Aug 19, 1993 - tetrachlorodibenzo-pdioxin (TCDD) in chick embryo ... at about 55 kDa in chick embryo liver microsomes (8). We recently ...... tom right panel ).
VOl. 269,
THEJOURNAL OF BIOICGICAL CHEMISTRY Q 1994 by T h e American society for Biochemistry and Molecular Biology, Inc.
No. 5 , Issue of February
4, pp. 3387-3396, 1994 Printed in U S A
Purification and Biochemical Characterization of T w o Major Cytochrome P-450Isoforms Induced by 2,3,7,8=Tetrachlorodibenzo=p=dioxin in Chick Embryo Liver* (Received for publication, August 19, 1993, and in revised form, October 1, 1993)
Arleen B. RifkindSST &io KanetoshiSII, Jason OrlinickS, Jorge
H.Capdevila**,and CharisLee*
From the Departments of $Pharmacology and $Medicine, Cornell University Medical College, New York, New York 10021 and the **Department of Medicine, Vanderbilt University, Nashville, Tknnessee 37232
Two cytochrome P-450isoforms induced by2,3,7,8tetrachlorodibenzo-pdioxin (TCDD) in chickembryo liver microsomes were purified.The P-450~exhibit catalytic selectivity either for arachidonic acid metabolism, particularly epoxygenation (P-450 TCDD55 m a ) , or for aryl hydrocarbon hydroxylase ( A H H ) and 7-ethoxyresorufin deethylase (7-EROD) (P-450 TCDD-, 64.5 m a ) . Turnover numbers for arachidonic acid epoxygenation, AHH, and 7-EROD, respectively, were 24.2, 0.23, and 0.45 for TCDD, and 0.67, 9.7, and Both P4SOs were low spin, with car35.5 for TCDD-. bon monoxide-binding peaks at 448 nm.Their N-terminal amino acid sequences showed 80% homology and contained the sequence: PXXXSATEXL, common to CYPlA P-rlSOs but not others. Polyclonal antibodies to TCDD, and TCDDcross-reacted with both P4SOs on Western blots and immunoinhibited all TCDDinduced liver microsomal arachidonic acid metabolism, A H H , and 7-EROD. Immunoquantitation using antibodies made monospecific by immunoadsorption against the heterologous P-450 showed that TCDD, and TCDDwere coinducedin liver in equal amounts and accounted for all of the TCDD-induced P-450. Enzyme assays and Western blots also showed expressionof both TCDD, and TCDDin kidney but only of TCDDin heart, indicating that these P-460s can be independently regulated and selectively induced. On Western blots non-immunopurified anti-TCDD, and antiTCDDantisera recognized rat and guinea pig CYPlAl and CYPlA2. Immunopurified TCDD, antiserum onlyrecognized rat CYPlA2, indicating that TCDD, is more closely related immunochemically to CYPlA2 than to CYPlAl. The evidence that pleotropic responses to Ah receptor ligands can include induction of a P-450 that can form biologically active products from the endogenous membrane lipid, arachidonic acid, suggests that organ- and cell-specificexpression of TCDD-induced P-460s could affect responses to TCDD.
