[CANCER RESEARCH 52. 3436-3442. June 15. 1992]

Dose-Response Relationships for Chronic Exposure to 2,3,7,8-Tetrachlorodibenzo-

/7-dioxin in a Rat Tumor Promotion Model: Quantification and

Immunolocalization of CYP1A1 and CYP1A2 in the LiverAngelika M. Tritscher,1 Joyce A. Goldstein, Christopher J. Portier, Zadock McCoy, George C. Clark,

and George W. LucierNational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina USA 27709

ABSTRACT'I lio mechanisms responsible for the broad spectrum of effects of

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are not entirely clear butseem to involve an initial interaction with the Ah receptor. A majoruncertainty in risk assessment for TCDD is the lack of adequate dose-response relationships following chronic exposure to TCDD. Inductionofcytochrome P-450 enzymes (CVP1 Al andCYPlA2)isoneof the mostsensitive responses to TCDD and its structural analogues. We have useda two-stage model for hepatocarcinogenesis in female Sprague-Dawleyrats to evaluate dose-response relationships for induction of CYP1A1and CYPIA2 in diethylnitrosamine-initiated as well as in noninitiatedrats. After initiation with a single dose of diethylnitrosamine, TCDD wasadministered biweekly by p.o. gavage at doses equivalent to 3.5, 10.7,35.7, and 125 ng/kg/day for 30 weeks. CYP1 Al and CYP1A2 concentrations were quantified in hepatic microsomes by radioimmunoassay andlocalized in hepatic tissue slices by immunohistochemical techniques.Radioimmunoassay data revealed a maximum induction of 200-fold forCYP1A1 and 10-fold for CYP1A2 and there were no statistically significant differences between initiated and noninitiated rats. Induction at thelowest dose (3.5 ng/kg/day) was 20-fold for CYP1A1 and 3-fold forCYP1A2. Mathematical analysis indicates that the best fit of the induction data are inconsistent with a threshold for this response. There wasa linear relationship between administered dose and TCDD liver concentration over the entire dose range of the study. This indicates thatinduction of CYP1A2 does not significantly alter the distribution ofTCDD in our chronic dosing regimen. Immunolocalization of CY'PIAI

and CYP1A2 revealed the same localization and induction pattern forboth isozymes in the cytoplasm of hepatocytes. However, the hepaticdistribution pattern was not uniform with the most intense stainingobserved around central veins. These studies help to clarify dose-responserelationships for dioxin-mediated effects and demonstrate different sensitivity of hepatocytes to the effects of TCDD.

INTRODUCTION

TCDD2 is the most toxic congener of a class of polyhalogen-

ated aromatic compounds (dioxins) that are ubiquitous environmental pollutants. The dioxins are biologically and ecologicallypersistent and are produced inadvertently during pesticide production, during bleaching processes, and through combustionin waste incinerators or diesel engines. TCDD is one of themost toxic chemicals tested in laboratory animals and it is apotent multisite carcinogen (1, 2).

In long-term bioassays, TCDD increases hepatic tumor incidence in female rats but not in male rats (1, 2). A positive dose-response relationship was apparent with a lowest observed effectat 10 ng/kg/day. TCDD is considered to be a nongenotoxic

Received 12/26/91; accepted 4/7/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' To whom requests for reprints should be addressed, at NIEHS, MD D4-01.P. O. Box 12233, Research Triangle Park. NC 27709.

•¿�The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-/>-dioxin;DEN. diethylnitrosamine; CYP1A1. cytochrome P4501A1; CYP1A2, cyto-chrome P4501A2: RIA, radioimmunoassay; EROD, ethoxyresorufin-O-deethyl-ase; BSA. bovine serum albumin; ED,,,, 50?c effective dose.

carcinogen since no DNA adducts are detected using methodsthat are capable of detecting 1 adduci in 10" normal nucleotides

(3) and it is negative in in vitro assays for genotoxicity (4).TCDD is a potent tumor promoter in rat liver (1,5) and mouseskin (6) and appears to be a carcinogen in humans although theissue of dose-response relationships has not been resolved (7).

