Document x1wz5v40EOExpEqqwM2DBxd1Q

Pi or. iYot. Ann!. Se.i. USA Yiil. 70, No. 5, pp. 1321-1325, May 1973 1\)1) chlorinaled Biphenyls: A New Type of Inducer of Cytochrome P-448 in the Liver (cytochrome P-4-50/ruts aryl hydrocarbon liydroxylasc/cri/.yinc iiiduction/microsoiual enzymes) \LV1T0 P. ALYARES*, DAVID R. BICKER.-. AND ATTALLA1I KAPPAS The feller UiiivcrMt.v, New York, N'.Y. 10021: tnd The Department of Medicine, Cornell Univerwlv Mcdienl CoIIokc, New York, X.Y. 10021 Communicated by Alexander G. Bearn, February 92, 1973 ABSTRACT The CO-diffcrcnec spectrum of micro tomes from rats treated with the |niIyclilorinaU-*l biphenyls mixture, Aioclnr 125-1, lias m> absorption maximum at 410 uni. Willi ctliylisocyflimnidc as the ligand for reduced niierosonics, Aroolor 1231 treatment causes a shift in the 455-iun peali to 433 mu and increases the ratio of alisorliHiiee of 455 mn to that at 430 rim from 0.53.obtained with not rented rats, to 1.24. These findings arc similar to those secti in ruts treated with the polycyclic hydrocarbon, 3-inclJiy Ichoinnt hrenc, hut differ from those that charactcri/.c cytochrome P-150 in control or phenoliaihilal| rented rats. Aroelor 1251 Irentnicnt results in a tripling of cytoclironic I'-14t) content and a 10-fold increase ill liciizo|]pjrciic liydioxylalion. Howcvci--unlike 3-nictliyIcliolanllirciic, JilH like the pliemdmiliilnl type of inducing ngciitf--Aroelor 1251 treatment causes a significant en hancement of et liy Imorphinc N-dcincthy la-c. T hese data suggest that Aroelor 1254-induecd cytoclironic P--140 may lie ciilnly tically <liffcrc.nl from the 3-nietliy Icliolaillhrcncinduccd P-44I1 or that the hcmopicilcin(s) induced by Aroclor 1251 may ho a mixture of cytochromes P-448 and P-4.5(1 exhibit tug cattily t ic properties of both cy toch routes. Polychlorinated biphenyls are industrial chemicals used as liihrii'iinfs, bent-exchange fluids, insulators, and as plasticizers in paints, synthetic resins, and plastics used,in a wide variety of commercial and domestic products. Residues of poly chlorinated biphenyls have been widely found in tissues of fish and wildlife (1-4). Contamination of hun.-.tn adipose, tissue and of bovine and human milk has been retorted (5-8). The polychlorinated biphenyl mixtures mammacured in the Cnifed Stnles are marketed under the trade name Aroclor. The Arorlors are designated by four-digit numbers, the last two digits of which define the percent of chlorine content by weight, o.g., Aroclor 1254 contains 54% chlorine. Previous studies have shown that polychlorinated biphenyls arc inducers of steroid hydroxylases in birds tl. 9),'and of drug-metabolizing; enzymes in animals (10, ID. The micro somal hemoprolein, cytochrome P-450, lias bee-:. implicated as the terminal oxidase in the hepatic enzyme systems that metabolize many drugs, other foreign compound-, and normal body constituents. Inducers of drug-metni -mV.zi:. j: enzymes in liver have been categorized into two groups 12, 13). One groups of inducers, of which pheuobarbital is a pm-otype, en hances llie metabolism of n large variety of substmtes by liver cells; a second group stimulates the metabolism o: only a few Abbreviation: 3-McCho), 3-Melhy lcholnnihrerie. " Please address reprint requests to: Alvilo P. Aivarcs, The Rockfellci University, New York, N.Y, 10021. substrates. Polycyclic hydrocarbon carcinogens, such as benzo- [a)pyrene and 3-methykTu:,'.j.nthrenc (3-MeChol) comprise the second group of connxmnd?. In addition, the effects of these two groups of inducers on cytochrome 1M50 differ. Treatment of rats with phen'/oarbital or 3-McCliol increases the concentration of the he::.;,protein present in liver nticro- somes (14, 15). However. 