TOMCOLOGICAL SCIENCES 44, 1-13 (1998)

ARTICLE NO. TX982467

Use of Model-Based Compartmental Analysis to Study Effects of2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics in Rats1'2

Sean K. Kelley,*t Charlotte B. Nilsson,* Michael H. Green,f Joanne Balmer Green,t and Helen Hakansson*3

*The Institute of Environmental Medicine, The Karolinska Institute, Stockholm, Sweden; and '[Nutrition Department,The Pennsylvania State University, University Park, Pennsylvania 16802

Received September 23, 1997; accepted March 30, 1998

Use of Model-Based Compartmental Analysis to Study Effectsof 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics inRats. Kelley, S. K., Nilsson, C. B., Green, M. H., Green, J. B., andHakansson, H. (1998). ToxicoL ScL 44, 1-13.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic,widespread environmental contaminant that has dramatic adverseeffects on the metabolism of vitamin A. We used model-basedcompartmental analysis to investigate sites and quantitative im-pacts of TCDD on vitamin A kinetics in rats given an oral loadingdose of TCDD in oil (3.5 ftg/kg) followed by weekly maintenancedoses (0.7 fig/kg) or oil only. [3H]Retinol in its plasma transportcomplex (experiment 1) or lymph containing chylomicrons labeledmainly with [3H]retinyl esters (experiment 2) were administerediv, and tracer kinetics in plasma, liver, carcass, urine, and feceswere measured for up to 42 days. TCDD treatment caused signif-icant reductions in liver vitamin A levels and significant changesin tracer kinetics and tracer excretion. A four-compartment modelwas used to fit tracer data for experiment 1; for experiment 2,compartments were added to describe the metabolism of newlyabsorbed vitamin A. The compartmental models predict thatTCDD caused a slight delay in plasma clearance (via an increasedrecycling to plasma), and in liver processing, of chylomicron-derived vitamin A. Models for both experiments predict thatTCDD exposure did not affect the fractional uptake of plasmaretinol from the rapidly turning-over extravascular pool, but it

1 This project was supported by funds from the Swedish EnvironmentalProtection Agency, Visiting Scientist grants from the Karolinska Institute, anda Fulbright Fellowship to S.K.K.

2 Presented in part at Experimental Biology 1996 [Kelley, S. K., Green,M. H., Green, J. B. Nilsson, C , and Hakansson, H. (19%). 2,3,7,8-Tetrachlo-rodibenzo-p-dioxin (TCDD) affects vitamin A homeostasis and kinetics in rats.FASEB J. 10, A467 (abstract 2694)] and 36th Annual Meeting of the Societyof Toxicology 1997 [Kelley, S. K., Green, M. H., Green, J. B., Nilsson, C , andHakansson, H. (1997) 2,3,7,8-Tetrachlorodibenzo-/wiioxin (TCDD) affectshepatic uptake and processing of vitamin A from chylomicrons. Fundam. AppLToxicoL 36(Suppl), 214 (abstract 1087)]. Part of this work constitutes adoctoral thesis (Sean Keith Kelley, The Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics in Rats: A Compartmental Model, The Penn-sylvania State University, University Park, PA, 1997).

3 To whom correspondence should be addressed at Institute of Environ-mental Medicine, Karolinska Institutes P.O. Box 210, S-171 77 Stockholm,Sweden. Fax: 46 8 33 44 67. E-mail: helen.hakansson@imm.ki.se.

doubled the fractional transfer of recycled retinol from slowlyturning-over pools of vitamin A to plasma. The residence time forvitamin A was reduced by 70% in TCDD-treated rats, transfer intourine and feces was tripled, and vitamin A utilization rates weresignificantly increased. Since our results do not indicate that ret-inol esterification is inhibited, we hypothesize that some of thesignificant effects of TCDD on vitamin A metabolism result fromincreased catabolism and mobilization of vitamin A from slowlyturning-over pools (especially the liver), o IWS society of Tmdcoiogy.

Key Words: 2,3,7,8-Tetrachlorodibenzo-p-dioxin; TCDD; rat; vi-tamin A; model-based compartmental analysis; kinetics.

Vitamin A4 is an essential nutrient that is required fornormal vision, growth, reproduction, cell differentiation, em-bryonic development, and immune function in mammals (seereview by Underwood, 1984). Much is known about the com-plex transport and metabolism of vitamin A (see review byBlaner and Olson, 1994). Recent research has established thatretinoids are a key regulator of gene expression, exerting theirbiological effects by interaction with nuclear receptors (seereview by Mangelsdorf et al., 1994).

Various studies have demonstrated that the normal metabo-lism of vitamin A is affected by alterations in vitamin A status,physiological state, or exogenous variables. One exogenousfactor that has profound effects on vitamin A metabolism inmany species is exposure to the persistent environmental con-taminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (see re-view by Zile, 1992). This widespread, highly toxic dioxincauses general body wasting, impaired growth and reproduc-tion, compromised immune function, epidermal effects, andhepatotoxicity (see review by Pohjanvirta and Tuomisto,1994). It is known that binding of TCDD to the nuclear arylhydrocarbon (Ah) receptor affects gene transcription. In the rat,TCDD exposure causes a decrease in liver vitamin A levels, anincrease in kidney vitamin A, and often an increase in plasmaretinol levels (Hakansson and Ahlborg, 1985; Bank et al.,1989; Hakansson et al, 1991a, b; van Birgelen et al., 1995).

4 Several compounds that show the biological activity of vitamin A arediscussed here (retinol, retinyl esters, and retinoic acid).

1 1096-6080/98 $25.00Copyright O 1998 by the Society of Toxicology.All righu of reproduction in any form reserved.

KELLEY ET AL.

TCDD also inhibits the normal storage of newly ingestedvitamin A in liver (Hakansson and Ahlborg, 1985), most no-tably in perisinusoidal stellate cells (Hakansson and Hanberg,1989), and it increases the mobilization of endogenous retin-oids from liver (Hakansson et al., 1988). In addition, TCDDexposure increases excretion of vitamin A metabolites in urineand feces (Hakansson and Ahlborg, 1985; Hakansson et al.,1988), increases the glucuronidation of retinoic acid in vitro(Bank et al., 1989), and results in increased retinoic acidcatabolism in microsomes from various tissues (Fiorella et al.,1995). At least some of the effects of TCDD on vitamin Ahomeostasis seem to be the consequence of changes in genetranscription and thus enzyme production (Zile, 1992; Pohjan-virta and Tuomisto, 1994). Furthermore, Weston et al. (1995)reported that the retinoic acid-dependent induction of genes forcellular retinoic acid-binding protein II and retinoic acid re-ceptor-/3 are inhibited by TCDD.

Here we investigated the mechanisms involved in the effectsof TCDD exposure on vitamin A metabolism by using model-based compartmental analysis to determine which whole-bodyvitamin A kinetic processes are perturbed by TCDD treatmentin the rat. Although model-based compartmental analysis hasnot been as widely used in toxicology and pharmacology asother approaches [e.g., physiologically based pharmacokineticmodeling (PB-PK; Andersen, 1995)], it has been fruitfullyapplied to data on the metabolism of vitamin A and othernutrients, metabolic fuels, hormones, and organ systems (seereferences in Green and Green, 1990a). Here we collected dataon vitamin A kinetics in plasma, tissues, and excreta of controlrats and those exposed to weekly doses of TCDD after admin-istration of a radioactive (tracer) dose of vitamin A. We hy-pothesized that TCDD might disrupt vitamin A homeostasis byaffecting vitamin A utilization and recycling of retinol amongplasma, liver, and extrahepatic tissues or by altering the uptakeand processing of newly absorbed retinyl esters. To examinethe first hypothesis, [3H]retinol tracer was administered in itsnormal vitamin A plasma transport complex (experiment 1) sothat effects of TCDD on utilization and recycling could bedetermined; to address the second hypothesis, [3H]retinyl es-ter-labeled lymph chylomicrons were administered (experi-ment 2) to trace the metabolism of newly ingested vitamin A.Then we applied model-based compartmental analysis (Fosterand Boston, 1983; Green and Green, 1990a) to tracer data inorder to quantify effects of TCDD exposure on the kineticbehavior of vitamin A. Our results suggest that previouslyobserved effects of TCDD on vitamin A metabolism are notrelated to decreases in plasma vitamin A transport to rapidlyand slowly turning-over extravascular vitamin A pools, nor todefects in esterification of vitamin A, but rather to increasedturnover of storage pools, resulting in net mobilization ofstored vitamin A, and to increases in irreversible utilization ofvitamin A.

MATERIALS AND METHODS

Animals and Diets

Studies were carried out at the Karolinska Institute, Stockholm, Sweden;animal experiments were approved by the Institute's Animal Care and UseCommittee. All procedures were done under light filtered through transparentfilms of titanium 35 (TESAB Solna AB, Solna, Sweden) to prevent photooxi-dation of vitamin A.

Weanling male Sprague-Dawley rats (B&K Universal, Sollentuna, Sweden)to be used as recipients of radioactive vitamin A were housed in shoebox cagesin an environmentally controlled animal facility which had a 12-h light cycle(lights on 0700—1900 h). Tap water and a commercial diet (catalogue No. R34;Lactamin, Stockholm, Sweden) were provided continuously. Assuming aver-age daily food intakes of 15-20 g/day, rats consumed 63 to 84 nmol vitaminA/day. This amount was chosen so that control rats would be in a slightpositive vitamin A balance.

TCDD Dosing Protocol

TCDD (Lot 851:144-11) was a generous gift from Dow Chemicals (Stock-holm, Sweden). A stock solution containing 100 fig TCDD/ml toluene wasstored in darkness. Two working solutions of TCDD in corn oil were prepared;one, used for initial loading doses, contained 3.5 jig TCDD/ml and the second,used for subsequent weekly maintenance doses, contained 0.7 jig TCDD/ml.As discussed by FlodstrOm et al. (1991), this dosing protocol was designed toproduce a quasi steady state with respect to TCDD, but was not lethal andwould presumably be effective in altering plasma and liver vitamin A levels.