In addition to cancer, exposure to TCDD or its structuralanalogues causes a variety of biochemical and toxic effects inmany species including immunotoxicity (8), teratogenicity (9),chloracne, and thymic involution (10, 11). Other TCDD-in-duced effects are changes in a number of cellular receptorsystems involved in mitogenesis including epidermal growthfactor receptor, estrogen receptor, glucocorticoid receptor ( 12-16), and induction ofcytochrome P-450 isozymes (10). It isgenerally accepted that these effects are dependent on the Ahreceptor (10, 13, 14). The Ah receptor system bears somesimilarities to the family of steroid hormone receptors and ispostulated to be a member of the erb-A family of genes ( 17).

The most investigated effect of TCDD and its structuralanalogues is induction of two cytochrome P-450 enzymes(CYP1A1 and CYP1A2) in animals and humans (10, 11, 18).Studies on the induction of CYP1A1 in in vitro and in vivosystems suggest that TCDD binds to the Ah receptor and thiscomplex binds to DNA-regulatory elements upstream from thestart site of the structural gene resulting in transcriptionalactivation of the CYP1A1 gene (19). CYP1A2 is induced onlyin liver whereas CYP1 Al is induced in hepatic and extrahepatictissues (20). There is considerable controversy regarding dose-response relationships for Ah receptor mediated events especially whether or not effects exhibit threshold behavior (21, 22).Since enzyme induction is a sensitive marker of exposure todioxins it has been considered as a possible surrogate for cancerrisk estimates (23). We investigated the induction of theseenzymes in the female rat liver as a function of TCDD dose ina rat liver tumor promotion model. This model was used in ourstudy because most risk estimates are based on tumor data inthe female rat liver (24).

Our study used a two stage model for hepatocarcinogenesiswith a single initiating dose of DEN and various doses of TCDDas promoter for 30 weeks. Dose-response relationships (3.5-125 ng TCDD/kg/day) were obtained in DEN-initiated as wellas in noninitiated rats. The induction of cytochrome P-450isozymes was quantified by radioimmunoassay in hepatic microsomes and the localization patterns of these isozymes infixed liver sections were examined by ¡mmunohistochemicalmethods.

MATERIALS AND METHODS

Animals and Treatment. Female Sprague-Dawley rats were givenDEN i.p. at a single dose of 175 mg/kg in saline as the vehicle (1 ml/kg of body weight) at 70 days of age. Control animals received salineonly. Beginning at 84 days of age, TCDD was administered in corn oil

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DOSE RESPONSE OF CYPIAI AND CYPIA2 INDUCTION BY TCDD

by gavage once every 2 weeks at doses of 50, 150, 500, and 1750 ng/kg for 30 weeks. These doses are equivalent to 3.5, 10.7, 35.7, and 125ng TCDD/kg/day; control animals received corn oil. The rats wererandomly divided into the following groups: S/C (controls receivingsaline and corn oil); S/TCDD (promoted only, four different TCDDdose groups); DEN/C (initiated only); and DEN/TCDD (initiated andpromoted, four different TCDD dose groups). There were at least ninerats in each of the treatment groups. One week after the last treatmentthe animals were euthanized with CO2, and the livers were removed,weighed, and sectioned. Portions of the sections were either frozen inliquid nitrogen or fixed for immunohistochemistry.

Preparation of Microsomes. Microsomes were prepared from approximately 2 g of frozen liver tissue by differential centrifugation. The liversamples were homogenized in 3 vols, of ice-cold buffer (150 HIMTris,pH 7.4) in a Potter-Elvehjem homogenizer with a Teflon pestle. Thehomogenate was centrifuged at 4°Cand 9,000 x g for 20 min; theresulting supernatant was centrifuged at 4°Cand 105,000 x g for 50

min. The microsomal pellet was washed once, ultracentrifuged againfor 50 min, and then resuspended in buffer containing 10% glyceroland frozen in small aliquots at —¿�70°C.The protein content of the

microsomes was determined with a Bio-Rad protein microassay (Bio-Rad Laboratories, Richmond, CA) using BSA as standard.

TCDD Analysis. One-g samples of livers from DEN/TCDD groupswere analyzed for TCDD concentration by gas chromatography-massspectrometry procedures with specific ion monitoring as previouslydescribed (5). TCDD concentrations in liver were determined on thebasis of both liver wet weight and total lipid (25). The TCDD contentin livers of noninitiated rats was not analyzed since there is no significant difference in liver TCDD levels between DEN-initiated and non-initiated rats (5).