3-MeChol induces tlie formation of cytochrome P-448, a hem-protein that differs in spectral and catalytic activities fro:., cytoclironic P-450 present in untreated rats or in rats tre-rovd with phenobnrbitul (14-17). Cytochrome P-448 has al-,;. r,een referred to as cytochrome 1V450 (15). ' Drugs and steroids inter:'. -, with microsomal hcinoprotein to give a lype-I difference sr-retrum, characterized by a peak at about 385 mn and a trough at about 420 imi, or a type-II difference spectrum, chan-.eterized by a peak at about 430 nm and a trough at about 390 mn (18, 19). We showed (10) that polychlorinated biphenyls stimulated the metabolism of clhybnorphine, a type-I substrate, and of aniline, a type-II substrate. In addition, ireu'.me'nt with the polychlorinated biphenyls resulted in n tripled content of cytochrome P-450 of liver microsomes (10). In the present study, we have further investigated the properties of the polychlorinated biphenyl-induced cyto chrome. The results of these studies indicate that polychlo rinated biphenyls arc a new type of inducer, causing the for mation of polycyclic hydrocarbon-induced cytochrome P-448, while eliciting the more general enzyme induction response evoked by drugs such as phtnobarbilal. METHODS Male Sprague-Diiwlcy rat.- weighing 100 -120 g were used. The polychlorinated biphenyl mixture used was Aroclor 1254, supplied by Monsanto Chemical Co. St. Louis, Mo. Aroelor 1254, dissolved in corn oil. was administered inlraperikmeally at a dosage of 25 mg/kg per day for G days, unless otherwise indicated. Control rats received corn oil only. Actinomycin D, dissolved in 10% cthi.i.was administered inlraperitonoally at u dose of 1 mg ke 1 hr before and G hr after the ad ministration of 100 mg k' e: Aroclor 1254 or corn oil. 3McChol was administer- b by the same route at a dose of 25 mg/kg per day for 4 day- and phcnobarbilal was admin istered intraperitoncully at a dose of 75 mg, kg per day for 4 days. The animals were hiked 24 hr after the injection of the last dose of Aroclor 1251. 3-AleChol, or phcnobarbilal. Livers were removed and microsomes were prepared (20). 1321 DSW 025657 v % ft .. I( i rt: yj 'K I rv*. & - s. $ % 'U V c> STLCOPCB4009612 1322 Medical .Sciences: Alvares et al. Proc. Nat. .lead. Act'. t'S.l 70 U07S) c-20 440 460 ' 4 30 4?0 440 460 .Vovelenglh (nm) Kio. 1. C.'a:oM! monoxide (left)- and ethyl isoeyanaide (r'.ft,l'r difference spectra of microsomes from untreated rat# and rat# treaied with Avoclor 1254. Aroclor 1254 was administered at a dosage of 23 mg/kg per day for 6 days. . Ktliylmorphk.e A'-dcmcthylnse activity was deternv.t rd as described 20 llenzolajpyrene hydroxylase activity ws determined in a reaction mixture containing 100 pir.oi of potassium pl.o-phate buffer (pH 7.4), 3 mnol of MgCl;. 1.0 pinol of XADPH. 100 nmol of bemo[u]pyrene (added in 5.05 ml of ncetoi.-- . and 0000 X ff supernatant derived fp..:r, 4 mg of liver, we-. weight, in a final volume of 1.0 ml. The ::.:x- ture was lucidat 373 for 5 min and the product was mea sured by the : -:.! of Xebert and Geiboin (21). In the Ixeuo- la]pyrene liy.ir--xyla.se assay, 0000 X g supernatant wa> .-ed because pir-i--undies (22) had shown that the MicLaAis constant u;i- -in.ilav whether 9000 X g supernatant or rr.ivro- somes were w d. Cytochrome 1M50 content was determir.ed by the meth-.-i of Omura and Sato (23), from the CO-difrer- ence, spcctn;::. of dithionite-mluced microsomes, using an extinction eceirlcient of 01 mM'1 cm-1 between 450 or 443 and 400 nm. The ethylisocyanide difference spectrum was determined in a similar manner, except that instead of bub bling CO through the solution, ethylisocyanide in a final con centration of 4.5 mM was added to the sample cuvette. The difference in .tt#orption at 430 and 500 um was used as an estimate of 430-nm peak, and the difference in absorp tion at 455 or 453 and 500 nm was used as ru estimate of the 455-nm peak. Protein contents were determined by the method of Sutherland elal. (24). RESULTS lifted- oi .1 rof.V 1251, Treatment on the Spectral Properties of Cytochrome P-1,50 in Liver Microsomes. The CO-difference spectra of liver microsomes from untreated rats and rats treated with Aroclor 1254, are shown in Fig. 1. A difference spectrum with a peak at 450 nm was observed with rr.icrosmues from ti:.treated rats, as expected (25, 26). However, when rats w.