Hepatic Vitamin A Depletion Is a Sensitive Marker of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Exposure in Four Rodent Species

Nicholas Fletcher, Annika Hanberg and Helen Håkansson,1

Institute of Environmental Medicine, Karolinska Institutet, PO Box 210, S-171 77 Stockholm, Sweden

Received December 21, 2000; accepted April 17, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-treated animals show altered retinoid homeostasis and exhibit signs of toxicity similar to those of vitamin A–deficient animals. In this study we established dose-response curves for sublethal oral doses of TCDD and hepatic vitamin A gain in four rodent species. This was done to evaluate any potential correlation between decreased hepatic vitamin A gain and other TCDD-induced effects, particularly depressed body weight gain and hepatic CYP1A induction. Young Hartley guinea pigs, Sprague-Dawley rats, C57BL/6 mice, and Golden Syrian hamsters were given single oral doses of TCDD at up to 2.5, 100, 1000, and 1000 µg/kg bw, respectively, and killed 28 days after treatment. Hepatic vitamin A gain was decreased 25% compared to controls at estimated doses of 0.1, 0.9, 1.1 and 3.6 µg/kg bw in guinea pigs, hamsters, rats, and mice, respectively. CYP1A induction and hepatic vitamin A gain were affected at similar dose levels and showed similar, but inverse dose-response curves in each of the four species, consistent with the hypothesis that altered vitamin A homeostasis is Ah-receptor mediated. In addition, there was an apparent correlation between the dose-response curves for decreased hepatic vitamin A gain and decreased body weight gain in all species. Taken together with the known importance of vitamin A in body weight regulation, this result was consistent with a contributing role for altered retinoid homeostasis in the wasting syndrome induced by TCDD.

Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD, vitamin A; retinoids; wasting; EROD; species; hepatic; renal.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The prototypical and most potent congener of the polychlorinated dibenzo-p-dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), elicits a wide range of biological and toxicological effects that are species, strain, age, and dose specific (Pohjanvirta and Tuomisto, 1994Go; Van den Berg et al., 1994Go). It is generally considered that dioxin-like compounds exert their toxicity through a common mechanism of action that involves binding to the aryl hydrocarbon receptor (AhR) and the subsequent persistent increase or decrease in transcription of AhR-regulated genes (Schmidt and Bradfield, 1996Go). Consistent with this hypothesis, several characteristic signs of dioxin toxicity (CYP1A1 induction, thymic involution, teratogenicity, and lethality) segregate with AhR-binding affinity in C57BL/6 and DBA/2 mice (Poland and Knutson, 1982Go). The latter strain show a point mutation in the ligand-binding domain that markedly reduces binding affinity for TCDD, as well as a decreased number of binding sites (Ema et al., 1994Go; Okey et al., 1989Go; Poland et al., 1994Go). A contrasting model exists for the Han/Wistar rat and Long-Evans rat, which show a difference of about 1000-fold to the lethal effects of TCDD. Han/Wistar rats are exceptionally resistant to the lethal effects of TCDD, yet are responsive to several other well-known AhR-mediated effects such as CYP1A1 induction (Pohjanvirta et al., 1988Go). The AhR of the Han/Wistar rat is smaller than the receptor in other rat strains and shows an altered structure in the transactivation domain (Pohjanvirta et al., 1998Go; Pohjanvirta et al., 1999Go). Recent genetic cross studies with the more sensitive Long-Evans rat show that resistance is inherited as a dominant trait, suggesting that the altered AhR is a major factor in the resistance of Han/Wistar rats to TCDD (Tuomisto et al., 1999Go). In addition to these two models, Ahr-null mice show only limited signs of toxicity at 2000 µg/kg bw TCDD, a dose 10-fold higher than that required to produce severe toxicity in wild-type mice and heterozygous littermates (Fernandez-Salguero et al., 1996Go), further implying a role for the AhR in the toxicity of TCDD.