After rats had been maintained on the experimental diet for 7 weeks(experiment 1) or 6 weeks (experiment 2), they were assigned by body weightto one of two groups. The loading dose (3.5 /xg TCDD/kg body weight in 1 mlcorn oil/kg; TCDD group) or an equal amount of corn oil (control group) wasadministered intragastrically. Rats to be used in long-term kinetic studies (seebelow) were put into metabolic cages (Techniplast Model 1700; Scanbur,Koge, Denmark); rats in short-term studies in experiment 2 were returned toshoebox cages. Maintenance doses of TCDD (0.7 fig, TCDD/kg body weightin 1 ml corn oiiykg; TCDD group) or equal amounts of corn oil (control group)were given 7 days after the initial loading dose and weekly thereafter.

Experiment I

Preparation of [3H]vitamin A-labeled plasma. [3H]Retinol-labeledplasma ([3H]retinol/retinol-binding protein (RBP)/transthyretin (TTR)} wasprepared in vivo as previously described (Green and Green, 1990b). Briefly,weanling male Sprague-Dawley rats (B&K Universal; n = 2) were fed avitamin A-free diet for 7 weeks to deplete hepatic vitamin A stores. Then,[ll,12(N>[3H]retinol (sp act = 1.4 TBq/mmol; NEN, Boston, MA; 18.5MBq/donor) in an aqueous suspension with Tween 40 (Sigma Chemical Co.,St. Louis, MO) was injected iv into these donor rats. After 100 min, blood wastaken from the abdominal aorta into heparinized syringes. Plasma presumablycontaining [3H]retinol/RBP/TTR (Green et al., 1985) was stored under anatmosphere of nitrogen at 4°C and used for kinetic studies over the next 2 days.Weighed replicate aliquots of the dose were taken for analysis of radioactivity(see below).

Kinetic study. One day before administration of retinol-labeled plasma torecipient rats, three rats in each group were killed and livers were obtained foranalysis of vitamin A ("time 0"). Rats were anesthetized with CO2 and killedby extracting a large blood sample from the right cardiac ventricle. Livers wereexcised, blotted, weighed, frozen in liquid nitrogen, freeze-dried, and storedunder an atmosphere of nitrogen at - 16°C for later analysis (see below).

The kinetic study was started 2 days after administration of the first main-tenance dose of TCDD. See Green and Green (1990b) for additional details onprotocols used in the kinetic study. Seven rats from each experimental group(control and TCDD) were injected intrajugularly with an accurately weighedamount (—0.4 g containing ~ 180 kBq) of [3H]retinol-labeled plasma. Over the

EFFECTS OF TCDD ON VITAMIN A KINETICS

next 42 days, serial blood samples (n = 29; 0.1-0.25 ml) were taken from acaudal vein into microcentrifuge tubes containing 25 IU heparin; samplingtimes were chosen based on a geometric progression. Plasma aliquots werefrozen in minivials under nitrogen for later analysis of tritium. At selectedtimes throughout the turnover study (8 samples/rat), additional aliquots weretaken for analysis of plasma retinol concentration (see below).

For each rat, urine and feces were collected as pools during day 1 (0-24 hafter administration of isotope), days 1-4 (24-96 h after dosing), days 4-7(96-168 h), and days 7-10 (168-240 h following isotope administration). Ateach of these times, the rat was moved to a clean metabolic cage (or, on day10, to a shoebox cage). Urine that had accumulated in the collection cylinderwas transferred into a preweighed disposable vial; the cage was rinsed twicewith a total of 5-6 ml ethanol:distilled water (1:1), and rinses were combinedwith urine. The sample was reweighed and aliquots (3X —1 g) were weighedinto minivials and frozen for later analysis of tritium (see below). Feces weretransferred from their collection cylinder into sample bags, weighed, frozen,lyophilized, and stored at - 16°C for later analysis of radioactivity (see below).

Forty-two days after administration of labeled plasma, rats were killed asdescribed above. In addition to removing livers, the thymus glands wereexcised and weighed (so that effects of TCDD on thymus weight could bedetermined) and then returned to the carcass. Carcasses were weighed andfrozen at - 16°C until analyzed for radioactivity (see below).

Experiment 2

Preparation of[3HJvitamin A-Iabeled lymph chylomicrons. Rats (n = 2)were anesthetized and the main mesenteric lymph duct was cannulated by theprocedure of Hanberg and Trossvik (1995). On the morning after surgery, 66TBq of [ll,12(N)3H]retinol (NEN) and ~1 /xg of unlabeled retinyl acetate wasdissolved in com oil (0.5 ml). Labeled oil (0.25 ml/rat) was administeredorally. Lymph was collected for 6 h after dosing into tubes containing 10 /ilNa3EDTA (0.4 M, pH 7.4); it was filtered through sterile gauze and dilutedwith unlabeled lymph. Replicate aliquots were taken for analysis of totalradioactivity and to determine the fraction of the radioactivity associated withretinol versus retinyl esters (see below). Lymph was stored at 4°C under anitrogen atmosphere and used within 2 days of collection for in vivo studies.

Kinetic study. Procedures were similar to those described for experiment1. Beginning 2 days after administration of the first TCDD maintenance dose,rats were injected iv with an accurately weighed amount of labeled lymph(~0.25 g containing —120 kBq). Rats were designated as either short-term orlong-term recipients. For rats in the long-term group (TCDD, n = 7; control,n = 8), serial blood samples (n = 32) were collected from a caudal vein for42 days after dose administration. Plasma aliquots were frozen under nitrogenfor later analysis of tritium and, at selected times, for retinol concentration (seebelow). In addition, aliquots of plasma collected during the first 2 h afterdosing were frozen for analysis of tritium in retinol versus retinyl esters (seebelow). Urine and feces were collected as individual animal pools for 4 daysafter dosing; rats were then returned to shoebox cages. On day 42, long-termrecipients were anesthetized and killed.

For short-term recipients in experiment 2 (3 rats/group/time), several bloodsamples were taken after administration of labeled lymph before rats werekilled at 25 min, 8 h, and 2 days. In addition to the procedures done forexperiment 1, small intestines and contents were excised, frozen in liquidnitrogen, Iyophili2ed, and refrozen for analysis of tritium (see below). Smallintestines were examined separately from the rest of the carcass to verify thatour dose was physiological (Green and Green, 1996) and to check whetherTCDD caused a rapid metabolism and excretion of the administered labelinto bile.

Plasma and Tissue Analyses

Plasma samples (40-200 /J) and aliquots of the vitamin A-labeled doseswere analyzed for tritium (Model 1409; Wallac Sverige AB, Upplands Vasby,Sweden) after addition of 5 ml of scintillation solution (Ecoscint A; NationalDiagnostics, Hintze, Stockholm, Sweden). Samples were counted twice to a

final 2(r error of 1%. After background correction, net counts/min (cpm) wereconverted to disintegrations/min (dpm) using an external standard channelsratio method.

The amount of vitamin A in plasma was determined by HPLC in eight of theserial plasma samples collected from each rat in the 42-day studies. Using amodification of the procedure of Thompson el aL (1971), retinol was extractedfrom 50% ethanol into hexane containing 23 jtM butylated hydroxytoluene(BHT); retinyl acetate was used as an internal standard. Solvent-free extractswere resuspended in ethanol and chromatographed on a Nucleosil 5 ft C18Resolve HPLC column (150 X 4.6 mm; Phenomenex, Torrance, CA) usingmethanol:wateT (90/10 v/v, 1 ml/min) as the mobile phase. Retinol and retinylacetate peaks were detected by UV absorbance at 328 am (Model 486; WatersAssoc., Milford, MA). The areas under the peaks for retinol and retinyl acetatewere obtained by integration (MiniChrome vl.66; Fisons Instr., VG DataSystems, Cheshire, UK) and an internal standard method was used to quanti-tate the amount of retinol in each sample.

Radioactivity in retinol and retinyl esters was determined in aliquots of thelymph chylomicron dose, and in plasma samples (n = 9) collected during thefirst 120 min after dose administration for long-term rats in experiment 2.Plasma samples were extracted as above. The hexane extracts were loaded ontocolumns of neutral aluminum oxide (Aldrich/LabKemi, Stockholm, Sweden)deactivated with 5% water (Ross, 1982). Retinyl esters were eluted with 3%diethylether in hexane and retinol with 50% diethylether. Fractions wereanalyzed for tritium (LS 1801; Beckman instruments, Stockholm, Sweden)using Ecoscint O (National Diagnostics) as scintillation solution.

Triplicate aliquots of freeze-dried liver (0.15 g) were saponified in ethanolicKOH and lipids were extracted into hexane containing 23 /xM BHT (Green etal., 1985). A portion of the extract was analyzed for tritium as described forplasma, using Ecoscint O as scintillation solution. Other aliquots were ana-lyzed for vitamin A content by HPLC as described above.

Additionally, radioactivity in liver retinol and retinyl esters was determinedfor short-term animals in experiment 2. Retinoids were extracted using thehexane:isopropanol:sodium sulfate method of Hara and Radin (1978) as de-scribed by Adams et al. (1995). Radioactivity in extracted retinol and retinylesters was determined by column chromatography and liquid scintillationspectrometry as described above for plasma.

The tritium content of urine was measured (Model 1215 Rackbeta II;LKB/Wallac, Uppsala, Sweden) after addition of Ecoscint A to triplicatealiquots of urine. Conversion of net cpm to dpm for each sample was doneusing an external standard channels ratio method.

Fecal radioactivity was determined using a modification of the method ofHakansson and Ahlborg (1985). Briefly, freeze-dried feces were ground in amortar. Quadruplicate aliquots (0.07-0.1 g) were incubated for 2 h in 4 mlmethanol while mixing (200 rpm) at 56°C. After incubation, samples werevigorously vortexed for 15 min and then centrifuged for 6 min at 3000 rpm.The methanol was aspirated into glass scintillation vials and the extraction wasrepeated twice, using 2 ml methanol each time and 15-min incubations. Aftersolvent had evaporated, extracts were analyzed for radioactivity using EcoscintO as the scintillation solution.