-re treated with Aroclor, a peak shift occurred and the CO-bii.ding pigment now exhibited an absorption maximum at 448 um instead of 450 nm. V. hen ethylisocyanide, instead of CO, was used ns the ligand the reduced micro somal hemoprotein, spectral peaks at 430 and 455 nm were observed, as reported (27). The ethyii-oeyanide difference spectrum of microsomes from rats injected "itli At odor 1254 exhibited a spectral shift from 455 to 453 nm; no such shift occurred with the 430-nm peak (Fig. Is. A- can a bo be seen in Fig. 1, treatment with Aroclor increav;-: the absorbance al 430 nm, as well as at 453 um. Howev-dm inerca-e in ab sorbance at 453 nm was greater than increase nh-erved with the 430-nm peak, resulting in an ml! increase in the ratio of the 455-nm to 430-nm peaks. T; v tiiidiiig.- suggested the possibility that Aroclor 1251 or !- n.otabolitds) may be bound to the microsomal hcmoprotT:. and that this com plex could possess spectral properties dirf-.-rent from the native cytochrome. This binding could accou.v. for the change in the ratio of the 455-nm to 430-nm peak.- - 'i>s.?rved in Aroclortreated rats. Alternatively, the observed change* coul.l be due to the synthesis of a new liemoi : :;cin having spectral and catalytic activities different from tiv native cytochrome. lifted of Aclinomycin D on Changes in Properties of Cyto chrome P-4-50 Induced by Aroclor 1251,. To te-t the :ibo\e alter natives the following experiments wetc performed: (i) addi tion of Aroclor to microsomes before i -d.wdr.n and bubbling of CO or nddition of ethylisocyanide resulted in spectra that were identical to those obtained with minosomes from un treated rats assayed in the absence of Aroclor. 'if) When whole homogenate from untreated rrr.s was incubated with Aroclor and an NADPH-gencraling system fur 0 5 hr at 373. and microsomes were then prepared, '.kesc microsomes ex hibited spectral properties identical to those of micro.-omt-s obtained from homogenate incubated in the al-ence of Arocior. Drug-induced increases in miero-oj aal enzyme activity can be prevented by certain inhibitors of protein synthesis DSW 025658 STLCOPCB4009613 Taijlf, 1. Effect of actinomyein D on Aroclor 1854-induced changes in microsomal hemoprotein and benzo[a]pyrene hydroxylase and elhylmorphine iV-demelhylase activities Treatment Controls . Actinomyein D Aroclor 1254 Actinomyein D HAroclor 1254 Benzola]pyrene Ethylmorphine hydroxylase .Y-demethylase activity** * activity t 3.90 0.22 3.37 0.28 22.42$ 1.36 6.29 1.35 0.287 0.018 0.270 0.006 0.505 0 023 0.331 0.028 Cytochrome P-450J 0.79 dt 0.02 0.73 0.04 1-286 0.04 0.79 rfc 0.07 Ethvlisocvanide difference spectra --------------- '------- ----------------------------------- 455 peak 430 peak Ratio of 455-nm to AA A j4o-mo 430-nm peaks 0.062 = 0 002 0.060 = 0.005 0.087 = 0.003 0.057 0.010 0.114 0.006 0.105 0.008 0.100 0.003 0.100 0.007 0.54 0.01 0.57 0.02 0.87 0.03 0.57 0.07 A roclor 1254, 100 mg/kg, was administered by a single injection. Actinomyein D, 1 mg/kg, was administered 1 hr before and 0 hr after the administration of Aroclor 1254 or corn oil. Animals were killed 24 hr after the administration of Aroclor 1254 or corn oil. Each value represent* mean SEM from five rats. * nmol of hydroxybenzpyrine formed per mg of protein per hr. t pinol of HCHO formed per mg of protein per hr. f nmol of cytochrmno P-450 per mg of protein. Value significantly different from respective control value (P < 0.05). or DNA-dependent RNA synthesis. To determine if Aroclor was indeed causing the induction of a spectr-ally-different lieinoprok'in, the effect of actinomyein D on the induction of benzo[a jpyienc hydroxylase and ethylmorphine Ar-demethylase, the levels of cytochrome P-450, and the ctbylisoeyanide difference spectral peaks (455-nm and 430-nm peaks) was studied (Table 1). 