* The Institute of Environmental Medicine, Karolinska Institutet, P.O. Box 210, S-177 77 Stockholm, Sweden; and
Graduate Program in Physiology and Nutrition Department, Pennsylvania State University, University Park, Pennsylvania 16802
Part of this work was presented in the doctoral theses of Sean Keith Kelley ("The Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics in Rats: A Compartmental Model," Pennsylvania State University, University Park, PA, 1997) and Charlotte B. Nilsson ("Studies on the Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Homeostasis," Karolinska Institutet, Stockholm, Sweden, 1999).
Received November 9, 1999; accepted February 14, 2000
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ABSTRACT |
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Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; rat; vitamin A; model-based compartmental analysis; kinetics.
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INTRODUCTION |
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To investigate the sites and quantitative impacts of TCDD on vitamin A dynamics, we recently applied model-based compartmental analysis (Foster and Boston, 1983; Green and Green, 1990a
) to data on the kinetics of [3H]vitamin A in rats treated with repeated oral doses of TCDD (Kelley et al., 1998
). Our models predict that repeated exposure to TCDD causes a more rapid movement of vitamin A into and out of storage pools in the liver and possibly other tissues (presumably due to increases in esterification and hydrolysis of retinol), as well as an increase in the vitamin A disposal rate. We wanted to discern whether increased retinyl ester hydrolysis was driving the observed increase in vitamin A degradation, whether increased degradation led to a subsequent increase in retinyl ester hydrolysis, or whether both responses occurred simultaneously. To distinguish between these possibilities, we administered TCDD to perturb the kinetics of previously injected [3H]vitamin A in rats and observed the resultant changes in plasma tracer ([3H]) response. We hypothesized that if the initial effect of TCDD was to mobilize vitamin A from liver stores, this would be reflected by an increase in plasma tracer concentrations. If, on the other hand, increased degradation was the earliest effect, we would observe an initial fall in plasma tracer concentrations after TCDD treatment. By applying model-based compartmental analysis to data collected after the TCDD perturbation, we were able to identify and quantitate the kinetic processes that were initially affected by TCDD administration.
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MATERIALS AND METHODS |
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This study was conducted at the same time as two related experiments that have been recently described in detail (Kelley et al., 1998), using extra rats that were not needed for that project. Animals described here were treated identically to control rats in the larger experiments (except for TCDD treatment) and analytical procedures were the same.
Weanling male Sprague-Dawley rats (n = 6; B&K Universal, Sollentuna, Sweden) were housed in shoebox cages and had free access to tap water and a commercial diet (catalogue # R34; Lactamin, Stockholm, Sweden) containing 4.2 µmol retinyl palmitate/kg for 6 to 7 weeks before initiation of kinetic studies. Assuming average daily food intakes of 1520 g/day, rats consumed 63 to 84 nmol vitamin A/day, an amount that we correctly presumed would put rats in a slight positive vitamin A balance (Kelley et al., 1998).
Kinetic studies.
The experimental protocol is outlined in Figure 1. To overview, changes in plasma tracer concentration were monitored for 21 days after administration of tritiated vitamin A to six rats. At 21 days, when changes in plasma tracer concentrations had entered a terminal slope, TCDD was administered to perturb vitamin A kinetics, and plasma tracer concentrations were monitored for an additional 21 days. Compartmental analysis was then used to identify primary effects of TCDD on vitamin A metabolism.
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Twenty-one days after administration of [3H]vitamin A, an oral dose of 7.0 µg TCDD (provided by Dow Chemicals, Stockholm, Sweden) in 2 ml corn oil/kg body weight was administered to perturb vitamin A metabolism. In order to maintain a TCDD quasi-steady state as discussed by Flodström et al. (1991), rats also received two additional maintenance doses of TCDD (0.7 µg/kg body weight) on days 28 and 35. Blood was sampled as described above at 4, 8, 12, and 24 h, and 2 days (group 1 only) after TCDD administration, and then at 4-day intervals until day 42 of the study.
Forty-two days after administration of labeled plasma or lymph, rats were euthanized with CO2. Livers were excised, weighed, and frozen for later analysis (see below). The thymus glands were excised, weighed, and then returned to the carcass. Carcasses were weighed and frozen at 16°C until they were analyzed for radioactivity (see below).
Plasma and tissue analyses.
Plasma samples (40200 µl) and aliquots of the vitamin A-labeled doses were analyzed for tritium (model 1409; Wallac Sverige AB, Upplands Väsby, Sweden) using Ecoscint A (National Diagnostics, Hintze, Stockholm, Sweden) as scintillation solution. Samples were counted twice to a final 2 error of 1%. After background correction, net counts/minute (cpm) were converted to disintegrations/min (dpm) using an external standard channels ratio method.
