Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and … · 2017-05-03 · of four cytochrome P-450 isozymes, Forms 2, 3, 4, and 6, in the kidney, lung, and liver of adult male rabbits

[CANCER RESEARCH 42, 1423-1432, April 1982]0008-5472/82/0042-OOOOS02.00

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and PhÃ©nobarbitalon theOccurrence and Distribution of Four Cytochrome P-450 Isozymes in RabbitKidney, Lung, and Liver1

Jane H. Dees,2 Bettie Sue Siler Masters, Ursula Muller-Eberhard,3 and Eric F. Johnson4

Department of Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235 [J. H. D., B. S. S. M], and Department of Biochemistry,Scripps Clinic and Research Foundation, La Jolla, California 92037[U. M-E., E. F. J.]

ABSTRACT

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

and of phÃ©nobarbital (PB) on the distribution and occurrenceof four cytochrome P-450 isozymes, Forms 2, 3, 4, and 6, in

the kidney, lung, and liver of adult male rabbits was investigatedusing immunofluorescence. In the kidney, Forms 2 and 3 werelocalized in the proximal tubules of both untreated and PB-

treated animals, while antibodies to Forms 4 and 6 showedweak to negative staining. In TCDD-pretreated animals, Forms

4 and 6 appeared in the renal endothelium, in addition tostaining the proximal tubular epithelium intensely. Form 2 wasthe only isoenzyme of those studied found to be present in thelungs of normal and PB-pretreated rabbits; it was also presentin lungs of TCDD-pretreated rabbits. Form 3 was not detected

in any of the rabbit lungs examined. Forms 4 and 6, while notapparent in the lungs of normal or PB-treated animals, werefound in the lungs of TCDD-treated animals and also appeared

in the endothelium of the pulmonary arteries and veins. Allforms tested were present in control liver. The staining for Form2 was intense in the livers of PB-pretreated animals, as wasthe staining for Forms 4 and 6 in the livers of TCDD-pretreated

animals. Our results indicate that, while PB altered the intensityof staining for Form 2 in the liver and kidney, TCDD alteredboth the staining intensity and distribution of the isozymes inkidney, lung, and liver, producing, for example, a localizationof Forms 4 and 6 in the endothelium of both the kidney andlung which was not seen in either untreated or PB-pretreated

rabbits.

INTRODUCTION

The cytochrome P-450 isozymes (16) have been implicated

in the metabolic activation of a wide variety of chemical carcinogens (14, 29, 40). They also catalyze detoxification pathwaysfor many of these chemicals, and each cytochrome may catalyze these divergent pathways to a different degree. For thisreason, the relative occurrence of the isozymes in tissue cellpopulations will influence the disposition of a given carcinogenwithin that tissue. Little is known regarding either the differentiallocalization of the isozymes within complex tissues or themanner in which this pattern of occurrence is modified byagents such as TCDD5 or PB, which are known to be selective

Received June 12, 1981 ; accepted December 2, 1981.' This work was supported by USPHS Grants HLBI 13619 and GM 16488.2 Recipient of USPHS Research Fellowship 5F32 HL05684-03. To whom

requests for reprints should be addressed.3 Recipient of USPHS Grant HD 04445.4 Recipient of USPHS Grant CA 24146.5 The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PB,

phÃ©nobarbital; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline

inducers of certain isozymes. In this study, affinity-purifiedantibodies to 4 forms of cytochrome P-450 (Forms 2, 3, 4 and6)6 were used to localize these isozymes by indirect immuno

fluorescence (45) in kidney, lung, and liver from untreatedrabbits and rabbits pretreated with either TCDD (33) or PB.Although others have used immunofluorescence techniques todemonstrate multiple forms of cytochrome P-450 in the liver(3) and lung (39), heretofore, no such broad-scale immunocy-tochemical demonstration of the localization of the cyto-

chromes has been reported. Our results indicate that therelative distribution of the isozymes can differ within a tissueand that this balance can be altered by PB and TCDD.

MATERIALS AND METHODS

Preparation of Antibodies. The development of goat antisera toeach of the cytochromes and the preparation of IgG fractions fromthese antisera are described in earlier publications (17, 20, 22, 31,36). Each of the antibodies was further purified by affinity chromatog-

raphy as described by Noshiro and Omura (32), using the appropriatepurified cytochrome linked to Sepharose 4B (Pharmacia). Their procedure was modified somewhat to include 0.4 M potassium phosphate,pH 7.7, in the coupling buffer and a ratio of 0.6 mg of the purifiedantigen per ml of Sepharose. Ouchterlony Â¡mmunodiffusion was usedto monitor the adsorption of antibody from the IgG fraction and itselution from the column.

Animals. Adult male New Zealand White rabbits were treated witheither PB (J. T. Baker Chemical Co.) or TCDD (a gift from Dow ChemicalCo.). PB was administered in the drinking water [0.1% PB (w/v)] adlibitum for 5 days; the animals were fasted during the last 2 days oftreatment to deplete the liver of glycogen and were killed on the sixthday. TCDD was administered by a single injection (i.p.) of 30 nmolTCDD per kg body weight as a 0.3 rnM solution in dioxane 5 days priorto killing. Control animals were either given drinking water containingno additives or were given injections of 0.9% NaCI solution.