Tritium in carcasses was determined using the method of Adams et aL(1995). Carcasses were coarsely ground using a meat grinder and five aliquots(—1.5 g each) were extracted using hexane:isopropanol:sodium sulfate. Sol-vent was evaporated and extracts were analyzed for radioactivity usingEcoscint O as the scintillation solution. Freeze-dried small intestines fromshort-term rats in experiment 2 were similarly extracted and counted.

Kinetic Analysis

Model-based compartmental analysis (Foster and Boston, 1983; Green andGreen, 1990b) was used to develop the simplest model that fit tracer data forexperiment 1. As described by Green and Green (1990b), the fraction of thedose of injected radioactivity (fdose) remaining in plasma at each samplingtime for each rat was calculated as dpm/ml of plasma divided by dpminjected/estimated plasma volume, where plasma volume was approximated asmean body weight (g) during the 42-day kinetic study X 0.038 ml plasma/g

KELLEY ET AL.

body weight. For each rat, the fraction of the injected dose of radioactivity inliver, carcass, urine, and feces was calculated as tissue dpm divided by the totaldpm injected. The fraction of the dose that was irreversibly lost by the end ofeach rat's study was calculated as 1 - (fdose liver + fdose plasma + fdosecarcass) for experiment 1 and as 1 — (fdose liver + fdose plasma + fdosecarcass + fdose small intestine) for experiment 2.

Individual animal data on plasma fdose versus time were fit to a multiexponen-tial equation using CONS AM the conversational version 31 (Berman etal, 1983),the conversational form of the Simulation, Analysis and Modeling computerprogram (SAAM; Berman and Weiss, 1978). Programs were run on an IBM80486 microcomputer. For each rat, the sum of the intercepts was used to adjustthe initial estimate of plasma volume that had been calculated from body weightat the time of dose administration. Then we used CONSAM to compare normal-ized data for plasma, urine, feces, and irreversible loss for each rat to die three-compartment model for vitamin A metabolism proposed by Green and Green(1994). We found that a four-compartment model provided the best fit to tracerdata from experiment 1 (see Results). Weighted, nonlinear regression analysis wasdone using CONSAM in order to determine values for the model parameters(fractional transfer coefficients [LflJ)s] or the fraction of compartment J's tracertransferred to compartment I each day) for each rat. For weighting purposes, afractional standard deviation of 0.05 was assigned to each datum for plasma andirreversible loss, while a fractional standard deviation of 0.1 was given to urine andfeces data, to reflect the potentially larger error in collection and analysis ofexcreta. Goodness-of-fit of the proposed model was determined by visual inspec-tion of model-predicted versus observed data and by calculation of the estimatedfractional standard deviation for each L(IJ). Parameters were considered wellidentified if their estimated variability was less than 0.5 (Jacquez, 1996). Next, themultiple studies feature of SAAM (Lyne et at, 1992) was used to calculateaverage fractional transfer coefficients for each group and the population estimateof the standard deviation.

Data from experiment 2 were handled similarly, using the final model fromexperiment 1 as a starting point and after addition of compartments related tochylomicron metabolism, except that estimates of plasma volume were notadjusted because of the rapid and variable removal of chylomicrons, data forurine and feces were not weighted since only one collection was made (a 4-daypool after isotope administration), and early data for fraction of dose in plasmaretinyl esters and retinol were given fractional standard deviations of 0.05. Aseven-compartment model with a delay element was needed to fit these data.Differences between the models presented for the two studies reflect not onlythe complexity needed to account for metabolism of vitamin A in chylomi-crons in experiment 2, but also a difference in the number of slowly turning-over extravascular compartments needed to fit the data (one in experiment 2versus two in experiment 1).

Using the model-predicted fractional transfer coefficients, several additionalkinetic parameters were calculated for each experiment; see Green and Green(1990b) for more information on these calculations. Transit time [f(I)] is thelength of time an average molecule of tracer spends in compartment I duringa single transit It was calculated as 1/2L(J,I). T(IJ), or mean residence time,is the total time an average retinol molecule spends in compartment I beforeirreversible exit from compartment I after entering the system via compartmentJ; it is equal to the area under the tracer response curve integrated from 0 toinfinity [AUC(IrT)]. Plasma fractional catabolic rate [FCRp], or the fraction ofthe plasma rctinol pool that is irreversibly utilized each day, was calculated asin~(I). The number of times that a retinol molecule recycles through compart-ment I before irreversible loss is the recycling number [i<I)]. It was calculatedas [T(I)/r(I)] - 1. T(SYS), or system mean residence time, expressed by theequation T(SYS) = S f (IJ), is defined as the total time a vitamin A moleculespends in the system before irreversible loss. The time that it takes for theaverage molecule which leaves compartment I to cycle back to compartment Iis known as the recycling time [ff(T)]; it was calculated as [T(SYS) — f (I)]/u(r).Finally, to calculate the system disposal rate for vitamin A (DR), individualanimal mean plasma retinol pool sizes (mean plasma retinol concentrationmeasured during the kinetic study * estimated plasma volume) were multipliedbyFCR,,.

Statistical Analysis

Data are presented as arithmetic group mean ± 1 standard deviation. Fordescriptive data and kinetic parameters calculated from the model L(U)s, astandardized t test with an alpha level of 0.05 was used (Ryan et at, 1985) totest for significant differences between groups. For group population meanL(IJ)s in each experiment, a t test (SigmaStat) was used to test for significantdifferences between groups.

RESULTS

Descriptive Data

Body weights at the time of administration of vitamin A-la-beled doses averaged 426 ± 34 g for rats in experiment 1 and413 ± 35 g for those in experiment 2. Body weight gain wassignificantly lower for TCDD-treated versus control rats inexperiment 2, and it tended to be lower in experiment 1 (Table1). As shown in Table 1, liver weights were not significantlyaffected by TCDD, but relative liver weights were significantlyhigher in TCDD-treated rats. Thymus weights and relativethymus weights were significantly lower in TCDD-treated ver-sus control animals in experiment 1 and tended to be lower inexperiment 2.

In addition, TCDD treatment was associated with changes invitamin A levels in plasma and liver (Table 1). Plasma retinolconcentrations were higher in TCDD-treated rats, although thedifference was not significant in experiment 2 (p < 0.066).Liver vitamin A levels were reduced by 60% (experiment 1)and 44% (experiment 2) in TCDD-treated versus control rats 8days after administration of the TCDD loading dose (t0). Dif-ferences were even greater (97 and 95%) 42 days later (t,). Asindicated in Table 1, we estimate that control rats in bothexperiments were in a positive vitamin A balance, whereasTCDD-treated rats were in negative balance.

Experiment 1: Kinetic Data

In order to determine which aspects of whole-body vitaminA kinetics were affected by TCDD exposure, we administered[3H]retinol in its physiological plasma transport complex tocontrol and TCDD-treated rats and used model-based compart-mental analysis to quantify vitamin A disposal rate and retinolrecycling. Disappearance of injected [3H]retinol/RBP/TTRfrom plasma versus time is shown as a semi-log plot in Fig. 1for one representative control and one TCDD-treated rat.Curves for other rats were very similar. As in earlier experi-ments (Green and Green, 1994), the plasma pool of retinolacted kinetically as a single compartment and, in control rats,about 50% of the injected radioactivity was rapidly removedfrom plasma during the first 50-60 min after administration oflabel (Fig. 1, bottom). The early bend (1-2 h) in the curveindicates recycling of labeled retinol to plasma, primarily froma rapidly turning-over extravascular compartment. Betweendays 2 and 10 (Fig. 1, top), there was a slow, gradual bend inthe plasma decay curve, characteristic of exchange of label

EFFECTS OF TCDD ON VITAMIN A KINETICS

TABLE 1Body and Organ Weights and Plasma and Liver Vitamin A Levels in Control and TCDD-Treated Rats

Measurement

BWt gain (g)°Liver wt (g)Liver wt/kg BWtThymus wt (g)Thymus wt/kg BWt[ROH]p (Annol/liter)6

<o Liver A (nmol)f, Liver A (nmol)Balance (nmol/day)c

Control

134 + 6418.3 ± 3.432.3 ± 1.40.47 ±0.170.84 ± 0.241.60 + 0.17

2365 + 1653772 + 588

+ 32

Experiment 1

TCDD

100 ± 2 221.0 ± 1.640.0 ± 2.1*0.30 ± 0.06*0.58 ±0.12*2.12 ±0.26*964 ± 216*

71.6 + 39.3*- 2 0

Control

139 + 1820.5 + 0.634.3 + 2.40.43 +0.170.77 + 0.261.22 ±0.191943 + 2744141 +339

+51

Experiment 2

TCDD

103 ± 25*22.7 ± 1.639.5 ± 2.5*0.32 ±0.110.62 ±0.151.46 ±0.281082 ± 191*98.4 + 51.8*

- 2 2

Note. Data are means ± SD. In experiment 1, n = 7 for both controls and TCDD-treated; in experiment 2, n = 8 for controls and n = 7 for TCDD. For t0

liver vitamin A, n = 3. BWt, body weight; [R0H]p, plasma retinol concentration; tg, time zero (1 day before tracer administration and 8 days after the TCDDloading dose); („ end of kinetic study (42 days after tracer administration).

" Body weight gain was calculated over the 42-day kinetic study.* Values are mean plasma retinol concentration during the 42-day kinetic study based on eight measurements/rat There were no significant effects of time on

plasma rctinol concentrations.c Liver vitamin A balance was estimated as (/, liver vitamin A — t0 liver vitamin A)/43 days.* Significantly different (p < 0.05) from controls in the same experiment.

between plasma and slowly turning-over extravascular pools ofvitamin A; the shape and timing of the bend is related to livervitamin A levels (Green et al., 1987). After 10-14 days, thecurve reached a constant, terminal slope. For TCDD-treatedrats (Fig. 1, dotted line), the same general features in plasmatracer response curves were apparent but responses for the twogroups diverged as early as 21 min after administration of thelabeled dose. The earlier bend indicates a more rapid recyclingof labeled retinol to plasma from rapidly turning-over extravas-cular pools of vitamin A in TCDD-treated rats. The steeperterminal slope indicates a higher fractional rate of loss oflabeled vitamin A from the system in TCDD-treated rats com-pared to controls.