hydrocarbons such as p-naphthoflavone (P-NF) and 3-methylcholanthrene leads to the transcriptionally regulated induction of cytochromes P-450 in the CYPlA family (1, 2). The Ah receptor also appears to be required for the toxicity syndrome caused by TCDD and relatedpolyhalogenated aromatic hydrocarbons (1,3).This toxicity syndrome includes abnormalitiesof hormone regulation, fluid balance, and cardiac contractile responses to inotropic stimuli, hyperkeratinization, immunedysfunction, tumor promotion, weight loss, and death (3, 4). The relationship between the P-450 induction and the toxicity of these compounds is not understood. The chick embryo close to hatching, which occurs at 21 days of development, is highly sensitive to TCDD toxicity and to P-450 induction (3,5,6). In chick embryo, as in rodentliver, Ah receptor ligands such as TCDD and P-NF selectively increase aryl hydrocarbonhydroxylase (AHH) and 7-ethoxyresorufin deethylase (7-EROD), mixed function oxidases primarily catalyzed by CYPlAl in rodent liver (7). Treatment with these inducers results in increased staining on SDS-PAGE of two bands, representing CYPlAl andCYPlA2 at about 55 and 52 kDa in rat liver microsomes (71, but only of a single broadband at about 55 kDa in chick embryo liver microsomes (8). We recently reported thattwo Ah receptor ligands, P-NF and TCDD, and anAh receptor-independent P-450 inducer, phenobarbital, markedly increase cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver (9). As P-450generated arachidonic acid metabolites are biologically active in ways that could be related to changes inTCDD toxicity (8,9), their role in Ahreceptor-mediated toxicity is of interest. Reconstitution studies, using P-450s purified from livers of chick embryos treated with either P-NF or phenobarbital showed that different P-450 isoforms catalyze the arachidonic acid epoxygenase activity induced by P-NF and by phenobarbital (8).Furthermore, differentP-450 isoforms with similarmolecular weights appeared to beresponsible for the increases in arachidonic acid epoxygenation and in AHH and 7-EROD induced by P-NF (8). We report here the purification of the arachidonic acidepoxyBinding to the aryl hydrocarbon ( A h ) ’ receptor by 2,3,7,8- genaseandthearyl hydrocarbonhydroxylaseinduced by tetrachlorodibenzo-p-dioxin(TCDD) and other planar aromatic TCDD in chick embryo liver. We present reconstitution, Nterminal aminoacid sequence, and immunochemical data dem* This work was supported by United States Public Health Service Grants ES-03606 (to A. B. R) and GM-37922 (to J. H. C.) The costs of onstrating that these P-450s, designated TCDDM2 and publication of this article were defrayed in part by the payment of page TCDDMH, are catalytically distinct but relatedisozymes and, charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact. ll To whom correspondence should be addressed:Dept. of Pharmacol- resorufn; 7-EROD, 7-ethoxyresorufindeethylase;HCA-100S, hydroxylogy, Cornell University Medical College, 1300 York Ave., New York, NY apatite 100s;HETEs, monohydroxyeicosatetraenoic acids; HPLC, high 10021. performance liquid chromatography; PAGE, polyacrylamide gel electro11 Present address: Hokkaido Institute of Public Health, Sapporo060, phoresis; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; *OH, whydroxy; Japan. 0-1-OH, wl-hydroxy; w2-OH, 0-2-hydroxy; Dm, dithiothreitol; BSA, The abbreviations used are: Ah, aryl hydrocarbon; AHH, aryl hy- bovine serum albumin;PFB, pentafluorobenzyl. drocarbon hydroxylase or benzo(a)pyrene hydroxylase; P-NF, P-450suntil We suggest these temporary designations for these acids; 7-ER, 7-ethoxy- they are cloned and fully sequenced. P-naphthoflavone; EETs, cis-epoxyeicosatrienoic
3387
3388
TCDD-induced Avian P-450s
Whennore, that each P-450is identical to its counterpart induced by @-NF,indicating that the pleiotropic response to Ah receptor ligands in chick embryo liver includes induction of these two P-450s.