100 mg/kg of Aroclor 1254 was administered and tbc rats were killed 24 In later. The shift in the 450-nm peak of the CO-difference spectrum or of the 455-iun peak of the clhylisocyanidc difference spectrum was not apparent 24 hr after a single injection of Aroclor. How ever, administration of Aroclor did cause a significant en hancement of benzo(aJpyrene hydroxylase, ethylmorphine A'-demethylasc, and cytochrome P-450 content, as well as an increase in the ratio of the 455-nm to 430-nm peaks from 0.54 to 0.87. Administration of actinomyein D, 1 hr before and 0 hr after the administration of Aroclor. prevented the marked stimulation of enzyme activities, the increase in cytochrome P-450, and t'r.e increase in the ratio of the ab sorbance at the 455-nm to 430-nm peaks. Actinomyein 1) administered alone had i.o effect on the basal levels of enzyme activity or the microsomal hemoprotein. Comparative Effects of Phenobarbital, 8-McChol, and Aroclor 1254 on Hepatic Microsomal Hemoprotein, ncnzo[a)pyrene Hydroxylase, and Ethylmorphine N-Dcmelhylase. As shown in Table 2, Aroclor 12-54 caused a 10-fold induction of benzo[ajpyrene hydroxylase, whereas 3-MeChol and phonobarbitul caused 13- and 4-fold induction of the hydroxylase, respec tively'. The three inducers increased the cytochrome P-450 levels; however, the CO difference spectra showed absorp tion maxima at 448, 448. and 450 nm with Aroclor, 3-MoOhol and phenobavbilal, respectively. Similarly, like 3-MeChol, Aroclor treatment resulted in a marked increase in the ratio of the 455-nm to 430-nrn iteaks. The increases in benzo[a]pyrene hydroxylase activity and in the ratio of the 455-nm Taui.k 2. Comparative effects of Aroclor 1854, 8-McChol and phenobarbetal on hepatic microsomal hemoprotein and benzo[a]pyrene hydroxylase and elhylmorphine N-demethylase activities Treatment Controls Aroclor 1254 3-MeChol I'benoburbitid Benzo[a]pyrene Ethylmorphine hydroxylase A'-demethylose activity* activity! 4.21 0.74 42,08$ i3.47 55.84$ 1.51 16.38$ 0.34 0.433 0.026 0.951$ 0.031 0.475 0.027 1.586$ =h 0.055 Cytochrome P-4 501 0.73 0.02 2.01$ 0.10 1.70$ 0.05 2.32$ 0.03 Ethyl isoryanide difference spectra 455 peak A Aish-un 430 peak A A 410-500 Ratio of 455-nm to 430-nm peaks 0.062 0.002 0.230$ 0.036 0.224$ 0.010 0.211$ = 0.010 0.117 0.008 0.192 0.033 0.129 0.009 0.354$ =fc 0.008 0.53 ' 0.02 1.24$ 0 10 1.74$ 0 05 0.59$ =fc 0.02 Hats were administered Aroclor 1254, 25 mg kg per day for 0 duys or 3-McChol, 25 mg/kg per day for 4 days, or phenobarbitnl, 75 ing/kg per dny for 4 days. Enel) value represents mean SEM from five rats. * nmol of liydroxybenzpyrene formed per mg of protein per hr. t mnol of HCHO formed per mg of protein per hr. t nmol of cytochrome P-450 per mg of protein. Value .significantly different from respective control value (P < 0.05). DSW 025659 STLCOPCB4009614 1324 Medical .Sciences: Alvnres et al. Proc. Nat. Acad. Sci. USA 70 {1973) to 430-nm peak.' observed with Aroclor were of a lesser mag nitude than those observed with 3-MeChol. In contrast, phcnobnrhital prcueatmeiTt resulted in a 3-fold increase in cytochrome P-450. and the rutio of the 455-nm to 430-nrn peaks was similar to that observed with untreated rats. As expected (15. 2s . 3-MeChol had no effect on the activity' of clhylmorphin..- .V-demethyluse; however, Aroclor trealmem (Tuble. 