At 42 days after dose administration, TCDD treatment wasassociated with a dramatic reduction in recovery of the label inliver (1.6% versus 30% in control rats), whereas the fraction ofthe dose in carcass was not affected by TCDD treatment (Table2). In both control and TCDD-treated rats, substantially moreradioactivity was recovered in methanol-soluble extracts offeces than urine (Table 2). Cumulative excretion of radioac-tivity in urine and feces was higher in TCDD-treated versuscontrol rats so that, at 10 days, cumulative urinary recoverywas 100% higher in TCDD-treated rats and that in feces was28% higher.

Experiment 1: Model Development and Kinetic Parameters

In view of some similarities in experimental design, livervitamin A levels, and the geometry of the plasma tracer re-sponse profiles, we began model development using the three-compartment model proposed by Green and Green (1994). In

that model, dietary vitamin A enters the system via a centralplasma compartment; this is also the site of introduction oftracer. Plasma retinol in compartment 1 could then exchangewith vitamin A in both a small, rapidly turning-over extravas-cular pool and a larger, slowly turning-over extravascular poolbefore leaving the system from the more slowly turning-overpool. The rapidly turning-over pool was postulated to representa pool of intracellular retinol and retinol in interstitial fluid andpossibly vitamin A filtered by the kidney which is in theprocess of being reabsorbed. The more slowly turning-overcompartment would include vitamin A in retinyl ester-contain-ing storage pools, primarily in the liver.

Plasma tracer data for individual rats in experiment 1 werecompared to the three-compartment starting model. Even withthe addition of separate outputs to accommodate data for urineand feces, that model did not adequately fit the current data forplasma tracer between days 2 and 10. Specifically, simulationsof the three-compartment model predicted a much lower recy-cling of the label into plasma from the slowly turning-over poolthan was observed here. Thus, we added a fourth compartmentexchanging with the more slowly turning-over extravascularcompartment (Fig. 2), hypothesizing that this compartmentincludes the least dynamic pools of body vitamin A (e.g.,retinyl esters in lipid droplets of hepatic perisinusoidal stellatecells and in other large, slowly turning-over extrahepatic vita-min A stores). Addition of a fourth compartment resulted in asignificant improvement in the sum of squares as determinedby an F statistic (Landaw and DiStefano, 1984); thus, thisstructure was accepted as our working model.

Values for the model parameters [i.e., the fractional transfer

KELLEY ET AL.

1.0

0.1

0.01

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I M I I I I I I I I ) M I I I I I

0.001 -I I I I I I I I I I I I I I I I I I I I I I t I I I I I I I: l I I I I I I I I I I I I I I -

20

Time (days)

30 40

1.0

0.1

- ^ ^

—

' 1 , , , ! 1

1 < 1 1 1

1 1 1 1 1

. 1 1 1 , 1 . 1 1

1 . . . .

' -

:

i

0.00 0.25 0.50

Time (days)

0.75 1.00

FIG. 1. Tracer response profiles (fraction of injected [3H]retinol-labeledplasma remaining versus time after dose administration) in one representativecontrol and one representative TCDD-treated rat in experiment 1. Top showsobserved data (symbols) and model-predicted values Oines) for plasma fractionof dose over 42 days (control, • ; TCDD A) and for irreversible loss [1 —(fraction of dose in plasma + liver + carcass)] on day 42 (control, O; TCDD,V). The bottom shows the first day's plasma data for the same rats on anexpanded scale. The model-predicted lines are the responses predicted by thecompartmental model shown in Fig. 2.

TABLE 2Recovery of Administered Radioactivity (Fraction of Dose) in

liver, Carcass, and Excreta of Control and TCDD-Treated Ratsin Experiment 1

Measurement

LiverCarcassCumulative urine (day)

14710

Cumulative feces (day)14710

Control

0.300 ± 0.0380.139 ± 0.049

0.009 ± 0.0040.023 ± 0.0050.031 ± 0.0070.037 ± 0.009

0.024 ± 0.0090.140 ±0.0190.194 + 0.0270.235 ± 0.036

TCDD

0.016 ± 0.039*0.151 ± 0.022

0.019 ± 0.006*0.048 ±0.011*0.062 ± 0.014*0.075 ± 0.017*

0.036 ± 0.0180.185 ± 0.0290.258 + 0.039*0.300 ± 0.046*

Note. Values are means ± SD (n = 7) for fraction of administered radio-activity recovered in liver and carcass at the end of the kinetic study (42 daysafter administration of [3H]retinol-labeled plasma) to control or TCDD-treatedrats and in pools of urine and feces collected on days 1, 4, 7, or 10 after traceradministration.

* Significantly different from controls (p < 0.05).

plasma from this pool [L(l,2)] was slightly but not signifi-cantly higher in the same group. The most dramatic effects ofTCDD were seen on the more slowly turning-over vitamin Acompartments (compartments 3 and 4): fractional release of

U(i)

L(43) /^~\ UJ.l) / ^ ^ \ UAi; w<-^^-f 3 )< / lX^~^L<3'4) / \ U U ) U U )

U31.3)

coefficients or L(I,J)s] for each rat were adjusted to obtain thebest fit of the data to the proposed model. The model's abilityto fit the data is indicated by comparing the model-predictedvalues for plasma tracer response versus time (solid and dottedlines; Fig. 1) to the observed values (symbols). A good matchwas also obtained between observed and predicted data forirreversible loss on day 42 (Fig. 1) and for tracer excretion inurine and feces (data not shown). All model parameters werewell identified, based on the criterion that the estimated frac-tional standard deviations for each L(IJ) in each rat's modelwas less than 0.5 and in most cases, they were less than 0.15.

Using the multiple studies feature of CONSAM (see Mate-rials and Mediods), group mean fractional transfer coefficients[L(I J)s] and their statistical uncertainties were calculated (Ta-ble 3). L(2,l), the fractional transfer of retinol from plasma tothe rapidly turning-over extravascular compartment, was sig-nificantly higher (43%) in TCDD-treated rats; die transfer to

Urine Feces

FIG. 2. Working compartmental model developed to fit tracer data forplasma, carcass, irreversible loss [1 — (fraction of dose in plasma + liver +carcass)], and excreta (urine and feces) of control and TCDD-treated ratsinjected with [3H]retinol-labeled plasma in experiment 1. Circles representcompartments and interconnectivities are the adjustable model parameters{fractional transfer coefficients [L(LJ)s] or the fraction of compartment J'stracer transferred to compartment I each day}. Compartment 1 representsplasma retinol and compartments 2-4 are extravascular pools of vitamin A;compartment 31 is the site of urinary output of tracer and compartment 32 isfeces. The asterisk indicates the site of tracer introduction and U(l) representsdietary input into the system. By adjusting the values for the L(I J)s for eachindividual rat, this model structure provided a good fit to the data for the 7controls and 7 TCDD-treated rats. As an example, the differential equationused to describe the change in labeled vitamin A present in compartment 1 isdQ,/dt = L(1,2)*Q(2) + L(1,3)*Q(3) - [{L(3,1)*Q(1)} + {L(2,1)*Q(1)}],where Q, equals the amount of labeled vitamin A in compartment I and L(I J)sare fractional transfer coefficients.

EFFECTS OF TCDD ON VITAMIN A KINETICS

TABLE 3Model-Predicted Fractional Transfer Coefficients [L(I>-Ds] for

the Proposed Model of Vitamin A Metabolism in Control andTCDD-Treated Rats in Experiment 1

L(U)(day-')

U2,DL<1,2)U3,l)Ul,3)U4,3)WA)U31.3)L(32,3)

Control

13.9 ± 3.914.6 ± 4.84.73 ± 1.36

0.0985 ± 0.03660.189 ±0.103

0.0849 ± 0.04940.00789 ± 0.004220.0499 ± 0.0185

TCDD

19.6 ± 3.9*18.2 ± 5.34.03 ± 0.62

0.182 ± 0.087*0.489 ± 0.217*0.224 ± 0.066*

0.0284 ±0.0114*0.145 ± 0.0480*

Note. Values are mean fractional transfer coefficients [L(LJ)s], or the frac-tion of compartment J's tracer transferred to compartment I each day ±estimated standard deviation for control (n = 7) and TCDD-treated rats (n =7) after administration of [3H]retinol-labeled plasma in experiment 1. Valueswere obtained by using the multiple studies feature of CONSAM: the L(I,J)sfor each individual rat were used to calculate the population mean ± SD forcontrols versus TCDD-treated rats. L(IJ)s describe the model presented inFig. 2.

• Significantly different from controls (p < 0.05).

label from compartment 3 to plasma [L(l,3)] was doubled withTCDD treatment, but there was no influence on uptake fromthe plasma [L(3,l)]. L{4,3) and L(3,4) were each 2.6 timeshigher in TCDD-treated than in control rats, indicating agreatly increased turnover of vitamin A between these pools.

Using model-predicted L(I,J)s for each rat, additional kineticparameters (Table 4) were calculated as described under Ma-terials and Methods. Although TCDD exposure did not signif-icantly affect the transit or residence times for retinol incompartment 1 (plasma) or in the rapidly turning-over ex-travascular compartment (compartment 2), TCDD caused areduction in the mean transit time for vitamin A in compart-ments 3 and 4 [7(3) and 7(4)] by 60 and 68%, respectively,compared to controls. Mean residence times in compartments 3and 4 [f(3) and f(4)] were reduced by 65 and 73%, respec-tively, with TCDD treatment, so that the time that the averagevitamin A molecule spent in the entire system before leavingirreversibly [f(SYS)] was reduced by 70% in TCDD-treatedrats. After TCDD exposure, the average vitamin A moleculetook one-third the time to recycle to plasma [H(l)] of the timein controls, and it recycled about three more times before beingirreversibly utilized [u(l)]. Although the plasma tracer re-sponse curves for TCDD-treated rats showed a steeper terminalslope (Fig. 1), the plasma fractional catabolic rate (FCRp) wasnot significantly affected by TCDD (Table 4). This seemingcontradiction is due to the relative contributions of early andlater responses to the area under the curve and the increasedretinol recycling to plasma in TCDD-treated rats. The vitaminA disposal rate (DR) was significantly higher (27%) in TCDD-treated than control rats.