Preparation of Tissues for Immunofluorescence. Kidneys, lungs,and liver from control, PB-, or TCDD-pretreated rabbits were excised

immediately after the animal was killed, and a portion of each organwas removed by dissection, placed in a plastic vial, and snap frozen inliquid nitrogen. In more recent experiments, gelatin was injected intothe lungs prior to freezing (41 ) in order to keep the alveoli open, sinceunsupported lungs tend to compress upon sectioning in the cryostat.Frozen sections (5 /im) were cut, mounted on glass slides, air driedunder a fan, wrapped in aluminum foil, and stored in a freezer (nolonger than 3 to 4 days) until the immunofluorescent staining wasperformed.

solution (2.7 row KCI-1.5 mM KH2PO4-8 mM Na2HPCv0.137 M NaCI); 3-MC, 3-methylcholanthrene; AAF, 2-acetylaminofluorene.

6 Form 2 or P-450LM2(13, 42, 43) is induciblo in rabbit liver microsomes byPB; Form 3 or P-450LÂ«3ois constitutive and not inducible by PB or TCDD in adultrabbit liver microsomes (17); Form 4 or P-450i.M4 is inducible in adult rabbit livermicrosomes by /3-naphthoflavone (7), 3-MC (25, 35), and TCDD (19, 20); andForm 6 is the predominant form induced by TCDD in neonatal rabbit livermicrosomes (19, 20).

APRIL 1982 1423

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J. H. Dees et al.

Indirect Immunofluorescence. In the first step of the procedure, 2to 3 drops of unlabeled, affinity-purified goat antibodies to each formof cytochrome P-450 were placed on the thawed cryostat sections,

and the slides were incubated for 30 min at room temperature in amoist chamber. All affinity-purified antibodies were used at a concen

tration of 0.05 mg/ml. This concentration was selected for all 4antibodies as the result of preliminary experiments using the chessboard titration technique of Beutner (5). After two 10-min washes inPBS, the slides were incubated for 30 min with FITC-labeled, rabbitanti-goat IgG (Miles Laboratories, Elkhart, Ind.) diluted 1:10 with PBS.

The slides were again washed twice with PBS, coverslipped withglycerol:PBS (9:1), and examined.

Immunocytochemical Controls. Routinely, 3 immunocytochemicalcontrols were performed on each tissue: (a) an unstained section ofthe tissue was included to detect autofluorescence of the tissue; (to)the primary antibody was omitted to demonstrate the degree of nonspecific binding of the FITC-labeled, rabbit anti-goat IgG to the tissue;

and (c) nonimmune goat IgG was used as the primary antibody in orderto show that IgG from goats not previously exposed to the specificantigen does not bind appreciably to the tissue. In addition, specificblocking studies were performed in which the primary antibody waspreincubated with the autologous and heterologous purified forms ofcytochrome P-450 in order to show that preincubation with only the

autologous antigen provided blocking of the binding site of the primaryantibody.

Fluorescence Microscopy and Photography. Tissues were examined using a Leitz Orthoplan photomicroscope equipped with a mercurysource and epiillumination for fluorescence microscopy. In each experiment, the optimal exposure was determined for each tissue for theantibody-tissue combination providing the brightest fluorescence, and

then all other conditions for that tissue were photographed at thatexposure, providing a visual record of the relative degrees of fluorescence seen with each of the antibodies for that tissue. These comparative sequences were photographed on one roll of Tri-X Pan film,

(Kodak, ASA 400), developed in Microdol X (Kodak), and the entireroll was printed on the same grade paper at the same f-stop and

exposure time as determined for the brightest frame in that roll. Printsmade from other rolls of film processed separately were matched tothese comparative rolls in preparing the photomicrographs for thisreport.

RESULTS

Kidney. In the normal rabbit kidney (Fig. 1, d, g, j, and m),anti-Form 2 stained the pars recta of the proximal tubules and

lower portion of the pars convoluta intensely; however, themain portion of the pars convoluta did not stain (Fig. 1d). Incontrast to the rather limited distribution of Form 2, Form 3 waspresent in both the pars convoluta and the pars recta of theproximal tubules (Fig. 1g). In both cases, the glomeruli, distaltubules, and collecting ducts were negative (Fig. 1, d and g).Immunofluorescence with anti-Form 4 and anti-Form 6 was

very weak to negative throughout the normal kidney (Fig. 1, jand m), although occasional spotty staining of the P3M7 seg

ment could be seen. In the normal kidney, renal arteries showedautofluorescence of the internal elastic lamina, but no specificfluorescence was seen using antibody to any of the 4 forms of