Preparation of Antibodies. CYPIAI and CYP1A2 were isolated andpurified to apparent homogeneity as described previously (26). NewZealand White rabbits were given injections of either isozyme emulsified in complete Freund's adjuvant. Antibodies to CYP1A2 were also

raised in goats. The antisera were immunoabsorbed to solid phaseimmunoabsorbent containing the heterologous antigen to remove cross-reactivity. Immunopurified CYPIAI contains <0.5% cross-reactivityto CYP1A2, and CYP1A2 contained <3% cross-reactivity to CYPIAIas verified through dot-blot and Western blots. All methods are described in detail elsewhere (26, 27).

RIA. Concentrations of both P-450 enzymes were quantified inhepatic microsomes by a double-antibody RIA procedure using thepurified isozymes as standard as described previously (27). Microsomeswere solubilized and incubated with I25l-labeled purified antigen(CYP1A1 or CYP1A2) and primary antibody for 16 h at 4°C.Secondary

antibody and carrier IgG were added the next day and the samples werecentrifuged 4 h later. The supernatant was discarded and the radioactivity in the pellet counted with a Packard Multi-Prias 2 gammacounter.

EROD. Aliquots of the samples (1-1.5 mg microsomal protein) wereassayed according to the method of Pohl and Fouts (28). 7-Ethoxyre-sorufin (2 MM)was added to the sample, the reaction was started withNADPH (1 min), and after incubation for 45 min at 37°Cthe reaction

was stopped with methanol. The samples were centrifuged and thefluorescence of resorufin measured spectrofluorometrically at 550 nmexcitation and 585 nm emission. 7-Ethoxyresorufin and resorufin wereobtained from Pierce Chemical Company (Rockford, IL).

Immunohistochemistry. Sections of each liver lobe (3-5 mm thick)were fixed in cold phosphate-buffered 4% paraformaldehyde for 16 h,dehydrated, and embedded in paraffin. Immunolocalization of theisozymes was detected using the avidin-biotin antiperoxidase method.Sections (5 ¿im)were deparaffinized followed by trypsin treatment for5 min (0.2 mg/ml; Whittaker Bioproducts, Inc., Walkersville, MD) andnonspecific binding blocked with 5% normal goat serum or normalrabbit serum (Jackson ImmunoResearch Laboratories, Inc., WestGrove, PA) for 20 min. The sections were incubated with primaryantibody (1:1500 for anti-CYPlA2, 1:200 for anti-CYPlAl) or withnonimmune serum for 16 h at 4°C,then rinsed in phosphate-buffered

saline plus 1% BSA followed by incubation with biotinylated secondaryantibody (rabbit anti-goat IgG for CYP1A2 and goat anti-rabbit IgG

for CYPIAI, both 1:100; Vector Laboratories. Burlingame, CA) for 30min. Endogenous peroxidase activity was blocked by incubation with1% HjO: for 5 min. After a rinsing with phosphate-buffered saline plus1% BSA the avidin-biotin complex (ABC Elite kit; Vector) was appliedto the slides. Slides were rinsed after 20 min and then incubated with3,3'-diaminobenzidine (1 mg/ml):H3O: (0.005%; Sigma Chemical

Company, St. Louis, MO) for 10 min. After a rinsing with distilledwater the slides were counterstained with 0.05% Toluidine Blue O(Fisher Scientific Co., Fair Lawn, NJ), dehydrated, and permanentlymounted.

Data Analysis. All data are presented as mean ±SD. Differencesbetween groups were analyzed by trend analysis based upon the use ofgeneralized linear models and considered significant at P < 0.05. Curvefitting to the CYPIAI and CYP1A2 protein data was done using anadditive Hill model (29) and least squares regression.