While the biochemical processes subsequent to AhR-regulated gene transcription, which may be responsible for the varying signs of toxicity observed in different species, are not as well understood, several lines of evidence suggest a correlation between altered vitamin A homeostasis and ligand binding of the AhR. First, the AhR appears to be involved in basic retinoid homeostasis, as indicated by markedly increased liver levels of retinoic acid, retinol, and retinyl palmitate in the livers of Ahr null mice (Andreola et al., 1997Go). Second, several studies of AhR ligands in rats have shown a correlation between altered tissue vitamin A levels and AhR-mediated effects, such as CYP1A induction and subchronic toxicity (Chen et al., 1992Go; Chu et al., 1994Go; Chu et al., 1995Go; Fattore et al., 2000Go; Håkansson et al., 1990Go; Håkansson et al., 1994Go; Lecavalier et al., 1997Go). Third, altered vitamin A homeostasis is observed in studies with planar polychlorinated biphenyl (PCB) congeners that bind the AhR, while nonplanar congeners that have a low affinity for the AhR have generally been shown to have little effect on vitamin A tissue levels (Azais et al., 1987Go; Chen et al., 1992Go; Chu et al., 1995Go; Chu et al., 1996Go; Lecavalier et al., 1997Go).

Altered vitamin homeostasis has been observed following TCDD exposure in all species that have been examined (Håkansson et al., 1991aGo; Thunberg, 1984Go). However, detailed studies have been conducted only in the rat, where depleted hepatic vitamin A stores, increased kidney vitamin A content, and altered levels of both serum and tissue retinoic acid levels have been observed (Brouwer et al., 1989Go; Nilsson et al., 2000Go; Thunberg et al., 1979Go). To date, more than 300 genes are known to be under retinoid control and through retinoid X receptors (RXRs), retinoids can act in concert with other nuclear receptors, including thyroid hormone receptors, peroxisome proliferator receptors, and vitamin D3 receptors (Giguère, 1994Go), so acting as fundamental regulators of gene expression in most vertebrate groups. Therefore, the implications of altered retinoid homeostasis, particularly at the level of the signaling retinoids, including retinoic acid, are diverse.

Although disturbance of vitamin A homeostasis has not yet been proven to result in overt toxicity, the similar signs of toxicity in TCDD-treated and vitamin A–deficient animals suggests that these disturbances to the retinoid system are functional and contribute to the overall toxicity of TCDD (Thunberg et al., 1980Go; Zile, 1992Go). TCDD-treated rats display a peculiar wasting syndrome characterized by a 2–5 week period of decreased body weight gain and hypophagia that has been suggested to contribute to the overall lethality of TCDD (Peterson et al., 1984Go; Christian et al., 1986Go; Kelling et al., 1985Go). Similarly, it has long been known that vitamin A is essential for growth and that vitamin A deficiency results in hypophagia and decreased body weight gain (Anzano et al., 1979Go; Orr and Richards, 1934Go; Patterson et al., 1942Go). More recent studies have shown similar effects on enzymes of intermediary metabolism in vitamin A–deficient and TCDD-treated animals (Weber et al., 1991aGo, bGo; Shin and McGrane, 1997Go). Together, these data suggest that altered retinoid homeostasis may play an important role in body weight loss observed after acute TCDD exposure.

To further clarify the contribution of altered retinoid homeostasis to species differences in sensitivity to dioxin toxicity, we established dose-response relationships for TCDD and hepatic vitamin A content in guinea pigs, rats, mice, and hamsters, and evaluated whether there existed a correlation between AhR-mediated effects, altered vitamin A homeostasis, and decreased body weight gain. AhR-mediated effects were measured as CYP1A induction and altered vitamin A homeostasis was measured as hepatic vitamin A levels; both have previously been shown to be sensitive markers of TCDD exposure. Relative liver weight and relative thymus weights were recorded as common markers of TCDD exposure.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Stock solutions of 100 µg TCDD (a generous gift from Dow Chemical Co., Midland, MI) per milliliter benzene and 100 µg TCDD (CIL, Andover, MA) per milliliter toluene were stored in darkness at –20°C. To prepare the dosing solutions, TCDD was taken up in corn oil, and the solvents were evaporated under a stream of nitrogen at 40°C. The TCDD concentrations of the two solutions were confirmed at the Department of Environmental Chemistry, University of Umeå, Sweden.