' Maunsbach (28) described the segmentation of the proximal tubule of rat

kidney as follows: the Si segment is the initial portion of the proximal convolutedtubule, the S2 segment is the main portion of the pars convoluta and the beginningof the descending straight portion, and the S3 segment comprises the descendingstraight portion or pars recta. Jacobson and Jorgensen (15) further divided theS3 or, as they called it, P3 segment into the portion present in the medullary raysof the cortex (P3C), and the portion present in the outer stripe of the outer medulla(P3M). We will use the nomenclature P,, P2, P3C, and P3M in accordance withthese definitions. Unless specified, P3 will refer to both P3C and P3M.

cytochrome P-450 (arteries of normal kidney stained for Forms

4 and 6 shown in Fig. 4, b and c). Pretreatment of the animalwith PB (Fig. 1, e, h, k, and n) did not significantly alter thepattern of the immunofluorescence relative to untreated animals, resulting only in a slight increase Â¡nthe intensity ofstaining seen with anti-Form 2 (Fig. 1e). Immunocytochemical

control sections of the kidney were negative (Fig. 1, a to c).In kidneys from TCDD-pretreated animals (Fig. 1, f, i, /, and

o), staining for Forms 2 and 3 in the proximal tubules (Fig. 1,f and /, respectively) was weak to negative in contrast to controlanimals. No staining with either anti-Form 2 or anti-Form 3 was

seen in the glomeruli, distal tubules, collecting ducts, or renalvasculature. On the other hand, specific fluorescence forForms 4 and 6 was moderately intense and intense, respectively, in the pars convoluta and pars recta of the proximaltubules (Fig. 1, / and o, respectively), in contrast to tissue fromcontrol animals. The endothelium of the renal arteries stainedintensely for Forms 4 and 6 (Fig. 4, d, e, and f), and there wasalso a faint fluorescence present in cells of the glomeruli whenstained for Forms 4 or 6 (Fig. 1, / and o; Fig. 4f).

Lung. In the untreated (control) rabbit lung (Fig. 2, d, g, j,and m), parenchymal cells displayed strong immunofluorescence with anti-Form 2 (Fig. 2d). Identification of these celltypes was clarified in the gelatin-infiltrated lungs (Fig. 2d,

inset), where parallel thin lines of fluorescence outlining thealveolar airspace could be clearly discerned. This is stronglysuggestive of staining of the type I pneumocytes on either sideof the alveolar wall. In addition, occasional cells which protrudeinto the alveolar lumen showed specific fluorescence and mayrepresent staining of type II or granular pneumocytes (Fig. 2d,inset, arrow). The bronchioles revealed weak to moderatestaining with some nonspecific binding to the apex of thebronchiolar epithelial cells. The pulmonary endothelial cellswere negative for cytochrome P-450, Form 2 (Fig. 41). Stainingwith anti-Forms 3 (Fig. 2g), 4 (Fig. 2j), and 6 (Fig. 2m) was

essentially negative with normal lungs. PB pretreatment did notalter the pattern of occurrence for the 4 isozymes in the lung(Fig. 2, e, h, k, and n). Control sections of normal lung showedonly minimal fluorescence (Fig. 2, a to c).

In the lungs from TCDD-pretreated rabbits (Fig. 2, f, i, I, ando), anti-Form 2 stained the parenchyma in a manner similar to

that seen Â¡nthe normal lungs (Fig. 2, f and inset). The bronchioles stained weakly with some apical nonspecific fluorescence of the bronchiolar epithelium. Anti-Form 3 was negativein both the small airways and parenchyma (Fig. 2/'). Anti-Form

4 gave a positive fluorescence in the parenchyma (Fig. 21);however, in gelatin-inflated lungs, no evidence of a parallel

staining pattern was present, but rather a single row of fluorescence, suggesting staining of components of the interstitium ofthe alveolar septum, possibly of the capillary endothelium, wasseen (anti-Form 4 not shown; see Fig. 2o, inset, anti-Form 6,

for a similar pattern). The bronchi and bronchioles stained veryweakly with antibodies to Form 4 (Fig. 21). Anti-Form 6 gave a

fluorescent staining of the parenchyma similar to that seen withanti-Form 4 (Fig. 2o, inset), but it additionally stained thebronchioles and bronchi weakly to moderately (Fig. 2o). As inthe kidney, the endothelium of the pulmonary arteries and veinswas intensely fluorescent with both anti-Form 4 (Fig. 4, o andp) and anti-Form 6 (Fig. 2o; Fig. 4, q and r).

Liver. In the normal rabbit liver (Fig. 3, d, g, j, and m),staining with anti-Form 2 (Fig. 3d) and anti-Form 3 (Fig. 3g)

1424 CANCER RESEARCH VOL. 42



Cytochrome P-450 Isozymes in Rabbit Tissues

elicited strong and moderate specific fluorescence, respectively, throughout the liver lobule. Neither form was detected inthe vascular endothelium or the connective tissue. Forms 4(Fig. 3y) and 6 (Fig. 3m) were distributed unevenly in the liversof normal rabbits, showing greater fluorescence in the centri-

lobular and midzonal regions than in the periportal region,where the fluorescence was markedly less intense. The contrast between the centrilobular and periportal zones wasgreater with anti-Form 4 than with anti-Form 6. No staining of

the vascular endothelium or the connective tissue was seen inthe normal liver with antibodies to Forms 4 or 6 (Form 6 shownin Fig. 4, g and /). Immunocytochemical control sections ofnormal liver (Fig. 3, a to c) were essentially negative, as will bediscussed later.