RESULTS

Livers were analyzed for TCDD concentration and the resultsrevealed a linear relationship (r = 0.999) between administereddose of TCDD and the liver concentration in the dose rangefrom 3.5 to 125 ng/kg/day (Fig. 1). The mean liver concentration of TCDD on a wet weight basis at the lowest dose (3.5 ng/kg/day) was 0.48 ±0.19 ppb; it was 19.9 ±4.8 ppb at thehighest dose (125 ng/kg/day). The lipid-adjusted TCDD concentration also exhibits a linear relation to administered dose[r= 0.993 (Fig. 1)]. The inset in Fig. 1 is a magnification of thecurve in the low-dose range.

The results of the quantification of CYPIAI and CYP1A2proteins by RIA in hepatic microsomes are presented in Table1. The dose-response data showed no consistent differencebetween initiated and noninitiated animals for both isozymes

20-1

15-

TCDD Liver Concentration

D Wet Weight

•¿�liptd Adjusted

50 100

DOSE TCDD (ng/kg/day)

150

Fig. 1. Concentration of TCDD in the liver of female rats after biweekly gavageof increasing doses of TCDD for 30 weeks. Left, concentration on wet weightbasis (D); right, concentration on the lipid basis (•):inset, magnification of thecurve in the low-dose range indicated by the dotted lines.

Table 1 Hepatic concentrations of CYPIAI and CYPIAI quantified hy RIA

pmol/mg microsomal protein

TCDD dose(ng/kg/day)

CYPIAI" CYP1A2"

DENr Salinef DENr Saline'

Control3.510.735.7125.01.5±1.4"34.2

±11.4130.5±22.2244.4

±26.8330.7±48.612.9±

\l.ij56.4

±26.7111.5±30.3181.4

±18.4293.3±17.129.9

±o.Q''87.2

±21.7127.9±32.7233.1

±78.9387.5±127.663.5

±38.4*''88.3

±23.0161.0±55.7193.1

±60.2297.4±88.3

" n —¿�6, mean ±SD.* n = 9, mean ±SD.' Dose-response trend significantly positive: P< 0.01.d DEN-treated rats not significantly different from saline-treated rats by Stu

dent's t test (P < 0.05).

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DOSE RESPONSE OF CYPlAl AND CYP1A2 INDUCTION BY TCDD

10 20

TCDD Liver Cone, (ppb)

30

Fig. 2. Induction of CYPIAl and CYPIA2 by increasing doses of TCDD.Individual values of DEN/TCDD-treated rats are expressed as a function ofTCDD concentration in the liver.

but DEN-treated animals appear to have lower constitutiveconcentrations of both CYPlAl and CYP1A2 (not statisticallysignificant). Although the absolute liver concentrations ofCYPlAl and CYP1A2 were similar between DEN-initiated

and noninitiatied rats in all TCDD dose groups, the higherconstitutive expression in the noninitiated rats produce a lowermagnitude of induction by TCDD. Induction at the lowestTCDD dose (3.5 ng/kg/day) of the DEN-initiated animals was20-fold for CYPlAl and 3-fold for CYP1A2. In the salinegroup, induction of CYP1A2 was not statistically significant inrats receiving 3.5 ng/kg/day. At the highest dose (125 ng/kg/day) both enzymes are present in similar amounts (Table 1).Maximum induction was approximately 200-fold for CYPlAland approximately 10-fold for CYP1A2.

Fig. 2 illustrates the relationship between the amount of eachP-450 enzyme and liver TCDD concentration. The individualvalues for DEN-initiated animals in all dose groups indicatethat CYPlAl is almost maximally induced at a TCDD concentration of 10 ppb whereas the curve for CYP1A2 appears to bestill ascending although there is greater variability in the concentration of CYP1A2 in the higher dose groups. The dose-response trend for both isozymes is highly significant (P <0.01).

The comparison between the quantitation of CYPlAl protein by RIA and EROD activity (CYPlAl-dependent enzymeactivity) showed a very strong positive correlation (r = 0.85). A

strong relationship also existed between EROD activity andliver TCDD concentration (Fig. 3). The approximate ED50values for induction are 18 ng/kg/day for CYPlAl, 25 ng/kg/day for CYP1A2, and 10 ng/kg/day for the EROD activity.These values indicate similar responses to TCDD for the threeassays. All ED50 values are in the range of 2-4 ppb for TCDDliver concentration. However, the ascending curve for CYP1A2in the high-dose region (Fig. 2) complicates evaluation of comparative ED50 values.