Experiment 2: Kinetic Data

In order to examine effects of TCDD exposure on themetabolism of dietary vitamin A, rats in experiment 2 wereinjected with [3H]retinyl ester-labeled lymph chylomicrons.Plasma tracer response curves for a representative control andTCDD-treated rat versus time after administration of labeledchylomicrons are presented in Fig. 3. Curves for other animalsfollowed similar patterns. The majority (90.5%) of the radio-activity in the administered lymph chylomicron dose waspresent as retinyl esters (Fig. 3, bottom). In control animals,essentially all of the injected radioactivity was cleared from theplasma within the first 30 min (Fig. 3, middle, solid line),reflecting lipolysis of chylomicron triglycerides and subse-quent clearance of chylomicron remnants containing [3H] reti-nyl esters primarily by liver. Then, plasma radioactivity rosefrom ~30 min to a peak at 140 min, corresponding to liversecretion of [3H]retinol/RBP/TTR into plasma. Subtle differ-

TABLE 4Kinetic Parameters for Vitamin A Kinetics in Control and

TCDD-Treated Rats in Experiment 1

Parameter

41) (h)K2)(h)7(3) (days)7(4) (days)f ( l ) (day)f (2) (day)f (3) (days)f (4) (days)f (SYS) (days)

t<D"(1) (day)FCRp (days"1)DR (nmol/day)

Control

1.34 ± 0.341.75 ± 0.523.51 ± 1.8515.4 ± 7.9

0.613 ±0.1940.594 ±0.193

18.2 ± 5.3949.0 ± 28.668.4 ± 32.98.38 ± 1.705.41 ± 0.801.74 ± 0.4347.9 ± 8.4

TCDD

1.02 ±0.171.38 ±0.451.39 ±0 .61*4.94 ± 1.82*

0.514 ±0.0730.556 ± 0.0906.38 ± 2.36*13.1 ±3 .8*20.5 ± 4.8*11.1 ± 2.3*1.86 ±0.73*1.98 ±0.3260.8 ± 10.3*

Note. Values are group mean model-predicted kinetic parameters ± SD forcontrol (n = 7) and TCDD-treated rats (n = 7). The model is presented in Fig.2. Parameters are transit times [/(I)], or the mean of the distribution of timesthat retinol molecules entering compartment I remain in compartment I duringa single transit before leaving reversibly or irreversibly, where 7(1) = 1/2L(J,I);residence times [T(I,J)], or the mean of the distribution of times that retinolmolecules spend in compartment I after entering the system via compartmentJ before irreversibly leaving compartment L are equal to the area under thetracer response curve for compartment I integrated from 0 to infinity [AUC(I)];f (SYS), system residence time, or the length of time the average vitamin Amolecule spends in the system before irreversible loss, where T(SYS) =2T(I J); recycling number [v(I)], or the average number of times a molecule ofretinol recycles through compartment I before leaving compartment 1 irrevers-ibly, where u(I) = [f (I)/7(T)] - 1; recycling time [JnT)], or the time it takes forthe average retinol molecule leaving compartment I to return to compartmentI, where H(I) = [f(SYS) - f(I)]/u(I); FCRp, the plasma fractional catabolicrate, or the fraction of the plasma retinol pool that is irreversibly utilized eachday, where FCR(U) = U(J)/M(I), where U(J) is die rate of input of tracee intocompartment J; and DR, disposal rate, or the daily utilization rate for vitaminA, where DR(SYS) = FCR(U) * M(I).

* Significantly different from controls (p < 0.05).

KELLEY ET AL.

1.0

0.1

0.01

0.001

- 1

I1

l l i l

I /

• ^ *

ct

=- t~ t

l l i l

OB—

1 1

1 1 1 1

a n .

i J , i

, , | , , i

A cO C

1 1 1 1 1 1 1

1

- A

I ,

1

-° oA- A

I 1 I 1 | .

- A

, 1 I 1 1 I

o -

- E

A ~=

I

10 20

Time (days)

30 40

1.0

0.1

0.01

-

-

II

ii

-

A A

* A V - 9 ^ ^

ifi t i i

i • * •

A -A ,

I I I ,

i | i i i i

" A

1 1 1 , 1 1

111,

1

-

>i'l

lllll

1

~ ~ ~ ~ ~ ~ — — - _ "

V I I I

0.00 0.25 0.50

Time (days)

0.75 1.00

1.0 -

"S 0.1 -

0.01 -

' 1 '

! \A

** \'• ^

oo

1 1 1

1

A

\ n

\

-.0

1

1 '

A

\ O

v- - 6 "

1

A

|

1 1 1

A

,—

1 1 '

' A

—o

i 1 i

1 '

...oA

a

1 1

__ ' - "

i i i i

E-

...a

ml

--

A

—a- -

. . . . "

0.000 0.025 0.050

Time (days)

0.075 0.100

FIG. 3. Plasma tracer response profiles for a representative control ( • )and TCDD-treated rat (A) for 42 days (top) after administration of [3H]vitaminA-labeled lymph chylomicrons (experiment 2) and irreversible loss on day 42(control, O, TCDD V). Also shown are the first day's plasma response on anexpanded scale (middle) and fraction of dose in plasma retinol (control, O;TCDD, V) and retinyl esters (control, D; TCDD, A) during the first 2 h(bottom). Lines are the responses predicted by the compartmental modelshown in Fig. 4.

ences between controls and TCDD-treated rats were evidenteven at very early times after dosing. Specifically, there was anearlier bend in the plasma tracer response curve between 10and 30 min in the TCDD-treated rat (Fig. 3, middle, dottedline), a later peak in plasma retinol radioactivity (215-260min), and wider appearance of die "hump." At later times (Fig.

3, top), as in experiment 1, plasma response profiles forTCDD-treated rats showed an earlier bend (beginning at ~ 1day) and a much steeper terminal slope, compared to controls.

Essentially all of the administered radioactivity (>94%) wasrecovered in plasma + liver + small intestine + carcass at 25min in both groups. Recovery of the radioactive dose in smallintestines was low in the short-term rats from both groups,averaging 0.4-1% at 25 min and 1.8-2.6% at 8 h. This resultindicates that essentially none of the chylomicron dose hadbeen cleared via the liver reticuloendothelial system and ex-creted into bile and small intestine, as it might have been if thedose had contained a nonphysiological component.

Hepatic recovery of the radioactive dose is shown in Table5. At all times, the majority of the radioactivity in liver was inretinyl esters. For control rats, hepatic recovery at 8 h waslower than that at 25 min. At 8 h and 2 days, hepatic recoverieswere significantly lower in livers of TCDD-treated versuscontrol rats. By 42 days, differences between the groups wereeven more marked than in experiment 1, with only 0.6% of thedose recovered in livers of TCDD-treated rats versus 46% incontrols.

Recovery of the injected radioactive dose in carcass (Table5) was similar for TCDD-treated and control rats at 25 min,whereas recovery tended to be higher at 8 h (18%) and it was

TABLE 5Recovery of Administered Radioactivity (Fraction of Dose) in

Liver, Carcass, and Excreta of Control and TCDD-Treated Ratsin Experiment 2

Measurement Control TCDD

Liver25 min Total

RE8 h Total

RE2 days Total

RE42 days Total

Carcass25 min8 h2 days42 days

Cumulative urine4 days

Cumulative feces4 days

0.762 ±0.0180.580 ± 0.0140.438 ± 0.0670.382 ± 0.0570.633 ± 0.0340.576 ± 0.0320.462 ± 0.073

0.134 ± 0.0340.317 ± 0.0290.152 ±0.0180.079 ± 0.009

0.026 ± 0.004

0.050 ± 0.007

0.7660.6160.2580.1940.2350.2080.0061

0.0920.3750.2520.047

0.075

0.139

± 0.038± 0.035± 0.018*± 0.024*± 0.034*±0.002*± 0.0022*

±0.003±0.069± 0.030*± 0.013*

±0.009*

± 0.026*

Note. Data are means ± SD for fraction of administered dose of [3H]vitaminA-labeled lymph chylomicrons recovered in total liver, liver retinyl esters(RE), carcass, urine, and feces. Urine and feces were collected as one 4-daypool from the time of administration of label. N = 3 for control and TCDD-treated rats killed at 25 min, 8 h, and 2 days after administration of label, n =8 for controls killed at 42 days, and n = 7 for TCDD-treated rats at 42 days.

* Significantly different from controls (p < 0.05).

EFFECTS OF TCDD ON VITAMIN A KINETICS

significantly higher (66%) at 2 days in TCDD-treated than incontrol rats. By the end of the experiment, recovery of thelabeled dose in carcass had decreased to levels lower than thoseobserved in experiment 1 and was significantly lower inTCDD-treated rats than in controls.

As in experiment 1, TCDD treatment was associated withsignificant increases in urinary and fecal excretion of labeledvitamin A, and more of the dose radioactivity was excreted infeces than in urine (Table 5). At 4 days after administration ofthe labeled dose, cumulative recovery of radioactivity wasalmost three times higher in urine and feces of TCDD-treatedversus control rats. Although >94% of the administered radio-activity was recovered at 25 min, at later times, the recoverywas lower due to metabolism and excretion of the label. Forexample, recovery in plasma + liver + carcass + smallintestine at 2 days averaged 80.7 ± 1.6% (n = 3) in control ratsand 51.7 ± 3.6% (n = 2) in TCDD-treated animals. Assumingthat excretion of radioactivity in urine and feces at 2 days wasless than that observed at 4 days, some of the dose (~15% incontrols and 20% in TCDD-treated rats) had already beenconverted to nonextractable polar metabolites. The nonrecov-ery of such polar metabolites would not alter our kineticanalyses or results, since, once retinol is metabolized to reti-noic acid or other polar metabolites, it cannot recycle to plasmaretinol and is thus irreversibly utilized from the kinetic per-spective. Still, additional useful information could be obtainedin future studies by using a biological materials oxidizer tocombust samples of carcass and feces to 3H2O prior to liquidscintillation spectrometry.