After addition of retinyl acetate (internal standard) to selected plasma samples, vitamin A was extracted (Kelley et al., 1998; Thompson et al., 1971
) and analyzed by reverse phase HPLC using a Nucleosil 5µ C18 Resolve column (150 x 4.5 mm; Phenomenex, Torrance, CA) and methanol:water (90:10 v/v, 1 ml/min) as mobile phase. Retinol and retinyl acetate peaks were detected by UV absorbance at 328 nm (model 486; Waters Assoc., Milford, MA). Using an internal standard method, the retinol and retinyl acetate peak areas obtained from integration (MiniChrome v1.66; Fisons Instr., VG Data Systems, Cheshire, UK) were used, in conjunction with mass:area ratios for retinol and retinyl acetate standards, to determine the amount of retinol in samples.
Radioactivity in retinol and retinyl esters was determined in aliquots of the lymph chylomicron dose and in plasma samples collected during the first 120 min after administration of [3H]vitamin A to group 2 rats. Samples were extracted (Kelley et al., 1998; Thompson et al., 1971
), and retinol and retinyl esters were separated on columns of deactivated aluminum oxide (Ross, 1982
). Solvent-free fractions were analyzed for tritium using Ecoscint O (National Diagnostics).
Aliquots of freeze-dried liver were saponified and lipids were extracted into hexane (Green et al., 1985). Extracts were analyzed for tritium as described for plasma, using Ecoscint O as scintillation solution, and for vitamin A content by HPLC (see above). Tritium in carcasses was determined using the method of Adams et al. (1995). Aliquots of ground carcass were extracted using hexane:isopropanol:sodium sulfate. Solvent was allowed to evaporate, and solvent-free extracts were analyzed for tritium after addition of Ecoscint O.
The tritium content of urine was determined by liquid scintillation spectrometry after addition of Ecoscint A. Freeze-dried feces were ground and aliquots were extracted using methanol (Håkansson and Ahlborg, 1985, Kelley et al., 1998
). Solvent-free extracts were analyzed for radioactivity after addition of Ecoscint O.
Kinetic analysis.
For each animal in the current study, we calculated fraction of the administered dose of tritiated vitamin A in plasma versus time, in liver and carcass at 42 days, and in pools of urine and feces as described by Kelley et al. (1998). Data on fraction of the dose in plasma from the time of administration of [3H]vitamin A until day 21 were fit to previously obtained models (Kelley et al., 1998; see Results) using the Simulation, Analysis and Modeling computer program (SAAM31; Berman and Weiss, 1978) and its conversational version (CONSAM; Berman et al., 1983). The model parameters or fractional transfer coefficients (L[I,J]s, the fraction of compartment J's tracer transferred to compartment I per day) for each rat were determined by weighted nonlinear regression analysis in CONSAM using a fractional standard deviation (FSD) of 5% as weighting factor. These L(I,J)s were then fixed for each rat. To accommodate the perturbation of the existing vitamin A system due to administration of TCDD on day 21, we added a time-interrupt function into each input file just prior to the time at which a change in plasma radioactivity was observed. Then the appropriate model L(I,J)s were made adjustable one at a time in order to identify the minimal change(s) needed to fit data obtained after the TCDD perturbation. Finally, mean L(I,J)s ± SD were calculated for each group.
Statistical analysis.
Descriptive data and kinetic parameters are presented as arithmetic group mean ± 1 standard deviation. Using Minitab (Ryan et al., 1985), a standardized paired t-test with an alpha level of 0.05 was used to test for significant effects of TCDD treatment.
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RESULTS |
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As is evident in Figure 3, these models provide a good fit to observed plasma tracer data for rats in the current study during the first 21 days following administration of [3H]vitamin A. Model-derived fractional transfer coefficients [L(I,J)s] for the six rats in the current study (Table 2
) were very similar (Fig. 2
) to those determined for the larger groups of control rats studied for 42 days (Kelley et al. 1998
).
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DISCUSSION |
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The fact that plasma tracer levels increased rather than decreased following TCDD administration (Fig. 3) indicates that the initial effect of TCDD was to mobilize vitamin A from body sites that had appreciable amounts of radioactivity, rather than to increase degradation of vitamin A. The initial effect seemed to be followed by an increase in vitamin A degradation, as evidenced by the increased excretion of radioactivity during the 4 days following TCDD administration in group 2 rats.