2) rc-u!vd in a significant enhancement of Af-demethylase activity, though of a lesser magnitude than that observed in vats treated with phenubarbital. The data ob tained with 3-.Me('hol and phenobarbitul are similar to those previously reported '14,16, 28, 29). DISCUSSION The possible health hazard to humans presented by the en vironmental pollutant, polychlorinated biphenyls, is unclear, but recent disclosures of the presence of this contaminant in human adipose tissue (5, 7, 8) and iu human milk (6) raise serious question? us to the potential effects of these tissue contaminants on human health. In these studies Aroclor 1254 was used as a representative mixture of polychlorinated bi phenyl compound? since it is similar to the polychlorinated biphenyl residues found in some human adipose, tissues (7). The data presented in this paper show that Aroclor 1254 is a potent inducer of the microsomal liemoproteiu, cytochrome P-448, and of benznlajpyrene hydroxylase and ethyl morphine Ar-demetbyla?c. Harbitinalc' and polycyclic hydrocarbons appear to stim ulate drug-metabolizing enzymes of liver by different mech anisms. The. barbiturates induce the metabolism of many substrates. For example, phenobiubit.nl in rats is a potent inducer of cytochrome l'-450, and enhances the metabolism of drugs in vitro well as in vivo. However, it is a poor in ducer of the aryl hydrocarbon hydroxylnses. In contrast, polycyclic hydroniiboiis, such as 3-MeOhol, induce the for mation of a spectrally and catalytienlly different, hemoprotein, cytochrome P-445 14, 10). 3-MeChol is a potent inducer of aryl hydrocarbon hydroxylases (30), but does not enhance the A'-demethylauoii of elhylmorphine (28). Polychlorinated biphenyls, as shown in these studies, share the properties of both the polycyclic hydrocarbon and the phenobarbital types of inducer compounds. Aroclor 1254, for example, caused a 3-fold induct ion of cytochrome P-448, a Mold induction of Af-demethyliiK' activity, and a 10-fold induction of bcnzo[a]pyronc hydroxylase. In addition, the hemoprotein induced by Aroclor 1254 showed a typical type-1 difference spectrum with hexobnrbitel, similar to that observed with microsomes from untreated or phcnobarbital-treated rats (unpublished observations). Cytochrome IM48 from 3-MeChol-induced rats showed with hcxobarbital a modified type-11 difference spectrum, as previourly reported (31). Treatment of rats with phenobarbital enhances the metabolism attd decreases the duration of action of both zoxazolamine and hexobarbital in the intact animal. On the other hand, polycyclic hydro carbons accelerate the metabolism of zoxazolamine, but not of hexobarbital 32:-. IVe have shown that Aroclor 1254 ac celerates the tn'-iabolism of hexobarbital and zoxazolamine in vivo (10). Thus, these data strongly suggest that the Aroclor. 1254-induced cytochrome P-448 is catalytienlly different from the 3-MeClml-inducpd cytochrome P-448 or, alterna tively, that the heinoproleiti(s) induced by treatment with Aroclor 1254 may he a mixture of cytochromes P-448 and P-450 exhibitin',; catalytic properties of both cytochromes. Aroclor 1254 is ft mixture of .polychlorinated biphenyls comprising chemical congeners of both high and low chlorine content. Since Aroclor 125*1 shows the inducing properties of both the barbiturate and the polycyclic hydrocarbon classes of inducers, it is possible that the mixture of polychlorinated biphenyls contains one or more compounds having the bar biturate type of inductive properties and compounds having the properties of the polycyclic hydrocarbon class of inducers. The two types of induction produced by 3-MeChol and pheno barbital proceed independently of each other when these compounds are administered simultaneously, as shown by Bidleinan and Mantiering (33); these independent induction phenomena result in biochemical and spectra] findings similar to those obtained after the administration of Aroclor 1254. Our studies show that the ralio of the 455-nrn to the 430-nm peaks (1.24) produced with ethylisoeyanide in Aroclor 1254treated animals (Table 2) is almost exactly intermediate between the ratio seen with microsomes from untreated and phenobarbital-treated rats (0.53 and 0.59, respectively) and