Fig. 4 illustrates photomicrographs of tissue sections of liversfrom DEN/TCDD-treated animals of all dose groups. Thesections were incubated with either anti-CYPlAl (Fig. 4, A-E)or anti-CYPlA2 (Fig. 4, F-J). The staining for both isozymes

was done in serial sections and photomicrographs were takenfrom the same areas for each cytochrome. Positive immuno-reactivity was found in the cytoplasm of hepatocytes of all dosegroups including controls. Both isozymes indicate the samelocalization and induction pattern throughout the dose-response range. CYPlAl is detectable in control animals in onlya few cells around the central veins (Fig.4/1). CYP1A2 incontrol animals is also localized in the centrilobular region butin a greater number of cells than CYPlAl (Fig. 4F). At thelowest TCDD dose (3.5 ng/kg/day) increased staining is detectable in a higher number of centrilobular hepatocytes. At adose of 10.7 ng/kg/day, positive immunoreactivity can also bedetected in midzonal areas; the next higher dose (35.7 ng/kg/day) clearly shows midzonal staining with slight staining in theperiportal region. It is noticeable that even at the highest doseof TCDD treatment (125 ng/kg/day) the staining is not uniformand that there are some periportal areas where hepatocytesshow no positive immunoreaction to either isozyme. The increase in total staining after different doses of TCDD is consistent with the results obtained through quantification by RIA.The staining pattern and dose-response relationships in saline/TCDD-treated animals was similar (not shown). The positiveimmunoreactivity shown in Fig. 4 could be blocked by preab-sorption of the antibodies to the purified antigen prior toimmunostaining demonstrating the specificity of our stainingmethod (data not shown). Nonspecific staining was evaluatedby incubating serial liver sections with nonimmune serum. No

1100-1

0 10 20

TCDD Liver Cone, (ppb)

Fig. 3. Induction of EROD activity by increasing doses of TCDD. Individualvalues of DEN/TCDD treated rats are expressed as a function of TCDD concentration in the liver.

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CYP1A1 CYP1A2

CONTROL

Fig. 4. Immunohistochemical detection ofCYP1A1 and CYPIA2 within livers of femaleSprague-Dawley rats treated for 30 weeks withdifferent doses of TCDD after initiation withDEN. Serial tissue sections were incubatedwith either anti-CYPIAI or anti-CYPlA2;photomicrographs were taken from sameareas. CYPI Al: A-E, dose-response; A, untreated control. CYP1A2: F-J, dose-response;F, untreated control. Right ordinale, doses. C,central vein; P, portal triad, x 28.

\ L/UU

(ng/kg/day)

3.5

B

10.7

mrH

I

35.7

125

staining was detectable indicating that nonspecific binding isvery low.

DISCUSSION

Our studies have characterized the dose-response relationships for induction of CYPI Al and CYPI A2 by chronic TCDDexposure in a rat liver tumor promotion model. Although datareported here are obtained from animals receiving TCDD for30 weeks (no tumors at this time), continued treatment for 60weeks with 100 ng TCDD/kg/day produces a high incidence ofliver tumors (30). Induction of CYPI Al protein occurs by

transcriptional activation of the gene and is Ah receptor dependent (31). A similar mechanism is likely to be responsiblefor CYP1A2 induction. Our study evaluated dose-response

relationships as a function of both the administered dose andtarget tissue concentration. These studies are relevant for evaluation of dose-response relationships for TCDD which remainsone of the most controversial issues in risk assessments for thisubiquitous environmental contaminant (21, 22).