Because the increased levels of radioactivity in plasma did not occur until several days after TCDD administration (Fig. 3), we hypothesize that the increased mobilization of vitamin A from body sites with appreciable amounts of radioactivity occurs secondary to an alteration in protein synthesis and/or degradation. In support of this idea, it is known that TCDD induces numerous liver enzymes (see review by Pohjanvirta and Tuomisto, 1994). For example, greatly increased expression of cytochrome P450 1A1 and 1A2 is already evident 24 h after TCDD administration (Santostefano et al., 1998
), indicating rapid enzyme induction by TCDD. Several lines of evidence indicate that members of the cytochrome P450 family are involved in the catabolism of retinoic acid (Spear et al., 1988
; Sonneveld et al., 1998
). Thus, induction of cytochrome P450 enzymes by TCDD may result in altered vitamin A catabolism.
Using compartmental analysis, we determined which model parameter(s) (L[I,J]s) were most sensitive to TCDD treatment. For rats that received [3H]retinol-labeled plasma (group 1), L(3,4) (Fig. 2, left), the fractional transfer of label to compartment 3 from compartment 4, was the parameter most sensitive to TCDD treatment. By adjusting this one parameter, we obtained a good fit to plasma tracer data obtained after TCDD administration (Fig. 3
, left). The model predicts a 428% increase in L(3,4) after TCDD treatment (Table 2
). We speculate that compartment 4 represents large, slowly turning-over pools, predominantly retinyl esters in hepatic perisinusoidal stellate cells. Corresponding to the increase in L(3,4), it seems likely that the increased radioactivity in plasma after TCDD administration probably originated from labeled retinyl esters in these slowly turning-over vitamin A pools, as the model predicts that these sites contain enough tracer to cause such a sustained perturbation. Physiologically, mobilization of vitamin A from compartment 4 would represent an increased fractional rate of retinyl ester hydrolysis. A similar conclusion can be drawn from the model developed for rats that received labeled vitamin A in chylomicrons (group 2), in which a 133% increase in L(1,3) (Table 2
and Fig. 2
, right) was sufficient to explain the effect of TCDD on plasma tracer response. Here, compartment 3 is presumably dominated by large vitamin A stores in the hepatic perisinusoidal stellate cells.
As it has previously been reported that TCDD and dioxin-related substances either inhibit or have no effect on various hepatic retinyl ester hydrolase activities (Chen et al., 1992; Jensen et al., 1987; Powers et al., 1987; Mercier et al., 1990; Nilsson et al., submitted for publication), it may be that nonspecific carboxyl ester hydrolase activities are responsible for the increased mobilization of vitamin A stores after TCDD treatment. Further work is needed to identify which enzyme is responsible for the increased mobilization and to determine if this is the same enzyme involved in normal retinyl ester hydrolysis.
When both L(3,4) and L(4,3) were allowed to vary when fitting the post-TCDD plasma tracer data for group 1 [with L(3,4) increasing and L(4,3) decreasing], a visually better fit was obtained. Although including the second parameter was not statistically justified, this result may indicate that there is an additional, secondary factor contributing to the response observed after TCDD exposure. Biologically, L(4,3) is thought to represent the esterification of retinol by lecithin:retinol acyl transferase (LRAT) for storage in the slow turning-over stellate cell pool. Our observation supports the results of Nilsson et al. (1996), which showed that TCDD reduces hepatic perisinusoidal stellate cell LRAT activity. In contrast, in rats undergoing a rapid depletion in liver vitamin A levels due to TCDD treatment (Kelley et al., 1998), while L(4,3) increased compared with controls, the estimated rate of transfer [L(4,3)* mean liver vitamin A level] was less than 50% of that in control rats (253 versus 580 nmol/day in TCDD-treated versus controls).
It is worth noting that the fractional transfer coefficient describing irreversible loss in group 2 (L[0,3]) did not need to change following TCDD treatment, indicating that the initial effect of TCDD was not to increase the fraction of compartment 3 being excreted per day. Rather, the observed increased tracer excretion in group 2 was caused by a mass action effect (i.e., an increase in the pool in compartment 3, from which system output was modeled). A similar effect was predicted for group 1; no change was needed in the fractional transfer coefficients involved in irreversible loss, indicating that the predicted increase in excretion of tracer was due to a mass action consequence of the increased mobilization of vitamin A stores.
In conclusion, this work illustrates the usefulness of model-based compartmental analysis in discriminating between possible mechanisms by which a system perturbation affects isotope kinetics. Our results indicate that the major impact of TCDD on vitamin A dynamics is to increase mobilization of vitamin A from storage sites, most likely liver perisinusoidal stellate cells; subsequently, vitamin A degradation increases. Because the effect of TCDD on plasma tracer concentrations was not evident for several days after TCDD administration, it seems likely that the increased mobilization is secondary to changes in protein levels or other effects of TCDD on liver parenchymal- and/or stellate cells.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed. Fax: 46 8 33 44 67. E-mail: helen.hakansson{at}imm.ki.se.
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