0.01 M KPi, pH 7.0, containing EDTAand DTT, each a t 0.1 m~ and 0.2% E911. P-450 was eluted by a gradient of K P i from 10 m~ to 300 m. Fractions that appeared to contain the same P-450 by SDS-PAGE and CO binding peaks were combined. F1 (source ofTCDD-) and F4 (source ofTCDD,) from a first HCA-100s chromatography were concentrated using Centriprep 10 concentrators, diluted with 10 m~ EXPERIMENTALPROCEDURES containing 20% glycerol and EDTA and DIT each at 0.1 m~ (pH 7.0), and reapplied to the HCA-100s column.The column was first regenerMaterials ated using 1 M K P i , pH 8.0, without glycerol and was reequilibrated as Sources of materials were as follows. Fertilized eggs, White Leghorn described above. The final P-450 fractions were dialyzed against 20% strain, were from H n R Poultry Farms (Cochedon, N Y ) ; odyl-Sepha- glycerol containing 1m EDTA and 1m~ DTT a t pH 7.0. Emulgen 911 rose CL-4B was from Pharmacia LKB Biotechnology Inc.; DEAE-cellu- was removed by cholate exchange on hydroxylapatite (13) as follows; lose (DE52)was from Whatman; hydroxyalpatite (Bio-Gel HTP), acryl- after adjustment of pH to 7.0, the sample was applied to a hydroxylapaamide, and SDS were from Bio-Rad; nitrocellulose membranes (0.45 tite column (2.4 x 3.0 cm), previously equilibrated with 0.01 M KP, (pH pm) were from Hoefer Scientific Instruments; bovine serum albumin 7.0) containing EDTA and DIT at10 m. The column was washedwith (BSA) Fraction V and anti-rabbit IgG(wholemolecule)peroxidasethe same buffer containing 0.5% sodium cholate. The P-450 was eluted linked goat IgG, molecular weightmarker proteins, cyanogen bromide- with 0.3 M K P i containing 0.1 m EDTA, 0.1 m~ DIT, and0.5% sodium activated Sepharose were from Sigma; [l-14C]sodium arachidonate cholate, pH 7.4, and thenconcentrated, diluted with 50 m~ potassium (52.0 mCi/mmol) was from Du Pont NEN; arachidonic acid standards phosphate (pH 7.4) containing 1 m EDTA and re-concentrated. The were from Biomol; HPLC-grade organic solvents were from V W R Sci- purified P-450s were stored at -70 "C. entific; 7-ethoxyresorufin(7-ER)was from Pierce Chemical Co; CentriWestern Blotting prep 10 concentrators were fromAmicon Divison, W. R. Grace and Co. Hydroxylapatite (HCA-100s; Mitsui Toatsu Chemicals Inc.,Tokyo, JaPolyclonal antibodies were raised in rabbits (Pocono Rabbit Farms, pan) was a gift.TCDD was obtained from the NCI Chemical Carcinogen Antiserum raised against Repository (Kansas City, MO).Samples of arachidonic acid metabolites Canadensis, PA) as previously described (14). generated by purified rabbit CYP4A and CYP2E1 werekindly provided p-NFM (14) was used as the anti-TCDD, antiserum after molecular by Dr. RonaldLaethem and Dr. Dennis Koop, University of Washington. weight, N-terminal amino acid sequence, reconstitution, and immunochemical data, described below, showed that TCDD, and PNF, were the same P-450. TCDDwas used as theantigen for anti-TCDDDeatment of Chick Embryos and Tissue Processing antisera. Preimmune serum was obtained before injectionand immune Fertilized chicken eggs wereincubated at 37 "C. Sixteen-day-old em- serum at weekly or biweekly intervals from 6 weeks after the first bryos were treated, unless otherwise indicated, with TCDD at 1 nmoV immunization. Antisera were screened by immunoblotting against the egg in 0.005 ml of dioxane or with the solvent alone, by injection antigen. IgG was prepared from preimmune serum and thehighest titer through a hole in theshell into the fluids surrounding the embryo.After antisera, 9 weeks after immunization, by ammonium sulfate precipita48 h, organs (livers mainly, but also kidneys and hearts) were removed, tion and DE52-cellulosechromatography (15). pooled, weighed, homogenized in three volumes of ice-cold potassium For Western blotting (16) proteins were transferred from SDS-polychloride (1.15%,w/v) containing 10 m EDTA a t pH 7.4, and used to acrylamide