It has been hypothesized that there is a proportional relationship between receptor occupancy and biological response formany receptor-mediated events, although different dose-re

sponse curves are possible for different responses regulated by3439

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the same receptor (32). If there is a proportional relationshipbetween receptor occupancy and biological response, a linearrelationship would exist at low ligand concentrations, followedby receptor saturation at higher concentrations. Assuming aproportional relationship between liver TCDD concentrationsand Ah receptor occupancy, our data for enzyme induction areconsistent with this classical receptor occupancy response curve(33). This is strongly supported by the additive Hill model (29)which was applied to these data (Fig. 2). Even though the modelallows for sigmoidal relationship at low ligand concentration,the best fitting curve was linear for induction of both CYPIAland CYP1A2 at low TCDD concentrations. Therefore, extrapolation of our data to lower doses is consistent with the absenceof a threshold for TCDD-mediated induction of CYPIAl andCYP1A2. It is important to emphasize that these dose-responseanalyses were based on target tissue TCDD concentration, noton administered dose, and that there is little interindividualvariation in response (based on target tissue dose) in the low-dose region. Although CYP1A2 induction in the saline treated(noninitiated) rats is not statistically significant at the lowestTCDD dose, mathematical analysis of these data was consistentwith data from initiated rats in that a linear relationship between induction and administered dose gave the best fit (29).However, the possibility remains that in the low-dose regiondose-response relationships for CYP1A2 induction may bedifferent in initiated and noninitiated rats.

Although the lowest dose used in our study (3.5 ng/kg/day)is well below toxic doses, it is still substantially higher thanbackground exposure to polychlorinated dibcn/o /> dioxins anddibenzofurans in humans (estimated 1-3 pg/kg/day of TCDDtoxic equivalents) (34). Therefore, data on effects at lowerexposure levels in experimental studies are needed to morecarefully define the shape of the dose-response in the low-doseregion for the effects of TCDD. A recent study3 addressed this

issue using polymerase chain reaction techniques to quantifymRNA for CYPIAl in rats receiving varying doses of TCDD.These studies demonstrate that CYPIAl mRNA levels areincreased in rat livers at doses which approximate tissue concentrations present in humans.

Several factors should be considered when evaluating dose-response relationships for the effects of TCDD which are mediated through the Ah receptor, (a) TCDD exposure increasesthe concentration of Ah receptor binding sites (35). This response is maximal at a chronic dose of 30 ng/kg/day and dose-response relationships for Ah receptor induction are similar tothose for CYPIAl induction. Induction of the Ah-receptormight produce sublinearity. (b) It is likely that occupation ofdifferent numbers of dioxin-responsive elements in the upstream region of the CYPIAl gene by TCDD-Ah receptorcomplexes produces varying degrees of transcriptional activation of the CYPIAl gene (36). This could also produce sublinearity. (c) TCDD (unlike most ligands for receptors of thesteroid superfamily) is extraordinarily persistent, as demonstrated by a half-life of about 30 days in rats (37) and about 7years in humans (38) compared to a few h for steroids (32).Therefore, it is possible that a TCDD-Ah receptor complexcould produce transcriptional activation of dioxin-responsivegenes and then dissociate from DNA but unlike steroids, TCDDwould not be degraded rapidly. The same molecule of TCDDcould then bind another Ah receptor and repetitively initiatetranscription, thereby producing supralinearity in the low-doseregion. The combined impact of the above factors on dose-

response relationships is uncertain although they could conceivably produce supra- or sublinearity of response in the low-dose range.

The relationship between administered dose and hepatic concentration of TCDD (Fig. 1) appears to be linear in the doserange of our study (3.5-125 ng TCDD/kg/day). This finding issomewhat different from reports by Rose et al. (37) and Abraham et al. (39) which indicated that a higher proportion ofadministered dose is found in the liver with increasing doses.The reason for this discrepancy may reflect the differencebetween chronic and short term or single exposures. Nevertheless, there is considerable evidence that hepatic concentrationsof TCDD were in equilibrium in our study, (a) TCDD concentrations in livers of rats receiving approximately the same dailydoses of TCDD for 2 years (1) were similar to our values. (¿>)Rose et al. (37) reported that TCDD concentrations in liverachieve equilibrium by 13 weeks after daily treatments withvarying doses of TCDD. (c) Mathematical calculations basedon a 30-day half-life estimate that steady-state concentrationswould be achieved by 3 or 4 months of daily exposure." There

fore, we are confident that our data on CYPIAl and CYP1A2induction reflect a chronic exposure and are of more use inmaking animal-to-human comparison of responses than animaldata obtained after a single dose.