When livers from PB-treated animals were examined (Fig. 3,e, h, k, and n), the intensity of staining for Form 2 was greatlyincreased (Fig. 3e), whereas the patterns observed for Forms3, 4, and 6 were similar to that seen with normal liver (Fig. 3,h, k, and n, respectively). On the other hand, when the animalswere pretreated with TCDD (Fig. 3, /, /', /, and o), livers stained

with anti-Form 4 (Fig. 3/) or anti-Form 6 (Fig. 3o) showedstrong immunofluorescence throughout the liver lobules, Â¡ncontrast to their uneven distribution in the normal livers. Theyalso exhibited weak, but uniformly distributed, specific fluorescence when stained with anti-Form 2 (Fig. 30 or anti-Form 3

(Fig. 3/), in marked contrast to the strong to moderate specificfluorescence seen Â¡nthe normal livers. Staining with antibodiesto Forms 4 and 6 could be occasionally recognized in theendothelium of the portal vasculature and bile ducts (Fig. 4y);however, the endothelial staining was not as prominent as thatseen in the kidney and lungs of TCDD-pretreated rabbits. Faint

positive fluorescence for these forms was occasionally seen inthe endothelium of the central veins (Fig. 4ft); however, thesinusoidal endothelium did not appear to be positive.

With regard to immunocytochemical controls, unstained sections of all tissues were negative with only minimal autofluores-cence (Figs. 1a, 2a, and 3a). In the hepatocytes, occasionalautofluorescent granules were seen in the cytoplasm. In thekidney, autofluorescent granules were present Â¡nthe proximaltubular cells. In the lung, elastin was autofluorescent, appearing as wavy lines Â¡nthe alveolar septae and at the base of theairways (Fig. 2, a to c). In all tissues, the internal elastic laminaof arteries and arterioles was autofluorescent (Fig. 4, a to e,and k to r). Sections treated only with the FITC-conjugated

secondary antibody were also negative (Figs. 10, 2b, and 30),except that occasional cells, presumed to be macrophages,seemed to bind the antibody nonspecifically; this was mostnoticeable in the lung. Substitution of nonimmune goat IgG forthe primary antibody gave negligible staining in the kidney (Fig.1c), liver (Fig. 2c), and lung (Fig. 3c). In the lung, a smallamount of nonspecific binding to the apex of the bronchiolarcells was generally observed on both secondary antibody only(Fig. 2Â£>)and nonimmune IgG preparations (Fig. 2c). In theblocking experiment, in which primary antibodies were prein-

cubated with their autologous and heterologous antigens, adiminution of fluorescence intensity was seen only in the caseof primary antibody blocked with autologous antigen (notshown).

DISCUSSIONWe have shown that 4 forms of cytochrome P-450 are

localized Â¡nspecific cell types in kidney, lung, and liver andthat this distribution differs for the individual forms. Pretreatment of the animals with TCDD, and to a lesser extent with PB,dramatically alters the pattern of occurrence.

The importance of the distribution of cytochrome P-450isozymes in various tissues relates not only to the metabolismof steroids and other endogenous substances, as well as tothe metabolism of therapeutic drugs, but also to the detoxica-tion-activation of a number of toxic xenobiotic compounds (30,

34), including many carcinogens (14, 29). Recently, it hasbeen shown that different forms of cytochrome P-450 contribute to different extents to the metabolic activation of variouschemical carcinogens (4, 24, 47) and may catalyze divergentpathways of metabolism of these agents (18). Therefore, thebalance of cytochrome P-450 isoenzymes in a tissue or cell

type will influence the outcome of exposure of that tissue to agiven carcinogen.

Cytochrome P-450 and related drug-metabolizing enzymes

have been studied extensively in the liver of several animalspecies. Less is known about the localization of cytochrome P-450-mediated enzyme systems in extrahepatic tissues, partic

ularly the kidney. We have shown that the Form 2 and Form 3isoenzymes of cytochrome P-450 are present in the pars recta

and pars convoluta of the proximal tubules of kidneys fromuntreated and PB-pretreated animals. Forms 4 and 6 becomepredominant in the renal proximal tubules of TCDD-pretreated

rabbits. These results are supported by immunohistochemicaland biochemical studies in the same (48) and other species(8-10,27,44).