Animals.
Male Hartley guinea pigs (Sahlin, Malmö, Sweden), Sprague-Dawley rats (ALAB Laboratorietjänst), C57BL/6 mice (ALAB Laboratorietjänst), and Golden Syrian hamsters (Wallenberg Laboratory at Stockholm University, Stockholm, Sweden) were housed in wire-bottom cages and allowed 1 week to acclimatize prior to treatment with either TCDD or the vehicle. Guinea pigs, rats, and hamsters were housed two to a cage, while mice were housed five per cage. Illumination cycle (12 h light, 12 h dark) was automatically controlled and a temperature of 21 ± 1°C was maintained. Feed and tap water were supplied ad libitum throughout the acclimatization and experimental periods. Guinea pigs received K2 maintenance feed (EWOS AB, Södertälje, Sweden) and rats, mice, and hamsters received R3 brood stock diet (EWOS AB). The vitamin A content of these feeds was 11,000 IU/kg K2-diet and 12,000 IU/kg R3-diet. Initial body weights were 337 ± 21, 91 ± 7, 12 ± 2, and 52 ± 6 grams for guinea pigs, rats, mice, and hamsters, respectively.

Experimental design.
All procedures involving animals were approved by the local ethical committee on animal experiments. TCDD in corn oil was administered by gavage as a single oral dose to groups (n = 5) of 4- to 5-week-old guinea pigs, rats, mice, and hamsters at levels as shown in Tables 1–4GoGoGoGo. Control groups were identically maintained but received the vehicle only. Dose volumes were 2, 4, 10, and 10 ml/kg for guinea pigs, rats, mice, and hamsters, respectively. Body weight was measured prior to study initiation and at weekly intervals thereafter. The animals were killed by blood withdrawal under anesthesia (Mebumal®; 90 mg/kg bw; ip) 28 days after treatment when the effect on hepatic vitamin A gain was easily detectable in all the species studied (Håkansson et al., 1991aGo).


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TABLE 1 Body Weight Gain, Relative Liver and Thymus Weights, Hepatic EROD Activity, and Hepatic and Renal Vitamin A Content in Hartley Guinea Pigs 28 Days after a Single Oral Dose of TCDD
 

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TABLE 2 Body Weight Gain, Relative Liver and Thymus Weights, Hepatic EROD Activity, and Hepatic and Renal Vitamin A Content in Sprague-Dawley Rats 28 Days after a Single Oral Dose of TCDD
 

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TABLE 3 Body Weight Gain, Relative Liver and Thymus Weights, Hepatic EROD Activity, and Hepatic and Renal Vitamin A Content in C57B1/6 Mice 28 Days after a Single Oral Dose of TCDD
 

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TABLE 4 Body Weight Gain, Relative Liver and Thymus Weights, Hepatic EROD Activity, and Hepatic and Renal Vitamin A Content in Golden Syrian Hamsters 28 Days after a Single Oral Dose of TCDD
 
Biochemical analyses.
Hepatic vitamin A levels were measured in additional groups of five control guinea pigs, rats, mice, and hamsters on the day of dosing, and in all surviving animals at study termination. The method of extraction and analysis of vitamin A have been described previously (Håkansson and Ahlborg, 1985Go; Håkansson et al., 1987Go). Briefly, tissue retinyl esters were completely hydrolyzed to retinol, and total retinol was extracted with petroleum ether. Retinol was analyzed by HPLC using fluorescence detection. Hepatic CYP1A activity, measured as O-dealkylation of 7-ethoxyresorufin (EROD), was determined fluorometrically in the microsomes at 37°C using a Shimadzu RF-5000 spectrofluorometer essentially as reported by Burke et al., (1985).

Data analysis.
Data reported are the arithmetic mean ± standard deviation (SD) for individual groups of surviving animals. Statistical analysis for each species and parameter were made by one-way analysis of variance (ANOVA) using SigmaStat Statistical software (Jandel Scientific, Erkrath, Germany). Where significant differences between groups were indicated and the data were homogenous (Levene median test) and normally distributed (Kolmogorov-Smirnov test), Dunnett's test was used for multiple comparisons. When tests for normality or variance failed, the Kruskal-Wallis One Way ANOVA on ranks was used and significant differences were evaluated using Dunnett's test for multiple comparisons. In cases where significant (p < 0.05), dose-related changes were found, curve fit was performed by nonlinear regression analysis using Table Curve 2D for Windows (Jandel Scientific). To facilitate comparison between the species, doses that resulted in a 25% increase or decrease relative to the control values were estimated for body weight gain, relative liver weight, relative thymus weight, hepatic vitamin A, hepatic vitamin A gain, and renal vitamin A. In the text this dose was defined as the 25% effect level (EL25) value. The ED50 value was determined for EROD activity because although the EROD response varied considerably between species in terms of both basal levels and maximally induced levels compared to controls, the dose-response was relatively linear at this point in all species.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hartley Guinea Pigs (Table 1Go)
At a dose of 2.5 µg/kg bw, three out of five guinea pigs died. The deaths occurred 11, 15, and 20 days following TCDD treatment, when the respective body weights of the animals had decreased to 49, 55, and 55% of their pretreatment values. Body weight gain was observed in all surviving animals, although mean weight gain in 0.66 and 2.5 µg/kg bw groups was reduced 22 and 58% compared to controls, respectively.