Experiment 2: Model Development and Kinetic Parameters

We began model development for experiment 2 data usingthe four-compartment model developed in experiment 1, afteraddition of compartments needed to account for the introduc-tion of the label on lymph chylomicrons (Fig. 4). In view of thedistribution of label in lymph, 90.5% of the dietary input intothe system [U(I)] is shown into the plasma retinyl ester inputcompartment (compartment 5) and 9.5% into a chylomicronretinol compartment (compartment 11). In order to explain thecomplex geometry of the initial portion of the curves and basedon current understanding of the metabolism of vitamin A inchylomicrons, a fraction of labeled chylomicron retinyl esterswas transferred from compartment 5 to a rapidly turning-overextravascular compartment (compartment 7) that was neededto fit early data for [3H]retinyl esters in plasma. To fit the laterrise in labeled plasma retinol, a delay element (component 6),presumably in the liver, was also required. The model requiresthat, after processing through compartment 7 and component 6(which together probably represent liver uptake of chylomi-cron-derived retinyl esters, hydrolysis of the esters, and trans-fer of retinol to apoRBP), labeled retinol is released intoplasma (compartment 1) bound to RBP. Labeled retinol in theadministered dose (compartment 11) was processed through a

DT(6)

System Output

FIG. 4. Working compartmental model developed to fit tracer data forplasma, carcass, and irreversible loss in control and TCDD-treated rats injectedwith [3H]vitamin A-labeled lymph chylomicrons in experiment 2. Circlesrepresent compartments and interconnectivities are fractional transfer coeffi-cients [L(I,J)s] or the fraction of compartment J's tracer transferred to com-partment I each day. Compartments 5, 7, 9, and 11, and delay component 6,were used to fit data for plasma tracer disappearance and hepatic processing ofchylomicron vitamin A; compartment 1 represents plasma retinol; compart-ments 2 and 3 are extravascular pools of vitamin A. The asterisks indicate sitesof tracer introduction (90.5% into compartment 5 as retinyl esters and 9.5%into compartment 11 as retinol) and U(5) represents dietary input into thesystem. As an example, the differential equation used to describe the change inlabeled vitamin A present in compartment 1 is dQ,/dt = L(1,6)*Q(6) +L(1,2)*Q<2) + U1,3)*Q(3) + L(1,9)*Q<9) - [{L<3,1)*Q(1)) +(L(2,1)*Q(1)}], where Q| equals the amount of labeled vitamin A in compart-ment I and L(IJ)s are fractional transfer coefficients.

single extravascular compartment (compartment 9) beforetransfer into plasma compartment 1. As in the model developedfor experiment 1, once the label was secreted into plasma, itwas able to exchange with vitamin A in both a rapidly turning-over extravascular compartment (compartment 2) and a moreslowly turning-over extravascular compartment (compartment3) before leaving the system irreversibly through compartment3. Since less extensive information on urinary and fecal outputof radioactivity was collected in the second experiment, themodel shows only one site of output [L(0,3)]. Also, in contrastto the model developed for experiment 1, a second slowlyturning-over extravascular compartment was not statisticallyjustified.

As in experiment 1, tracer data for each rat were fit to thismodel by adjusting the values for the fractional transfer coef-ficients [L(IJ)s]. As is evident in Fig. 3 by comparing observed

10 KELLEY ET AL.

TABLE 6Model-Predicted Fractional Transfer Coefficients [L(I,J)s] for

Proposed Model of Vitamin A Metabolism in Control and TCDD-Treated Rats in Experiment 2

U\J) Control TCDD

L(7,5) (days"1)U5,7) (days"1)L(6,7) (days"1)DT(6) (min)L(9,ll)(days~')L(l,9) (days'1)U2.1) (days"1)L(U) (days"1)L(3,l) (days"1)L(l,3) (days"1)L(0,3) (days'1)

185 ±481.82 ±0.726.10 ± 0.8437.3 ± 3.4222 ± 676.10 ± 1.7912.6 ± 3.82.48 ± 1.4415.4 ± 3.9

0.0931 ± 0.01690.0200 ± 0.0042

240 ± 11813.8 ± 12.2*10.8 ±5.1*56.4 ± 11.3*258 ± 936.96 ± 3.7316.9 ± 8.15.86 ± 2.98*13.6 ± 3.4

0.210 ± 0.048*0.0743 ± 0.0101*

Note. Values are population mean fractional transfer coefficients [L(IJ)s], orthe fraction of compartment J's tracer transferred to compartment I each day ±estimated population standard deviation for control (n = 8) and TCDD-treatedrats (n = 7) after administration of [3H]vitamin A-labeled lymph chylomicronsin experiment 2. L(IJ)s describe the model presented in Fig. 4.

• Significantly different from controls (p < 0.05).

data (symbols) to the model predictions (lines), the modelprovided a good fit to plasma tracer data. All parameters werewell identified (estimated parameter FSDs < 0.5), with thehighest variability seen in L(5,7). Group mean fractional trans-fer coefficients and their statistical uncertainties are shown inTable 6. Several parameters associated with metabolism of thechylomicron dose [L(5,7), L(6,7), and DT(6)] were signifi-cantly higher in TCDD-treated than in control rats. The in-crease in L(5,7) suggests that a higher fraction of the label thatreaches the liver on absorptive lipoproteins is recycled toplasma before hepatic uptake in TCDD-treated rats (56% ver-sus 23% in controls). The increase in L(6,7) in the TCDDgroup indicates that, after intemalization, label was more rap-idly transferred to the delay component. Before secretion intoplasma as [3H]retinol bound to RBP, the processing of thelabeled diet-derived vitamin A in liver [DT(6)] took 51%longer in TCDD-treated rats. For the parameters related tometabolism of retinol/RBP, fractional transfers of retinol fromplasma compartment 1 to extravascular compartments 2 and 3[L(2,l) and L(3,l), Table 6] were unaffected by TCDD treat-ment but die L(IJ)s reflecting recycling to the plasma com-partment from compartments 2 and 3 were both more thandoubled in TCDD-treated rats. Finally, the fraction of label thatwas irreversibly lost from the system [L(0,3)j was 3.6 timeshigher in animals given TCDD than that in controls.

Additional kinetic parameters for experiment 2 are presentedin Table 7. The mean transit time for vitamin A in compart-ment 7 [f(7)] was 49% lower after TCDD exposure. Meantransit times in compartment 1 [f(l)] were similar to thosedetermined in experiment 1 and were not different for TCDD-treated versus control rats. TCDD caused significant reductions

(57 and 60%) in the transit times in compartments 2 and 3,respectively. The mean residence times [f (I)] in compartments1-3 were all significantly reduced with TCDD treatment. Ofthese perturbations, the most dramatic effect was seen in com-partment 3, where TCDD caused a 74% reduction in meanresidence time. The reduced residence time in compartment 3was the most significant contributor to the 73% lower systemresidence time [f (SYS)] in the TCDD-treated rats; this reduc-tion is comparable to that calculated for experiment 1. VitaminA recycling time [«(!)] was significantly lower (63%) inTCDD-treated rats, whereas the plasma fractional catabolicrate (FCRp) and the disposal rate (DR) were significantlyhigher (35 and 52%, respectively) in TCDD-treated versuscontrol rats.

TABLE 7Kinetic Parameters for Vitamin A Kinetics in Control and

TCDD-Treated Rats in Experiment 2

Parameter

7(5) (h)7(7) (h)7(9) (h)7(ll)(h)7(1) (h)7(2) (h)7(3) (days)f ( l ) (day)t(2) (days)f (3) (days)T(SYS) (days)

ni.1) (days)FCRp (days'1)DR (nmol/day)

Control

0.139 ±0.0423.07 ± 0.464.27 ± 1.28

0.120 ± 0.0480.894 ± 0.269

12.2 ± 5.89.05 ± 1.58

0.381 ± 0.0292.28 ± 0.7352.3 ± 11.755.2 ± 11.98.55 ± 2.625.61 ± 1.342.60 ±0.1955.3 ± 6.6

TCDD

0.130 ± 0.0721.50 ± 1.05*4.31 ± 2.00

0.105 ± 0.0370.837 ± 0.232

5.29 ± 3.00*3.62 ± 0.69*

0.295 ± 0.061*0.939 ± 0.484*

13.8 ± 2.2*15.2 ± 2.2*8.10 ±3.152.10 ± 0.85*3.50 ± 0.63*84.1 ± 17.1*

Note. Values are calculated kinetic parameters (means ± SD) for control(n = 8) and TCDD-treated rats (n = 7). The model is presented in Fig. 4.Parameters are transit times [7(1)], or the mean of the distribution of times thatretinol molecules entering compartment I remain in compartment I during asingle transit before leaving reversibly or irreversibly, where 7(1) = 1/2L(J J);residence times [T(I,J)], or the mean of the distribution of times that retinolmolecules spend in compartment I after entering the system via compartmentJ before irreversibly leaving compartment I, are equal to the area under thetracer response curve for compartment I integrated from 0 to infinity [AUC(I)];T(SYS), system residence time, or the length of time the average vitamin Amolecule spends in the system before irreversible loss, where T(SYS) =ST(IJ); recycling number MI)], or the average number of times a molecule ofretinol recycles through compartment I before leaving compartment I irrevers-ibly, where i<I) = [f (I)/7(I)] - 1; recycling time [ml)], or the time it takes forthe average retinol molecule leaving compartment I to return to compartmentI, where /r(I) = [f (sys) - f (I)]/i<I); FCRp, the plasma fractional catabolic rate,or the fraction of the plasma retinol pool that is irreversibly utilized each day,where FCR(IJ) = U(jyM(I), where U(J) is the rate of input of tracee intocompartment i; and DR, disposal rate, or the daily utilization rate for vitaminA where DR(SYS) = FCRflJ) • M(R.

* Significantly different from controls (p < 0.05).

