg control rata (receiving either corn oil or pi (10 >>g/kg). It Is estimated that the half dioxane). Aryl hydrocarbon hydroxylase ia o m e- maximal response is elicited by a dose of g.6 x 10 * mole/kg (0.265 ygAg). roughly one hundredth the dose that kills BOJfe of rata induced to the earne extent at 1 and 4 days by maximally inducing doses of each drug. However, by $ days, hydroxylase activity (5). 3'Methyicholanthrene, a polycylic hydro In the 3-methyfcbdbaidUrgP*-treated rata re ,t' carbon carcinogen is perhaps the moat widely turned to control leejj^gHUle induction per ty ie used compound sa an inducer of aryl hydro sisted in the TC&lMilEwd rate for over 1 carbon hydroxylase. TCDD was found to month. Accompanying gni induction of aryl n- be nearly 8 x 10* times as potent as 3- hydrocarbon hydroxylase by both drugs the in o- inethylcholanthrene at inducing hydroxylase following changes were noted: an increase in * activity. Both drugs produce the same maxi the total CO-binding microsomal cytochrome iy mal degree of enzyme induction in rat liver, (cytochrome P-450 and P-448), a shift in jn and the administration of both drugs to the CO-maximum peak by difference spectro gether, each at a dose which produces maxi scopy from 460 nm to 448 nm, and a ma] induction, elicits a response that is no shift in the ratios of the peaks observed with greater than that produced by either drug ethyl iaocyanlde as a ligand by difference I, alone. apectroecopy. These changes are interpreted to mean both S-methylcholantrene and TCDD * ir i i t r Frct-ic 5. Logarithmic dose response curve for the PlCUSX 8. Time count of aryl hydrocarbon hydroxy- r. (adoction of aryl hydrocarbon hydrexylaaa in rat lirrr. Mato Spragua-Dawloy rata weighing about lam induction In rat livor following TCDD and 3mothylcholonthrcm administration. Molt Spraruc- 80 g wen injected intraparitoneally with TCDD DawJay rati, weighing about B0 g wen givan a !n diuofved in p-dioxano or p-diexano olono (0-8 single injection of TCDD la y-dioxans (10 t/kf). *1 ml/kg), and hepatic aryl hydrocarbon hydroxylam 3-mcthylcboionthmw la corn oil (20 mgAg), or ,{ activity was asoayed 24 br later. Each point is the aolvanta alone. Each point raprasenta the mean the mean standard error of five animals. t standard error of four rets s September 1973 249 induce the formation of a new type of cyto chrome P-460 which contains its heme iron in a high-spin atate. We have also found that both TCDD and methylcholanthrene pro duce induction of aryl hydrocarbon hydroxy lase in a number of extrahepatic tissues. In summary. TCDD appears to b* simi lar to 3-methylcholanthrene as an Inducer of microsomal oxygenase differing primarily in potency (by four orders of magnitude) and duration of action- The persistent induc tion following TCDD administration is prob ably a result of its long biological half-life. Discussion The results of our Investigation demon strate TCDD is a potent inducer of ALA synthetase and aryl hydrocarbon hydroxy lase in the chick embryo liver. There is a perfect correlation between those dioxins which induce both enzymes and the toxicity data, to the extent the date are available on the various dioxina. The structure-activity relationship reveala that all dioxina which ire potent inducers have halogens at three the four lateral ring positions and at least me nonhalogenated carbon atom. The sen sitivity of induction of aryl hydrocarbon hydroxylase by TCDD and other toxic dioxins suggests this response might be a very valu able screening bioaaaay to detect the pret ence of the toxic dioxins in commercial pro ducts or environmental samples. It should be emphasized that the nonspeciflcity of the response makes it imperative that one ex tract the samples tested to remove polycylie hydrocarbons, end the teet is only collabora tive, not definitive for TCDD and related dibenzo-p-dioxins. Now, I should Hke to turn to the breeder question of the mechanism of toxic action produced by TCDD. Any proposed mecha nism of tosidty must account for several observation about TCDD (1). TCDD ia a nearly planar, highly lipophilic, and a rather chemically unreactive molecule, which pos sesses remarkable biologic potency, and hence specificity (2), There ere very large differ ences in susceptibility of different species to TCDD, ft* presented by Schwetz (5). The oral 250 LDv, in the guinea pig is one thousandth that of the dog (3). There is a very sharp struct ure-activity relationship among the dioxins. The oral LD* values of the 2,7-dichloro and octachloro derivatives are greater than 10* times that of TCDD in the rat (4). TCDD seems to product hepatic cell necrosis, and liver insufficency is the presumed cause of death in the rat. However, multiorgan in volvement in the rat has been reported at this symposium and elsewhere. Furthermore, as reported by Vos and Moore and colleagues (7, t), hepatic necrosis ig jptiiijna] in the mouse and guinea pig awl perhaps insuffic ient to account for death. Thus we must ac count for the different pattern of histologic damage in different species (6). TCDD is remarkably slow in its toxic action leading to death. Regardless of dosage, animals die weeks after a single administration of TCDD (6). TCDD ia an extraordinarily potent tera togen in a number of species (7). Finally, our investigations suggest that ail dioxins which are potent toxins, as acneogens, tera togens. producing mortality or chick edema, also are potant inducers of aryl hydrocarbon hydroxylase activity. This enzyme