A physiologically based pharmacokinetic model for TCDDhas been constructed and used to estimate risks from TCDDexposure (23). One of the implications of this model is thatsince CYP1A2 is a TCDD-binding protein (40), induction ofCYP1A2 would provide a mechanism for retaining higherproportions of TCDD in livers at inducing doses of TCDDcompared to noninducing doses. Our data are inconsistent withthis hypothesis in that we observed a linear relationship betweenadministered dose and TCDD liver concentration over a doserange which produced a minimal to near maximal induction ofCYPIAl and CYP1A2 (Figs. 1 and 2).

The immunolocalization of CYPIAl and CYP1A2 leads toseveral interesting conclusions. Induction of CYPIAl occursin the same liver cells as CYP1A2 and the liver is not homogeneous in the induction pattern. The difference in the responsiveness of centrilobular and periportal hepatocytes indicatesthat the regulation of CYPIAl and CYP1A2 expression in liveris much more complex than generally recognized. Our resultsraise the possibility that some hepatocytes may be maximallyinduced by low doses of TCDD and that it is inappropriate toassume that all hepatocytes exhibit the same dose-responserelationships, a finding which could have significant impact onrisk estimation. This heterogeneity in response is probably notdue to pharmacokinetic distribution of TCDD since steady-state conditions for total hepatic TCDD concentrations areachieved and the direction of the blood flow in the liver lobuleis from portal veins to centrilobular veins. Hepatocytes in thethree different regions within a liver lobe are different in theirbiochemistry and morphology. Our data are consistent with thefindings that centrilobular hepatocytes contain more smoothendoplasmatic reticulum which is associated with cytochromeP-450-dependent monooxygenases (41) and up to twice as muchcytochrome P-450 as cells closer to periportal veins (42). Preferential centrilobular localization of CYPIAl and CYP1A2was detected in untreated (43) as well as in 3-methylcholan-threne- and isosafrole-treated rats (44). Other metabolic enzymes also exhibit a primarily centrilobular localization (45-47).

3J. P. Vanden Hcuvel, personal communication. 4 C. J. Portier, unpublished data.

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The role of enzyme induction in toxicity and/or carcinogen-icity is uncertain. Effects secondary to enzyme induction mightbe important such as increased metabolism of xenobiotics andendogenous substances to DNA-reactive metabolites. For example CYP1A2 catalyzes 17/3-estradiol conversion to the 2-hydroxycatechol (48) which might lead to cellular damage viabinding to DNA or interaction with cellular proteins (49). Thesefindings are consistent with tumorigenicity data in rat liver inthat intact female rats but not male or ovariectomized rats haveincreased liver tumor incidence after chronic TCDD treatment(1, 5). These differences indicate a critical involvement ofovarian hormones and emphasize the importance of hormonalinteractions in the carcinogenicity of TCDD. We are currentlyinvestigating other cellular responses to chronic TCDD exposure and the relevance of these effects to toxicity/carcinogen-icity. We are comparing epidermal growth factor receptor status, cell proliferation, and putative preneoplastic lesions (fociof cellular alterations) with TCDD concentration in the targettissue (50) from the same animals that we analyzed for CYP1A1and CYP1A2 induction.

In summary our data suggest that, in a chronic exposureexperiment, there is no evidence of a threshold for the inductionof hepatic CYPIAI and CYP1A2. Moreover, induction ofCYP1A2 as binding protein for TCDD does not seem toinfluence liver retention of TCDD. Immunolocalization studiesrevealed identical patterns of induction for CYPIAI andCYP1A2 and suggest that all hepatocytes in the lobe do notrespond uniformly to TCDD.

ACKNOWLEDGMENTS

The authors acknowledge Suzanne Snedeker, Julie Foley, and RobertMaronpot for assistance in techniques for immunohistochemical studies and interpreting the achieved result. We also thank Michael Kohnand Robert Diedrek for their helpful suggestions on modeling thesedata and Louise Harris for technical assistance in dosing the animals.

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1992;52:3436-3442. Cancer Res   Angelika M. Tritscher, Joyce A. Goldstein, Christopher J. Portier, et al.   CYP1A2 in the LiverModel: Quantification and Immunolocalization of CYP1A1 and

-dioxin in a Rat Tumor Promotionp2,3,7,8-Tetrachlorodibenzo-Dose-Response Relationships for Chronic Exposure to

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