EFFECTS OF TCDD ON VITAMIN A KINETICS 11

DISCUSSION

As summarized in the introduction and in a review by Zile(1992), TCDD exposure has a variety of adverse effects onvitamin A metabolism in rats. Here we investigated the mech-anisms involved in the effects of TCDD exposure on vitamin Ametabolism by using model-based compartmental analysis todetermine which whole-body vitamin A kinetic processes areperturbed by TCDD treatment in the rat.

Based on work by HSkansson and Ahlborg (1985) andHakansson and Hanberg (1989), which showed that TCDDadministration affects the storage and excretion of newly ab-sorbed vitamin A, we speculated that TCDD might disruptvitamin A homeostasis by altering the uptake and processing ofnewly absorbed retinyl esters or by affecting the normal, ex-tensive recycling of retinol among plasma, liver, and extrahe-patic tissues that has been documented by previous compart-mental models (Green et al, 1985, 1987). Our results fromexperiment 2 indicate that there are subtle effects of TCDDtreatment on chylomicron clearance, as well as on the rate ofprocessing of chylomicron-derived vitamin A in the liver.However, by 1 day, the model (Fig. 4) predicts that in bothgroups, the liver had processed essentially all of the labeledincoming chylomicron vitamin A. Thus, it seems unlikely thatthe dramatic effects of TCDD on vitamin A metabolism can beattributed to alterations in the metabolism of chylomicronsand/or the initial hepatic handling of chylomicron-derived vi-tamin A. Additionally, results from both experiments indicatethat there is no effect of TCDD on the fractional uptake ofRBP-retinol by tissues [L(2,l) and L(3,l), Tables 3 and 6].

Although tissue uptake of vitamin A from plasma was un-affected by TCDD treatment, the models predict that TCDDincreases the fractional recycling of vitamin A from faster andmore slowly turning-over pools into plasma (Table 3). Livervitamin A balance data (Table 1) are compatible with the ideathat, as liver vitamin A levels decrease (by —90%) during theturnover study in TCDD-treated rats, a larger fraction of theremaining pool is mobilized to maintain a constant rate ofretinol secretion into plasma, resulting in stable (althoughslightly increased) plasma retinol concentrations. Althoughinteresting, this increased mobilization of vitamin A intoplasma does not explain how TCDD might be interfering withvitamin A storage in liver (Hakansson and Ahlborg, 1985;Hakansson et al, 1988) and liver stellate cells (Hakansson andHanberg, 1989; Nilsson et al., 1996).

Based on a study by Nilsson et al. (1996), which showed thatTCDD decreases both lecithin:retinol acyltransferase and acylCoA:retinol acyltransferase activity in hepatic stellate cells, weexpected to see a lower transfer of label into vitamin A storagepools in experiment 1. However, our kinetic results indicatethat vitamin A is moved into storage pools more rapidly withTCDD treatment and that it is also removed more quickly. Thatis, there may be a futile cycling of vitamin A between com-partments 3 and 4, involving increased formation or storage of

retinyl esters, and an increased net mobilization of vitamin A(most likely through increased hydrolysis in conjunction withincreased degradation). Although Brouwer et al. (1988) foundno influence of TCDD on activity of the bile salt-dependentretinyl ester hydrolase (REH) in rat liver homogenates, there isthe possibility that the membrane-bound, neutral REH activity,which is present in rat liver stellate cells as well as hepatocytes(Matsuura et al., 1997), or the acid REH could be involved. Inthis regard, Nilsson et al. (unpublished data) have found thatthe activity of membrane-bound, neutral REH activity in wholeliver, as well as isolated stellate cells and hepatocytes, is notaffected by TCDD treatment. It is worth noting that increasedhydrolysis need not require an increase in the amount of REHprotein, but might result from an increase in relative activitydue to the removal of product.

Regarding effects of TCDD on degradation of vitamin A,our data (Tables 2 and 5) and earlier studies (Hakansson andAhlborg, 1985; Hakansson et al, 1988) show that TCDDtreatment results in increased excretion of vitamin A metabo-lites in urine and feces. Our models predict significant eleva-tions in the irreversible utilization of vitamin A in TCDD-treated versus control rats (Tables 4 and 7). The idea (Moore,1957; Wolf, 1984; Hakansson et al., 1988; Green and Green,1996) that vitamin A utilization is composed of both a func-tional (related to the biological activity of vitamin A) and anonfunctional component (a mechanism for ridding the body ofvitamin A through degradation) may be relevant to these ob-served effects of TCDD exposure. First, it is difficult to imag-ine a functional process that could account for the magnitude ofthe depletion in liver vitamin A observed in TCDD-treated rats.Further, work by others (Bank et al., 1989; Fiorella et al.,1995) supports the idea of increases in nonfunctional catabo-lism, possibly related to TCDD induction of cytochrome P-450enzymes [see reviews by Zile (1992) and Pohjanvirta andTuomisto (1994)]. Such an increase in catabolism might resultnot only in a decrease in the availability of intracellular retinoicacid for vitamin A action, but also a need for further mobili-zation from retinyl ester stores.

Due to the significant overlap in the consequences of TCDDtoxicity and vitamin A deficiency, it has been suggested (Zile,1992) that some of the toxic symptoms of TCDD exposure maybe due to a functional vitamin A deficiency in target tissues.However, the plasma retinol turnover rate for TCDD-treatedrats in experiment 1 was ~10 times higher than that predictedfor die vitamin A-deficient rats studied by Lewis et al. (1990).In addition, rats with low vitamin A status had very lowvitamin A disposal rates (5.8 nmol/day versus >60 nmol/dayin TCDD-treated rats); and residence times for retinol inplasma and liver were much lower than those in TCDD-treatedrats. Our results suggest that, at the whole-body level, vitaminA dynamics are dramatically different in TCDD-treated versusvitamin A-deficient rats.

We used model-predicted fractional transfer coefficients[L(I J)s] to estimate compartment vitamin A masses [M(I)] and

12 KELLEY ET AL.

U(l)=82 (42)

+32

(-20)

U(l)-109 (66)

+51

(-22)

58

380 (31S)

271

269

269 (419)

FIG. 5. Estimated (model-predicted) rates of vitamin A transfer (nmol/day) calculated from population mean L(U)s (Tables 3 and 6) and estimatedplasma retinol pool sizes (experiment 1 controls, 28.4 nmol, and TCDD, 31.7nmol; experiment 2 controls, 21.4 nmol, and TCDD, 24.8 nmol) for experiment1 (top) and experiment 2 (bottom). Inputs, and rates of storage and mobiliza-tion, were adjusted to reflect the observed liver vitamin A balances (Table 1).Estimated rates for TCDD-treated rats are shown in parentheses next to thevalues for controls.

rates (nmol/day) of vitamin A transfer between compartmentsand into and out of the system. This was done by adjusting theinput rates and the transfer rates related to storage in order toreflect the observed vitamin A balances (net storage for controlrats or net mobilization for TCDD-treated rats) (Fig. 5). Thatis, we assumed a vitamin A steady state in all compartmentsexcept the most slowly turning-over storage compartments(compartment 4 in experiment 1 and compartment 3 in exper-iment 2). This analysis indicates that, although control andTCDD-treated rats consumed the same diet, TCDD-treatedanimals absorbed ~40 nmol/day less vitamin A than controls.TCDD exposure is frequently associated with a decrease infood intake (Pohjanvirta and Tuomisto, 1994) and in fact, ourdata on differences in body weight gain between TCDD-treatedand control rats (Table 1) may indicate that there was adecrease in food intake. Our calculations suggest that a smalldecrease in food intake, coupled with a decrease in vitamin Aabsorption efficiency, results in a vitamin A input into thesystem which is 40-50% lower in TCDD-treated than controlrats. In other work in this lab, vitamin A absorption tended tobe lower in lymph duct-cannulated rats given a single oral doseof TCDD (Hanberg et al., submitted for publication). In spiteof an estimated decrease in vitamin A absorption, we wouldhave predicted, based on estimates (Green et al., 1985, 1987)of vitamin A utilization in normal adult rats, that TCDD-treated rats in experiment 1 should have been in vitamin Abalance and those in experiment 2 in positive balance. Since, in

contrast, the data indicate that TCDD-treated rats in bothexperiments were in negative vitamin A balance, we concludethat TCDD affects vitamin A utilization (24-52% higher inTCDD-treated rats) independently of its effects on food intakeand vitamin A absorption efficiency.

The rate of movement of vitamin A into slow turning-overstorage compartments (compartment 4 in experiment 1 andcompartment 3 in experiment 2) was not dramatically affectedby TCDD treatment, especially in view of the predicted dif-ferences in vitamin A input. However, mobilization from thosepools was high, resulting in a negative vitamin A balance in theliver of TCDD-treated rats. As argued above, TCDD maydivert vitamin A into catabolic processes. Perhaps this diver-sion decreases the availability of retinol in a pool that isinvolved in the homeostatic regulation of retinyl ester hydro-lysis. Since dietary supplementation with retinoic acid (Keilsonet al., 1979) (and perhaps consequently cellular concentrationsof retinoic acid) and the ratio of apo/holo cellular retinol-binding protein (CRBP) (Herr and Ong, 1992) have beenshown to influence the mobilization of vitamin A from storagepools and the formation of retinyl esters, it would be interestingto study effects of TCDD on cellular retinoic acid, apo/holoCRBP ratios, and CRBP levels.

In conclusion, this paper presents the first application ofmodel-based compartmental analysis to exploring the disrup-tion of vitamin A homeostasis by TCDD. Our results indicatethat the primary sites of the dysregulation by TCDD are in-creased catabolism and mobilization of vitamin A from slowlyturning-over tissue stores of vitamin A (presumably mainly theliver). Based on our data, we are not able to say whether theincreased utilization results in increased mobilization orwhether the increased mobilization leads to increased utiliza-tion. Since past work and the current results suggest that theliver is a main target for TCDD's effects on vitamin A metab-olism, it would be informative to characterize in detail theeffects of TCDD on vitamin A dynamics in the liver, using anapproach similar to that described by Green et al. (1993). Itwould also be interesting to use kinetic approaches to studyTCDD-related changes in renal vitamin A dynamics, in orderto determine whether the reciprocal effects of TCDD on liverand kidney might be due to an alteration in signaling amongkidneys, plasma, and liver.