Utilizing antibody to liver microsomal NADPH-cytochrome c(P-450) reductase as an indicator of the presence of cytochrome P-450-mediated systems, Dees et al. (8, 9) demonstrated its localization in proximal tubules of rat and miniaturepig kidneys following 0.9% NaCI solution or PB pretreatment.Specific fluorescence was limited to the cortex and outer stripeof the outer medulla and was seen in all segments of theproximal tubule, appearing the strongest in the P2 and P3segments. Masters ef al. (27) reported the localization of thecytochrome P-450 isolated from the livers of PB-pretreatedminiature pigs and the cytochrome P-450 obtained from untreated pig kidney in sections of kidney from PB-pretreated

miniature pigs. The antibody to the miniature pig liver cytochrome P-450 stained the P2 and P3 segments of the proximaltubule, but the antibody to the pig kidney cytochrome P-450

stained the P,, P2, and P3 segments of the proximal tubule. Ourlocalization, in the present study, of Forms 2 and 3 in the P2and P3 segments of the proximal tubules of normal and PB-pretreated rabbits shows that the site of cytochrome P-450-

mediated enzyme systems in rabbit kidney is similar to thatdemonstrated for the rat and miniature pig kidney.

In 1962, Wattenberg and Leong (44) demonstrated the presence of reduced pyridine nucleotide-dependent polycyclic hydrocarbon-metabolizing systems in various tissues from several

animal species. They found that, although the fluorescenceemitted by the polycyclic hydrocarbon metabolites was onlyweakly demonstrable in normal rat kidneys, the histochemicalreaction was intensified in kidneys from 3-MC-pretreated rats

(44) and was localized in the distal portion of the proximaltubule, i.e., the pars recta. Since polycyclic hydrocarbons areknown inducers of Form 4 (and in the case of TCDD, Form 6),their results are compatible with the present findings that

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J. H. Dees et al.

antibodies to Forms 4 and 6 stain the kidneys of normal andPB-pretreated rabbits only minimally but stain the pars recta

and pars convoluta of the proximal tubules of kidneys fromTCDD-pretreated rabbits intensely. The work of Fowler ef al.

(10) is also compatible with our results. They showed in renaldissection studies that the induction of benzo(a)pyrene hydrox-

ylase activity by TCDD was highest in the renal cortex andouter stripe of the medulla, a location paralleling the distributionof the P3 segment of the proximal convoluted tubule. A markedproliferation of the smooth endoplasmic reticulum in cells ofthe P3 segment as a result of a single dose of TCDD was seenby electron microscopy (10). Zenser ef al. (48) demonstratedbiochemically that aryl hydrocarbon hydroxylase activity wasnot measurable in control rabbit kidney, but 3-MC pretreatment

induced its activity in cortical and outer medullary microsomes.Cytochrome P-450 content was detected in kidney cortex inboth untreated and 3-MC-pretreated rabbits but was identifiedin the outer medulla only in 3-MC-pretreated rabbits (48).

In contrast to the kidney, Form 2 was the only isoenzymedetected in the lungs of normal and PB-pretreated rabbits and

was seen in bronchial and bronchiolar epithelium as well as intype I and possibly type II alveolar pneumocytes. Boyd (6),using autoradiography, has suggested that the Clara cell is thesite of cytochrome P-450-dependent monooxygenase activityin the lung. Dees et al. (8, 9) detected NADPH-cytochrome c(P-450) reducÃase in bronchi and bronchioles of rat and mini

ature pig lungs as well as in a parenchymal cell type(s).Recently, Guengerich (11 ) and Philpot ef al. (46) have re

ported the purification of cytochrome P-450 from lungs ofuntreated rabbits. Philpot's laboratory (38, 39) reported that

one pulmonary form of cytochrome P-450 (P-450,), indistinguishable from the PB-induced hepatic form (37), i.e.. Form 2,

is present in Clara cells of rabbit lung but could not be localizedin either ciliated bronchiolar epithelial or alveolar epithelialcells. Antibodies to a second form of pulmonary cytochrome P-450 (P-450ii) also stained the Clara cell but gave additional

indications of alveolar type II pneumocyte localization (39).Masters ef al. (27) have shown that, although antibodies toliver cytochrome P-450 from PB-pretreated miniature pigs stainthe lung in a pattern similar to that of anti-NADPH-cytochromeP-450-reductase, the antibodies to cytochrome P-450 from

untreated pig kidneys do not stain bronchi or bronchioles butdo stain a parenchymal cell type(s). These studies suggest thatmultiple cytochrome P-450's are present in bronchioles and

alveolar lining cells of the lungs of various species, ana theyemphasize that a form similar to the PB-inducible form found in

the liver is present in the lungs of normal rats, rabbits, andminiature pigs.

The response of the rabbit lung to TCDD-pretreatment is

similar to that seen in the kidney, since Forms 4 and 6 appear.With regard to other work on the induction of cytochrome P-

450 by polycyclic hydrocarbons, Wattenberg and Leong (44)first observed a positive reaction for aryl hydrocarbon hydroxylase activity in the alveolar walls of rats pretreated with 3-MC

but not in normal rats. Liem ef al. (26) found that TCDD inducedonly Form 6 in adult rabbit kidney and lung, but the presentdata indicate a weak but positive induction of Form 4, also. Toeliminate any possible cross-reactivity of the antibodies toForm 4 and Form 6, affinity-purified anti-Form 4 was passed

over a column containing immobilized Form 6. No markeddifference in staining was noted between the immunoabsorbed

anti-Form 4 and the anti-Form 4 which had not been immunoabsorbed. It is probable, therefore, that our Â¡mmunohisto-chemical studies afford an increased sensitivity over the standard biochemical and and immunochemical assays previouslyused. The present study has shown that, while the PB-induciblecytochrome P-450 isozyme, Form 2, is the predominant formin the lung in normal and PB-treated rabbits, 3 forms areapparent in the lungs of TCDD-pretreated rabbits (Forms 2, 4,

and 6). It is probable that additional forms will be identified andlocalized in lung and other organs in future studies.