gels to nitrocellulose membranes at 75 V overnight and then prepare microsomes as previously reported (8). Microsomes were also for 1h at 100 V, both at 10 "C.Nitrocellulose membranes were washed prepared from livers of 300-350-g male Hartley guinea pigs and 150with 10 m~ Tris, pH 7.4, in 0.9% NaCl (TridNaCl). They wereincubated 200-g male Sprague-Dawley rats, treated for 5 and 7 days, respectively, successively with the same solution containing 3% BSAfor 1h at room with TCDD intraperitoneally, at 10 pgkg incorn oil,or with an equiv- temperature to block nonspecific binding of IgG and then with either alent volume of corn oil alone (controls). Microsomes were stored at non-immunopurified anti-TCDD,IgG at 2.5 Wml, anti-TCDD-70 "C at 250 mg(wet weight equivalent)/mlin 0.1M K P i , pH 7.4, when IgG at 5 pg/ml, or immunopurified anti-TCDDM or anti-TCDDmH used for mixedfunction oxidase assays or immunoblota and in the same serum (see below) at 1:800 dilution for 1 h at room temperature, each buffer with 1 m~ EDTA and 20% glycerol (v/v) when used for P-450 again in the 3% BSA, TridNaCl solution.ARer washing with TriefNaCl, purification. blotswere incubated with anti-rabbit IgG horseradish peroxidaselinked goat IgG at 1:lOOO dilution for 1 h at mom temperature in 3% Purification of P-450s fromLivers of TCDD-treated BSA, TridNaC1 and then washed with TriaNaCl. Immunoblots were Chick Embryos developed in 30 ml of 0.03% 4-chloro-1-naphthol and 0.03% hydrogen peroxide in 50 m~ Tris, pH 7.6 (17). The TCDD-induced P-450s were purified essentially as previously reported (8) with some modifications as described below.All buffers Zmmumpurification of Antibodies against TCDD, were a t pH 7.4 and contained 20% glycerol (w/v), and all steps were and TCDD,, carried out a t 4 "C unless otherwise indicated. Eluates were monitored Microsomes (1.5 mg of protein) from livers of chick embryos treated for heme at 417 nm and for protein at 280 nm, or at 295 nm when together with samples contained Emulgen 911(E911).Selected heme-containing with dioxane alone(control microsomes) were solubilized or TCDDAAin 4 ml of 0.1 M K P i containing 1.95% samples were assayed for P-450by CO binding spectra (10) or by SDS- 20 nmol of TCDDPAGE (11).SDS-PAGE was performed on slab gels, using 3 and 7.5% sodium cholate, 1 m DTT, 1 m EDTA, and 40% glycerol, pH 7.4, at acrylamide in the stacking and separating gels, respectively, unless 4 "C. Cyanogen bromide-activatedSepharose (0.3 g) was hydrated in 10 otherwise indicated; molecular weight markers were included. Proteins ml of 1m HCl for 15 min to a final volume of about 1ml, washed with were stained with CoomassieBlue. Protein was measured by the 0.5 M K P i , pH 7.5, on a sintered glass filter at mom temperature, transferred to the solubilized microsome-P-450solution and gently rocked at w r y et al. (12). method of h Liver microsomes from 330 TCDD-treated chick embryos,containing 4 "C for 12-16 h. The microsomes were included to minimize crossa total of 1.4 pmolof P-450 were solubilizedwith 1.95% sodiumcholate linking of the purified P-450 to the activated matrix (18). The settled matrix was washed successively with five volumeseach and applied to an octyl-Sepharose CL4B column (2.6 x 38 em); the P-450 was eluted as a single large peak with the same buffer containing of 0.5 M mi,pH 7.5, 1 M potassium chloride in 0.05 M K P i , pH 7.5, at 0.25%(w/v) E911 and dialyzed, all as previously described (8). The room temperature to remove noncovalently bound protein. Ethanoldialyzed eluate was applied to a DE52-cellulose column(2.6 x 35 em) amine (1M), pH 8.0 (10 volumes),was mixed with the matrix for 2 h to equilibrated with 0.01 M K P i with EDTA and dithiothreitol (DIT) at1 block remaining activated sites. The matrix was transferred to a glass m, 0.2% E911,and 0.5% sodium cholate. P-450-containingfractions F1 transfer pipette plugged with