A significant dose-related increase in relative liver weight was observed in guinea pigs from 0.047 to 0.66 µg/kg bw. Relative thymus weight was decreased compared to controls from 0.18 to 2.5 µg/kg bw, though the results were not statistically significant.

Hepatic EROD activity increased in a dose-related manner, but was only significantly different from controls at 0.66 µg/kg bw, where the induction was approximately 5-fold. Likewise, hepatic vitamin A levels were decreased compared to controls at all doses, though the results were only significant at 0.18 and 0.66 µg/kg bw. In addition, a dose-related decrease in hepatic vitamin A gain (15–75%) was observed over the studied period in animals treated from 0.012 to 0.18 µg/kg bw, whereas in the 0.66 and 2.5 µg/kg bw groups there was a mean loss of vitamin A from the liver.

Rats (Table 2Go)
No mortality was observed during the study. Body weight gain was significantly decreased compared to controls at 100 µg/kg bw. Increased relative liver weight was observed from 0.12 to 100 µg/kg bw, and relative thymus weight was decreased at the same doses.

A maximum increase of hepatic EROD activity was observed in the 19 µg/kg bw group, where the activity was 60 times that of control values. Hepatic vitamin A levels and hepatic vitamin A gain (15–97%) were significantly decreased after 4-weeks at doses of 0.12 to 100 µg/kg bw. Renal vitamin A content was slightly increased compared to controls at 3.5 and 100 µg/kg bw, though the maximal and only significant increase occurred at 19 µg/kg bw.

Mice (Table 3Go)
No mice died during the 4-week observation period. Body weight gain was decreased 60% compared to controls at 1000 µg/kg bw. Relative liver weight was significantly increased from doses of 8–1000 µg/kg bw, and relative thymus weight was decreased significantly at 200 and 1000 µg/kg bw.

Hepatic EROD activity was significantly increased compared to controls at all doses, with a maximal induction (49 times the control value) observed in the 40 µg/kg bw group. Dose-related and significant decreases in hepatic vitamin A levels and hepatic vitamin A gain (22–77%) were observed from 1.6 to 1000 µg/kg bw.

Hamsters (Table 4Go)
There were no deaths during the study period. Significant decreases of 15, 20, and 55% in body weight gain were observed at doses of 40, 200, and 1000 µg/kg bw, respectively. A dose-related increase in relative liver weight was observed from 1.6 to 1000 µg/kg bw, whereas relative thymus weight was significantly decreased from 40 µg/kg bw, with a maximal reduction of 90% observed at the high dose.

A dose-related increase in hepatic EROD activity was observed in all groups, though the results were only significant at doses above 1.6 µg/kg bw. Hepatic vitamin A levels were decreased from 1.6 to 1000 µg/kg bw. Hepatic vitamin A gain was also markedly (10–98%) and significantly reduced at all doses.

Species Comparisons
The estimated EL25 and ED50 values (EROD activity) for the measured parameters, as well as previously reported acute LD50 values, are shown in Table 5Go. Body weight gain was significantly decreased at the high doses in all of the examined species, with EL25 values of 0.9, 47.1, 114.9, and 151.4 in guinea pigs, rats, mice, and hamsters, respectively. These values were in agreement with the relative sensitivity of the species to the acute lethality of TCDD determined in previous studies. EL25 values for relative liver and relative thymus weights showed that guinea pigs were also the most sensitive species to these effects, but there was no correlation to acute lethality in the rat, mouse, or hamster.