In the normal liver, it is surprising that the immunofluorescentstaining for Form 2 was moderately strong, considering thatCoon ef a/. (7) reported that P-450LM2 is present only in trace

amounts in untreated adult rabbit liver. On the other hand, thepresence of Forms 3, 4, and 6 in normal rabbit liver can beexpected from the literature (12,17,19-21,31 ). The increasedimmunofluorescence for Form 2 in the livers of PB-pretreatedrabbits correlates well with the fact that Form 2 is the majorPB-inducible form of cytochrome P-450 (42, 43). Form 2

exhibits a fairly uniform distribution in the livers of normal andPB-pretreated rabbits, differing from the results of Baron ef al.(3) in rat livers in which antibodies to a PB-inducible livercytochrome P-450 showed more intense staining of the centri-

lobular regions than of the periportal zones. The differences inthese 2 animals may reflect a species variability or a differencein sensitivity of the methods used to quantitate the results.Recently, Masters ef al. (27) have reported that antibodies tocytochromes P-450 isolated from PB-pretreated miniature pigliver and untreated abattoir pig kidney, respectively, stain liversfrom PB-pretreated miniature pigs, indicating that both formsare present in the livers of these animals. NADPH-cytochromec (P-450) reducÃase has been localized by immunofluores-

cence techniques in rat and miniature pig tissues (2, 8, 9)throughout the liver lobule in untreated or PB-induced animals.The staining was more intense in the centrilobular and midzonalregions than in the periportal regions and increased in intensityin the PB-pretreated animals. Our results with TCDD-induced

rabbits show that in the liver, as in the kidney, Forms 4 and 6stain intensely, while Form 2 is markedly reduced. These dataare compatible with the results of Baron ef al. (3) for 3-MC-pretreated rats, in which the 3-MC-inducible form of cytochrome P-450 increased in all regions of the lobule, while thePB-inducible form decreased.

One of the most striking findings in the TCDD-induced animals was the induction of Forms 4 and 6 in the vascularendothelium of liver, lung, and kidney. This has some precedentin the literature in that Juchau ef al. (23) were able to detectcytochrome P-450 and aryl 4-monooxygenase activity in aortic

microsomes from rabbits, monkeys, and humans. They suggested that the presence of these enzymes in the aortic wallmight play a role in the pathogenesis of atherosclerosis, possibly through a mutagenic event causing a proliferation ofsmooth muscle cells in the aortic wall (23). However, they wereunable to identify the cell type(s) containing these enzymes(23). Baird ef al. (1) reported that cultured aortic endothelialand bovine lung fibroblast-like cells metabolize benzo(a)pyrene

to a proximate carcinogen and suggested that researchersmust take into account the possible metabolic activation ofhydrocarbons and other chemicals by endothelial cells in predicting the outcome of exposure to these agents in humansand animals (1).




Cytochrome P-450 Isozymes in Rabbit Tissues

In summary, our results indicate that Forms 2 and 3 arepredominant in normal and PB-pretreated liver and kidney,

while Forms 4 and 6 are not as prevalent in these tissues. Form2 appears to be constitutive in the lung. In animals exposed toTCDD, immunofluorescent staining for Forms 4 and 6 becomespredominant in kidney, lung, and liver. This is of particularinterest in view of the recent report by Johnson ef a/. (18), whohave shown that multiple forms of cytochrome P-450 catalyzedivergent pathways of AAF metabolism. A/-Hydroxylation, rep

resenting activation of AAF, is accomplished mainly by Form4, whereas ring hydroxylation leading to detoxication is carriedout by Forms 3 and 6. Form 2 does not appear to contribute tothe metabolism of AAF. They suggested that since Form 6 isthe major TCDD-inducible form in neonatal liver microsomes,AAF will be primarily detoxified by neonatal rabbit liver,whereas the presence of Form 4 as the major form of cytochrome P-450 in liver microsomes from TCDD-pretreated adult

animals is associated with increased activation of AAF in thelivers of these animals (18). Thus, the balance of activating anddetoxifying forms of cytochrome P-450 in different organs andunder different conditions may determine the tissue-specific

outcome of exposure of an animal to a given carcinogen.Information, such as presented here, on the presence or absence of these forms of cytochrome P-450 in specific regions

of an organ in the normal animal and in response to variousinducing agents may clarify the reasons why particular tissuesbecome target tissues for certain carcinogens and why tumorsoriginate in a particular cell type.