ACKNOWLEDGMENTS

We thank Ellu Manzoor and Christina Trossvik for their generous technicalcontributions to this project, Dr. Annika Hanberg and Christina Trossvik forperforming lymph duct cannulations, and Dr. Mats Rundgren for administeringTCDD.

REFERENCES

Adams, W. R., Smith, J. E., and Green, M. H. (1995). Effects of N-(4-hydroxyphenyl) retinamide on vitamin A metabolism in rats. Proc. Soc. Exp.Biol. Med. 208, 178-185.

EFFECTS OF TCDD ON VITAMIN A KINETICS 13

Andersen, M. E. (1995). Physiologically based pharrnacokinetic (PB-PK)models in the study of the disposition and biological effects of xenobioticsand drugs. Toxicol. Lett. 82/83, 341-348.

Bank, P. A., Salyers, K. L., and Zile, M. H. (1989). Effect of tetrachloro-dibenzo-p-dioxin (TCDD) on the glucuronidation of retinoic acid in the rat.Biochim. Biophys. Ada 993, 1-6.

Berman, M., Beltz, W. F., Greif, P. C , Chabay, R., and Boston, R. C. (1983).CONSAM User's Guide. U.S. Govt. Printing Office, Washington, DC. PHSPubl. 1983-421-132:3279.

Berman, M., and Weiss, M. F. (1978). SAAM Manual. U.S. Govt. PrintingOffice, Washington, DC. DHEW Publ. 78-180.

Blaner, W. S., and Olson, J. A. (1994). Rennol and retinoic acid metabolism. InThe Reiinoids: Biology, Chemistry, and Medicine (M. B. Spom, A. B. Roberts,and D. S. Goodman, Eds.), 2nd ed. pp. 229-255. Raven Press, New York.

Brouwer, A , Blaner, W. S., Verleg, H., Goettsch, W. G., Van den Berg, K. J., andKoeman, J. H. (1988). Effect of 3,4,3',4'-Tetrachlorobiphenyl (TCB) and2,3,7,8-Tetrachlorodibenzo(p)dioxin (TCDD) on Hepatic Retinyl Palmitate Hy-drolase (RPH) and Acyl-CoA-Retinol Acyltransferase (ARAT) Activities inWAG/RIJ Rats (abstract), p. 147. Dioxin '88: The 8th International Symposiumon Chlorinated Dioxins and Related Compounds. Umea, Sweden.

Fiorella, P. D., Olson, J. R., and Napoli, J. L. (1995). 2,3,7,8-Tetrachloro-dibenzo-p-dioxin induces diverse retinoic acid metabolites in multiple tis-sues of the Sprague-Dawley raL Toxicol. Appl. Pharmacol. 134, 222-228.

Flodstrom, S., Busk, L., Kronevi, T., and Ahlborg, U. G. (1991). Modulationof 23,7,8-tetrachlorodibenzo-p-dioxin and phenobarbital-induced promo-tion of hepatocarcinogenesis in rats by the type of diet and vitamin Adeficiency. Fundam. Appl. Toxicol. 16, 375—391.

Foster, D. M., and Boston, R. C. (1983). The use of computers in compart-mental analysis: The SAAM and CONSAM programs. In CompartmentalDistribution of Radiotracers (}. S. Robertson, Ed.), pp. 73-142. CRC Press,Boca Raton, FL.

Green, M. H., Uhl, L., and Green, J. B (1985). A multicompartmental modelof vitamin A kinetics in rats with marginal liver vitamin A stores. J. UpidRes. 26, 806-818.

Green, M. H., Green, J. B., and Lewis, K. C. (1987). Variation in retinolutilization rate with vitamin A status in the raL J. Nutr. 117, 694-703.

Green, M. H., and Green, J. B. (1990a). The application of compartmentalanalysis to research in nutrition. Annu. Rev. Nutr. 10, 41-61.

Green, M. H., and Green, J. B. (1990b). Experimental and kinetic methods forstudying vitamin A dynamics in vivo. Methods Enzymol. 190, 304-317.

Green, M. H., Green, J. B., Berg, T., Norum, K. R., and Blomhoff, R. (1993).Vitamin A metabolism in rat liver A kinetic model. Am. J. Physiol. 264,G509-521.

Green, M. H., and Green, J. B. (1994). Vitamin A intake and status influenceretinol balance, utilization and dynamics in rats. J. Nutr. 124, 2477-2485.

Green, M. H., and Green, J. B. (1996). Quantitative and conceptual contribu-tions of mathematical modeling to current views on vitamin A metabolism,biochemistry, and nutrition. Adv. Food Nutr. Res. 40, 3-24.

Hakansson, H., and Ahlborg, U. G. (1985). The effect of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) on the uptake, distribution and excretion of asingle oral dose of [11,12-3H)retinyl acetate and on the vitamin A status inthe rat J. Nutr. 115, 759-771.

Hakansson, H., Johansson, L., and Ahlborg, U. G. (1988). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on tissue levels of vitamin A and onthe distribution and excretion of the endogenous pool of vitamin A in themarginally vitamin A sufficient rat. Chemosphere 17, 1781-1793.

Hakansson, H., and Hanberg, A. (1989). The distribution of [l4C]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its effect on the vitamin A contentin parenchymal and stellate cells of rat liver. /. Nutr. 119, 573-580.

Hakansson, H., Manzoor, E., and Ahlborg, U. G. (1991a). Interaction between

dietary vitamin A and single oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the TCDD-induced toxicity and on the vitamin A statusin the rat. J. Nutr. Sci. Vitaminol. 37, 239-255.

Hakansson, H., Johansson, L., Manzoor, E., and Ahlborg, U. G. (1991b).Effects of 2,3,7,8-tetrachlorodibenzo-/>-dioxin (TCDD) on the vitamin Astatus of Hartley guinea pigs, Sprague-Dawley rats, C57B1/6 mice, DBA/2mice, and Golden Syrian hamsters. J. Nutr. Sci. Vitaminol. 37, 117-138.

Hanberg, A., and Trossvik, C. (1995). In situ collection of intestinal lymph inthe non-restrained rat. Scand. J. Lab. Anim. Sci. 22, 329-334.

Hara, A., and Radin, N. S. (1978). Lipid extraction of tissues with a low-toxicity solvent. Anal. Biochem. 90, 420-426.

Herr, F. M., and Ong, D. E. (1992). Differential interaction of lecithin-retinolacyltransferase with cellular retinol binding proteins. Biochemistry 31,6748-6755.

Jacquez, J. A. (19%). Identifiability. Adv. Food Nutr. Res. 40, 305-321.

Keilson, B., Underwood, B. A., and Loerch, J. D. (1979). Effects of retinoic acidon the mobilization of vitamin A from the liver in rats. J. Nutr. 109, 787—795.

Landaw, E., and DiStefano, J. III. (1984). Multiexponential, mutlicompart-mental, and noncompartmental modeling. II. Data analysis and statisticalconsiderations. Am. J. Physiol. 246, R665-677.

Lewis, K. C, Green, M. H., Green, J. B., and Zech, L. A. (1990). Retinolmetabolism in rats with low vitamin A status: A compartmental model. J.Upid Res. 31, 1535-1548.

Lyne, A., Boston, R. C, Pettigrew, K., and Zech, L. A. (1992). EMSA: A SAAMservice for the estimation of population parameters based on model fits toidentically replicated experiments. Comp. Methods Prog. Biomed. 38, 117-151.

Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994). The retinoidreceptors. In 77ie Retinoids: Biology, Chemistry, and Medicine (M. B.Spom, A. B. Roberts, and D. S. Goodman, Eds.), 2nd ed., pp. 319-349.Raven Press, New York.

Matsuura, T., Gad, M. Z., Harrison, E. H., and Ross, A. C. (1997). Lecithin:retinol acyltransferase and retinyl ester hydrolase activities are differentiallyregulated by retinoids and have distinct distnbutions between hepatocyteand nonparenchymal cell fractions of rat liver. J Nutr. 127, 218-224.

Moore, T. (1957). Vitamin A. Elsevier, New York.

Nilsson, C. B., Hanberg, A., Trossvik, C , and Hakansson, H. (19%). 2,3,7,8-Tetrachlorodibenzo-p-dioxin affects retinol esterification in rat hepatic stel-late cells and kidney. Environ. Toxicol Pharmacol. 2, 17-23.

Pohjanvirta, R., and Tuomisto, J. (1994). Short-term toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals: Effects, mechanisms,and animal models. Pharmacol. Rev. 46, 483-549.

Ross, A. C (1982). Retinol esterification by mammary gland microsomes fromthe lactating raL J. Upid Res. 23, 133-144.

Ryan, B. F., Joiner, B. L., and Ryan, T. A., Jr. (1985). Minitab Handbook, 2nded. PWS, Boston, MA.

Thompson, J. N., Erdody, P., Brien, R., and Murray, T. K. (1971). Fluorometricdetermination of vitamin A in human blood and liver. Biochem. Med 5,67-89.

UndeTwood, B. A. (1984). Vitamin A in animal and human nutrition. In TheRetinoids (M. B. Spom, A. B. Roberts and D. S. Goodman, Eds.), Vol. 1, pp.281-392. Academic Press, Orlando, FL.

van Birgelen, A. P. J. M., van der Kolk, J., Fase, K. M., Bol, I., Poiger, H.,Brouwer, A., and van den Berg, M. (1995). Subchronic dose-response studyof 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats. Toxi-col. Appl. Pharmacol. 132, 1-13.

Weston, W. M., Nugent, P., and Greene, R. M. (1995). Inhibition of retinoic-acid-induced gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Bio-chem. Biophys. Res. Commun. 207, 690-694.

Wolf, G. (1984). Multiple functions of vitamin A. Physiol. Rev. 64, 873-938.

Zile, M. H. (1992). Vitamin A homeostasis endangered by environmentalpollutants. Proc. Soc. Exp. BioL Med 201, 141-153.