complex is present and inducible in a number of tissues and is responsible for the aromatic hydroxylation of many xenobiotjea. It is useful to examine the proposed me chanism of toxicity for other aromatic or halogensted aromatic compounds that, like TCDD, are chemically relatively unreactive and highly lipophilic. The two most exten sively investigated models are the liver necro sis produced by halogensted benzenes and the carcinogenesis produced by polycyclic hydrocarbons {?). Briefly the literature can be summarised as follow*. The parent com pound ia metabolised to a very reactive arene oxide intermediate. This intermediate may then chemically rearrange to a phenol, be further metabolized to a dihydrodiol or glu tathione conjugate, or, react chemically to covalently bind to various cellular macromo lecules which act as nucleophiles. In the case of hromobenzene centrolobular liver necrosis, the epoxide is believed to attach to proteins, and in the esae of the polycyclic - .25*-* Environmental Heittli Perspectives - HONS 20O6J? that . ucU -tins, and , 10' ;dd and e of i in* d at lore, sue* the ifflc- ac- ogic ) is g to die DD era* illy, tins >ra- ma, bon dex of itic neor ike ive on* rotnd lie an ni ne ay to !:ohe er ch lie es hydrocarbon carcenogenesis the critical event ia believed to be the binding of the "K- region" epoxide to DNA. We propose a similar model for the toxi city of TCDD. The parent compound entara the cell and binds to some induction-recapt or site which initiates the events which ulti* metaly lead to the formation of more aryl hydrocarbon hydroxylase activity, TCDD ia recognised by a second site in ths cell, the enzyme-active center of microsomal oxygen ase, and converted to a reactive metabolite, possibly an epoxide. The conversion of TCDD to its reactive metabolite ia the rate-limit ing step in dioxin metabolism, and this step is increased by the induction of aryl hydro carbon hydroxylase. Some of these reactive metabolite molecules bind to cellular macro molecules producing some impairment of function which gradually produces cell death. It Is useful to examine this hypothesis; in light of the known facts concerning TCDD toxicity. The large difference in special susceptibility to TCDD might be explained by the differences in the rate of metabolism of TCDD. The multiple organ damage produc ed by TCDD and variable pattern of histo logic damage in different species might be explained by the relative rate of formation and further inactivation of the reactive metabolite in different organa. (3) As pointed out by Gehring (10), than la some evid ence. far from unequivocal, that the admin istration of C`*-TCDD results in unextractable radioactivity in rat liver. (4) The tera togenic effect of TCDD may be a result of mutsgenesis by intercalation of the parent compound into DNA (11) or by Intercala tion and cevaknt binding of tha metabolite, analogous to the acridine and amiitofiuorena companded*, i*). This hypoteeis is highly speculative and presented only to encourage further inveetigation. The major assumptions remain un supported: (1) TCDD ia metabolized, and (2) the metabolite covelently binds to some cellular constituent. The demonstration that both these events do or do not occur must wait the synthesis of radioactive TCDD of high specific activity. , The chlorinated dibenzo-p-dioxins ire worthy of much greater investigation, not only because the potential public health haz ard they pose, but also the remarkable poten cy and sharply defined structure-activity re lationship they demonstrate suggests an un common spedfldty of action. Ultimately, TCDD, like other potest toxins, (i.e., botulinua toxin, tfftrodobmfft, Organic phosphates) may become a useful biologic tool. unMNCSi 1. BWW|, J, e it, IndutHdb acqwlred por phyria. Arch. Dermetology N: TM (IMS). 2. Polend, A, ot el, A health survey ef workers la 2,4-D sad 2,4,8-T plant. Arch. Environ. Health, Sit Ilf (1971). S. Poland. A, sad Glover, X. 2,2,7,9-Tetraehloredibenie-pHiloxin: potent ladnear of S-aminolevullnk add synthetase. Science 179: 47# (1973). 4. Clelen, J., Goujon, P., and Nobert, D. Genetic regulation of aryl hydrocarbon hydroxylase In duction. II. Simple mondolian expression in moose Usenet be vtoo. 1. BioL Chom. U7: (1972). 3. Behwetn, B. CUoradibenao-p-dioxin toxicology. Environ. Health Penpect. No. 87 1973. 8, Woods, J. Stadia* of the effect! of TCDD on mammalian hepatic t-aminolevollnlc acid syn thetase. Environ. Health Penpact. No. S: 221 (1978). 7. Harris, M., Moon, J.. and Voe, J. General bio logical efforts of TCDD In laboratory animals. Environ. Health Penpect No. 8; 101 1973. 5. Voe, J. G, Moon, J. A and Zlhkl I. J. Effect* of TCDD on the Immune system of laboratory animate. Environ. Health Par*poet Na. *. 149 (1979). 9. Daly. J, Joins. D, and Wltkop, B. Arana oxides and the NIH shift: tha metabolism, toxicity end carcinogenicity of aromatie eompoonde. Experientia 28: 1188 (1*71). 10. Piper, W. N., Inca, J. Q, and Gehring, P. J. Excretion and tissue distribetion of 24,74-tetra* chlerodibonao-p-dioxia in the rat Environ. Health Penpect. No, I: 24! (1971). 11. Hussain, S., t eL Mutagenic effect* of TCDD on bacterial system*. Ambio 1: 82 (1972). 1* Am**, B., *t el-. Derivative* of 2-seetrleminofluonne end other aromatle tmin* carcinogen*. Pw. Net Acad. 6ci.. 4*. 3128 (1872). 13. Creech, H., et aU Antitamor end mutagenic properties of e variety Of heterocyclic nitrogen end sulfur mustards- J. Med. Chem., IS: 739 (1972). September 1973 231 MOMS 200*3t