the ratio seen* with microsomes from 3-MeChol-trealcd rats . (1.74). However,, a second possibility exists, namely, that a single component of the polychlorinated biphenyl mixture possesses the combined properties of the two classes of in ducers and that the characteristic inducing action of Aroclor 1254 described here is intrinsic to the polychlorinated bi phenyl moiety itself. Drug-induced increases in enzyme activity can be pre vented by inhibitors of nucleic acid and protein synthesis. In the present studies, actinomycin D, an inhibitor of 1)XAdependent RNA synthesis, was administered before Aroclor 1254 and this treatment prevented (a) the change in the ratio of the 455-nm to the 430-nm peaks of the ethylisocynnide difference spectrum of reduced microsomes, and (6) the in crease in benzo[a]pyrene hydroxylnse and ethylmorphine .V-demethylase activities induced by the polychlorinated biphenyls. This inhibitory effect on the Aroclor 1254-induced spectral changes in microsomes supports the view that Aroclor 1254 enhances the synthesis of a distinct microsomal hemoprotein having spectral properties different from that pres ent in the livers of untreated rats. The aryl hydrocarbon hydroxylase system has come under increasing investigation in the field of oncology. Polycyclic hydrocarbon carcinogens are the most widely occurring en vironmental chemical carcinogens known and occur as atmo spheric pollutants resulting from combustion of fuels and other organic materials; they are also present in cigarette smoke. In addition, they are potent inducers of aryl hydro carbon hydroxylase in liver and nonhepatic tissues in Rnimals and man, and several of them have been shown to induce cytochrome P-448 in the liver. Since the polychlorinated biphenyls possess certain of the characteristic inducing prop erties of benzo[a]pyrene and 3-MeChol, and are themselves ubiquitously distribute'.; in tbe environment, it may Vie of considerable importance to examine the carcinogenic po tentialities of these biphenyl mixtures. In thi? regard, it is of considerable interest that recent studies by Alien and Xorback (34) have shown that ingestion of polychlorinated bi phenyls by rhesus monkeys results in hyperplastic, dysplnstic, and invasive changes suggestive of eventual neoplastic trans formation of gastric mucosa. The technical assistance of Mrs. Susnu Peters and Mi's. Evelyn Grossman is gratefully acknowledged. This work was supported in DSM 025660 STLCOPCB4009615 r- . pnrt My grants'from Iloffmmi I.a Roche, the Council for Tobacco Research, U.S.A., USPIIS Grant ES-00621, anti the Scaife Family Charitable Trusts. Dr. Rickers was supported by N1H Training Gntill AM 0-5320 awarded to the Department of Dermatology, Xcw York University Medical Center. 1. Itisebrough, It, W., Rieche, P., Peakall. D. B., Herman, S. G. <!k Kirvcn, M. N. (1908) Nature220, 101K-1102-. 2. Jensen, S., Johncls, A, G., Olsson, M. <t Otterlind, G. (1909) Nature 224, 247-250. 3. Kocinnn, J. H., do Brnmv, M. C. T. N. <c Vos, R. If. (1909) _...... Nature 221, 1120. 4. Bnchc, C. A., Scrum, J. W., Youngs, W. D. & Bisk, D. J. (1972) Science 177, 1191. ,r>. Biros, F. J., Walker, A. C. &, Medbcry, A. (1970) /lull. En viron. Contain. Toxicol. 5, 317-323. (1. Acker, L. & Scluilte, 10. (1979) .Xaluncis-enschaftcn 57, 497. 7. Price, 11. A. & Welch, It. L. (1972) Environ. Health Perspecl. ' 1, 73-78. ft. Yobs, A. It. (1972) Environ. Health Perspecl. 1,78-81. 9. Liuccr, J. L. & Peakall, D, 11. (1970) Mature. 228, 783-784. 10. Bickers, D. It., Ifarber, L. C., Kappas, A. & Alvares, A. P. (1972) Rrs. Common. Ckem. Path. 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II., Davidson, C., Gastel, R. & Burns, J. J. (1900)/. Pharmacol. Exp. Thcr. 130, 1-8. 33. Bidlcman, K. & Mannering, G. J. (1970) Mol. Pharmacol. 6,697-701. 34. Allen, J. It. & Norback, D. H. (1973) Science 179, 493-499. sw 025661 STLCOPCB4009616