ACKNOWLEDGMENTS

We are grateful to Dr. James Gilliam, Chairman, Division of Dermatology,Department of Internal Medicine, The University of Texas Health Science Centerat Dallas, for the use of his cryostat and fluorescence microscope. The expertiseof Kendis Cox and Dr. Heng H. Liem are gratefully acknowledged.

REFERENCES

1. Baird, W. M., Chemerys, R., Grinspan, J. B., Mueller, S. N., and Levine, E.M. BenzcKa)pyrene metabolism in bovine aortic endothelial and bovine lungfibroblast-like cell cultures. Cancer Res., 40: 1781-1786, 1980.

2. Baron, J., Redick, J. A., Greenspan, P., and Taira, Y. Immunohistochemicallocalization of NADPH-cytochrome c reducÃase in rat liver. Life Sci., 22:1097-1102, 1978.

3. Baron, J., Redick, J. A., Kapke, G. F., and Guengerich, F. P. Effects ofphÃ©nobarbital and 3-methylcholanthrene on the distributions of cytochromesP-450 in rat liver. Fed. Proc., 38. 660, 1979.

4. Belvedere, G., Miller, H., Vatsis, K. P., Coon, M. J., and Gelboin, H. V.Hydroxylation of benzo(a)pyrene and binding of (â€”)trans-7-8-dihydroxy-7,8-

dihydrobenzcKa)pyrene metabolites to deoxyribonucleic acid catalyzed bypurified forms of rabbit liver microsomal cytochrome P-450. Biochem. Phar-macol., 29. 1693-1702, 1980.

5. Beutner, E. H. Defined immunofluorescent staining: past progress, presentstatus, and future prospects for defined conjugates. Ann. N. Y. Acad. Sci.,J77: 506-526. 1971.

6. Boyd, M. R. Evidence for the Clara cell as a site of cytochrome-P-450-dependent mixed function oxidase activity in the lung. Nature (Lond.), 269:713-715, 1977.

7. Coon, M. J., Vermilion, J. L, Vatsis, K. P., French, J. S., Dean, W. L, andHaugen, D. A. Biochemical studies on drug metabolism: isolation of multipleforms of liver microsomal cytochrome P-450. In: D. M. Jerina (ed.), DrugMetabolism Concepts, ACS Symposium Series. Vol. 44, pp. 46-71. Washington, D. C.: American Chemical Society, 1977.

8. Dees, J. H., Coe, L. D., Yasukochi, Y., and Masters, B. S. S. Immunofluo-rescence of NADPH-cytochrome c (P-450) reducÃase in rat and minipigtissues injected with phÃ©nobarbital. Science (Wash. D. C.), 208: 1473-

1475, 1980.9. Dees, J. H., Yasukochi, Y., Okita, R. T., Parkhill, L. K., Coe, L. D., Key, B.

A., Patrizi, V. W., and Masters, B. S. S. Immunohistochemical localization ofNADPH-dependent cytochrome c (P-450) reducÃasein liver, lung and kidneyof phÃ©nobarbital- versus saline-pretreated rats: correlation with ultrastruc

tural and biochemical observations. In: M. J. Coon, A. H. Conney, R. W.Estabrook, H. V. Gelboin, J. R. Gillette, and P. J. O'Brien (eds.), Microsomes,

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induction of renal microsomal enzyme systems: ultrastructural effects onpars recta (S3) proximal tubule cells of the rat kidney. J. Pharmacol. Exp.Ther., 203. 712-721, 1977.

11. Guengerich, F. P. Preparation and properties of highly purified cytochromeP-450 and NADPH-cytochrome P-450 reducÃase from pulmonary microsomes of untreated rabbits. Mol. Pharmacol., ÃŽ3:911-923, 1977.

12. Haugen, D. A., and Coon, M. J. Properties of electrophoretically homogeneous phenobarbital-inducible and /9-napthoflavone-inducible forms of livermicrosomal cytochrome P-450. J. Biol. Chem., 25). 7929-7939, 1976.

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descendens of the proximal tubule in the kidney of the male rat. Z. Zellforsch.Mikrosk. Anat., 136: 479-499, 1973.

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18. Johnson, E. F., Levitt, D. S., Muller-Eberhard. U., and Thorgeirsson. S. S.Evidence that multiple forms of cytochrome P-450 catalyze divergent pathways of 2-acetylaminofluorene metabolism. Cancer Res., 40: 4456-4459,1980.

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Fig. 1. Kidney. Top row, control sections of normal rabbit kidney: a. unstained; b, stained with secondary antibody only; c. stained with nonimmune goat IgG,followed by FITC-conjugated rabbit anti-goat IgG. Beginning with Row 2, the left column is from untreated rabbits (d, g, /, m). the middle column is from PB-pretreatedrabbits (e, h, k, n), and the right column is from TCDD-pretreated rabbit kidneys (f, i. I, o). flow 2 (d, e, O was stained for Form 2, flow 3 (g, h, /) was stained for Form3, Row 4 (i, k, /) was stained for Form 4. and flow 5 (m, n, o) was stained for Form 6. P2, pars convoluta of the proximal tubule; P3 pars recta of the proximal tubule;G, glomerulus; A, arteriole.