glass wool and washed successivelywith 10 volumes each of 10 m Tris, pH 7.5;0.1 M glycine, pH 2.5;10 m~ m s , and F2 were eluted from the void volume of DE52-cellulose. F3 was eluted by washing the column with the equilibration buffer and FA, F4, pH 8.8;0.1 M triethylamine, pH 11.5;and 10 m~ Tris, pH 7.5, all at 4 "c. Anti-TCDD, or anti-TCDDserum, 0.5 ml, was diluted 10-foldin and FB by a linear gradient from 0 to 0.25 M NaCl in the equilibration 10 m Tris, pH 7.5, at 4 "C and passed three times over the Sepharosebuffer. F3 was not purified further but was stored a t -70 "C after reTCDDmatrix or the Sepharose-TCDDM matrix, respectively, at moval of Emulgen 911 as described below. m n i .Each matrix was washed with one After dialysis (except forF1) and adjusting pH to 7.0,F1, FA, F4, and flow rates of about 0.2 d FBfromDE52were each applied to a hydroxylapatite (HCA-100s) column volumeof 10 m~ Tris, pH 7.5, and thewash volume was added column (2.1 x 4.5 cm) previouslyequilibrated with 0.01 M K P i (pH 7.0) to the combined eluate. The total volume of the eluate was used as a containing 0.1 m DTT and 0.2% E911. The column was washed with measure of theextent to which the original serum was diluted. K F ' )
TCDD-induced Avian P-450s Immunoadsorbed sera were tested for cross-reactivityby Western blots. Matrices were regenerated t o immunopurify additional batches of sera or IgG by first removing adsorbed cross-reactive antibody with 10 volumes of a chaotropic agent, 2 M potassium thiocyanate, and then reequilibrating with 10 column volumesof 10 n w Tris, pH 7.5. Reconditioned matrices could be used many times. Zmmunoquantitation TCDD, and TCDDin microsomes were measured by immunodensitometry on Western blots using the immunopurified anti-TCDD, and anti-TCDDantisera. SDS-polyacrylamidegelsforimmunoquantitation contained 7.5 or 6.5% polyacrylamide. Each blot for immunoquantitation included microsomes at 5 pg/lane (first shown to be within the linear range for densitometry) and a standard curve containComing 0.25-3 pmol of the immunopurified TCDD, or TCDD-. pleteness of transfer of microsomal proteins and standards from SDSgels was monitored by post-transfer staining with Coomassie Blue. A nitrocellulose backing sheet included in the transfers was blotted to ensure that thetransferred proteins had not passed through the nitrocellulose. Immunoquantitation was performed by laser densitometry on photographic negatives of the blots, a t least two per blot, using an UltroScan I1 laser densitometer (Pharmacia LKB Biotechnology Inc.). N-terminal Amino Acid Sequence Analysis Amino acidsequencing was performed by Dr.Liane Mende Mullerat the ProteidNucleic Acid Shared Facility of the Medical Collegeof Wisconsin. Purified P-450 was subjected to SDS-PAGE (10% gel) electroblotted to polyvinylidene difluoride (Immobilon) membranes stained with Amido Black, cut out, and sequenced on an AppLied Biosystems model 477A pulsedliquid-phase protein sequenator. Metabolism of Arachidonic Acid, AHH, a n d 7-EROD by Microsomes a n d Reconstituted P-450s Reactions were a t 37 “C in a shaking water bath in air and in 0.1M unless otherwise indicated, in total volumes of 0.25 ml. P-450 concentrations and incubation times were within the linear range for product formation. For microsomes, assays were at 5 minfor 7-EROD, 10 min forAHH, and 10-20 min for arachidonic acid metabolism; for reconstituted systems, assays were at 1-2 min for 7-EROD, 2-5 min for AHH, and 1 6 min for arachidonic acid metabolism. Reactions were started with pyridine nucleotides after preincubation at 37 “C for 1 min. Microsomal AHHand 7-EROD were measured as previously reported (8,9).Arachidonicacid metabolism was assayed essentially as described (8, 19). Reaction mixtures (0.25 ml) contained 35-93pgof microsomal protein, 30 [l-**C]arachidonicacid, and 1 m NADPH. Non-enzymatic arachidonic acidbreakdown (usually