APRIL 1982 1429



Fig. 2. Lung. Top row, control sections of normal rabbit lung: a, unstained; fa, stained with secondary antibody only; c, stained with nonimmune goat IgG, followedby FITC-conjugated rabbit-anti-goat IgG. In the section stained with nonimmune IgG (c), there is some nonspecific staining of the arteriolar smooth muscle and theapex of the bronchiolar cells. Row 2 (d, e, f) was stained for Form 2. flow 3 (g, h, /) was stained for Form 3 and is negative. Row 4 (j, k, /)was stained for Form 4,and flow 5 (m, n, o) was stained for Form 6. Beginning at flow 2, left column is from lungs of untreated rabbits (d, g, j, m), middle column (e, h. H, n) is from PB-pretreated rabbits, and right column ( f, i, I, o) is from lungs of TCDD-pretreated rabbits. B, bronchioles; P, parenchyma; A, arteriole; V, venule. Insets are from gelatin-inflated lung preparations. Note that in the normal lung stained for Form 2 (Inset d) the fine, parallel staining pattern is probably indicative of staining of type Ipneumocytes. Lungs of PB-pretreated (gelatin preparation not shown) and TCDD-pretreated (Inset f) rabbits show a similar staining pattern when stained for Form 2.In the lungs of TCDD-pretreated rabbits stained for Forms 4 (gelatin preparation not shown) and 6 (Inset o), the pattern of fluorescence is no longer a parallel one butrather seems to stain the interstitial region of the alveolar septum.

1430



* V T* .^Wfc&T** :

Fig. 3. Liver. Top row, control sections of normal rabbit liver: a, unstained; b, stained with secondary antibody only; c. stained with nonimmune goat IgG, followedby FITC-conjugated rabbit anti-goat IgG. Beginning at Row 2. the left column is from untreated rabbits (d, g. /, m), the middle column is from PB-pretreated rabbits(e, h, k. n), and the right column is from rabbits pretreated with TCDD ( I, i. I, o). Row 2 of sections (d, e. O was stained with anti-Form 2, Row 3 (g, h, i) was stainedwith anti-Form 3. Row 4 (j, k, 0 was stained with anti-Form 4, and Row 5 (m, n, o) was stained with anti-Form 6. CV, central vein; PP, periportal zone; MZ, midzonalregion.

APRIL 1982 1431



J. H. Dees et al.

Fig. 4. Vascular staining in kidney, liver, and lung. Top row, kidney, a, control section of a kidney from a TCDD-pretreated rabbit, stained with secondary antibodyonly, showing autofluorescence of the internal elastic lamina (arrowhead) but no specific fluorescence of the endothelium (arrow), b and c, sections from kidneys ofcontrol rabbits, stained for Forms 4 and 6, respectively. Although the internal elastic lamina is autofluorescent, no specific staining of the endothelium is seen, d ande. sections from kidneys of TCDD-pretreated rabbits, stained for Forms 4 and 6, respectively. Staining of the endothelial cells (arrows) is evident on the luminal sideof the internal elastic lamina (arrowheads'), f, section of kidney from a TCDD-pretreated rabbit, stained for cytochrome P-450 Form 6. Here the endothelial stainingin the afferent and efferent arterioles (A1) is quite prominent and can be seen contiguous with the glomerulus (G'). Middle row, liver, g, section of normal liver stainedfor Form 6; no endothelial staining is seen, h, section from the liver of a TCDD-pretreated rabbit stained for Form 6; faint immunofluorescent staining of theendothelium of the central vein (CV) is seen (arrows). ;', periportal region of a normal rabbit liver stained for Form 6; no staining is seen in the portal tract, portal vein

(PV). j, periportal region of liver from a TCDD-pretreated rabbit stained for Form 6; specific fluorescence is seen in the endothelium of the portal vein (PV), in a portalarteriole (A), and in some of the epithelial cells lining the bile duct (6D). Bottom 2 rows, lung. Although the internal elastic lamina appears autofluorescent(arrowheads), no endothelial fluorescence is seen in unstained sections of control lung (k), or in normal rabbit lung stained for Form 2 (/), Form 3 (see Fig. 2g), Form4 (m), or Form 6 (n). However, when lung tissue from TCDD-pretreated rabbits is stained for Form 4 (o and p) and Form 6 (q and r), prominent staining of the

endothelium (arrows) is seen in both arteries (A) and veins (V), separate from the autofluorescence of the internal elastic lamina (arrowheads).




1982;42:1423-1432. Cancer Res Jane H. Dees, Bettie Sue Siler Masters, Ursula Muller-Eberhard, et al. Isozymes in Rabbit Kidney, Lung, and Liveron the Occurrence and Distribution of Four Cytochrome P-450

-dioxin and PhenobarbitalpEffect of 2,3,7,8-Tetrachlorodibenzo-

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Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and … · 2017-05-03 · of four cytochrome P-450 isozymes, Forms 2, 3, 4, and 6, in the kidney, lung, and liver of adult male rabbits