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TABLE 5 Literature LD50 Values and Estimated EL25 and ED50 (µg/kg bw) Values in Hartley Guinea Pigs, Sprague-Dawley Rats, C57BL/6 Mice and Golden Syrian Hamsters 28 Days after a Single Oral Dose of TCDD
 
Maximal induction of EROD activity differed greatly between species, with rats and mice showing values 50–60 times those of controls, whereas guinea pigs and hamsters showed levels 5 and 9 times those of controls, respectively. Guinea pig, rat, and mouse control animals had similar basal EROD activities (0.04–0.06 nmol/mg protein/min), whereas in hamsters the basal activity was higher. Accordingly, although the maximal EROD induction in the hamster was only 9 times the control level, the absolute maximal activity was almost as high as that in rats and mice (1.5 versus 3.9 and 1.8 nmol/mg protein/min). In guinea pigs, however, the maximal activity was about 10-fold lower and the ED50 value was also markedly lower than in the other species.

The EL25 value for decreased hepatic vitamin A content was lowest in the guinea pig, followed by the rat, hamster, and then the mouse. Hepatic vitamin A gain EL25 values indicate that this parameter was a very sensitive measure of TCDD exposure in all of the species investigated in this study, and that the guinea pig was more sensitive than rats, mice, and hamsters. However, the most remarkable difference between species appeared to be renal vitamin A content, which was markedly increased in the rat (EL25 2.5 µg/kg bw), but not clearly affected in guinea pigs, mice, or hamsters.

Dose-response relationships for percentage body weight gain, hepatic vitamin A gain, and EROD activity compared to controls are shown in Figures 1–4GoGoGoGo for guinea pigs, rats, mice, and hamsters, respectively. Curves for increased EROD activity and decreased hepatic vitamin A gain showed an inverse relationship, particularly in the guinea pig and hamster. Regression analyses (p < 0.01) resulted in coefficient of determination values (r2) of 0.78 for guinea pigs, 0.85 for rats, 0.66 for mice, and 0.67 for hamsters. The shapes of the body weight gain and hepatic vitamin A gain curves were similar, especially for the guinea pig and mouse, whereas in the rat, hepatic vitamin A gain was markedly depressed at lower doses than any effects on body weight gain were observed. Regression analysis (p < 0.01) of body weight gain and hepatic vitamin A gain gave r2 values of 0.65, 0.83, 0.53, and 0.64 in the guinea pig, rat, mouse, and hamster, respectively.



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FIG. 1. Body weight gain (square), hepatic vitamin A gain (circle), and EROD activity (triangle) for Hartley guinea pigs 28 days following a single oral dose of 0.012, 0.047, 0.18, 0.66, or 2.5 µg/kg bw TCDD. All values are expressed as a percentage of control values. Standard deviations are shown in Table 1Go.

 


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FIG. 2. Body weight gain (square), hepatic vitamin A gain (circle), and EROD activity (triangle) for Sprague-Dawley rats 28 days following a single oral dose of 0.12, 0.66, 3.5, 19, or 100 µg/kg bw TCDD. All values are expressed as a percentage of control values. Standard deviations are shown in Table 2Go.

 


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FIG. 3. Body weight gain (square), hepatic vitamin A gain (circle), and EROD activity (triangle) for C57BL/6 mice 28 days following a single oral dose of 1.6, 8, 40, 200, or 1000 µg/kg bw TCDD. All values are expressed as a percentage of control values. Standard deviations are shown in Table 3Go.

 


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FIG. 4. Body weight gain (square), hepatic vitamin A gain (circle), and EROD activity (triangle) for Golden Syrian hamsters 28 days following a single oral dose of 1.6, 8, 40, 200, or 1000 µg/kg bw TCDD. All values are expressed as a percentage of control values. Standard deviations are shown in Table 4Go.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TCDD-induced depletion of hepatic retinoids has been relatively well characterized in the Sprague-Dawley rat (reviewed in Nilsson and Håkansson, in press). TCDD dosing has been shown to cause decreased storage and increased mobilization of retinyl esters, as well as a net decrease of hepatic concentrations of retinyl esters, particularly in the hepatic stellate cells (Håkansson and Ahlborg, 1985Go; Håkansson and Hanberg, 1989Go; Håkansson et al., 1988Go; Kelley et al., 1998Go; Nilsson et al., 1996Go). In contrast to the established effects of TCDD on retinoid homeostasis in the rat, few data were available for the effects of TCDD on hepatic vitamin A levels in other species. The results of the present study have confirmed the well-described and dose-dependent decrease in hepatic vitamin A levels following acute exposure in the rat (Håkansson et al., 1991bGo; Pohjanvirta et al., 1990Go), and further, have shown dose-related decreases in hepatic vitamin A gain in other rodent species that vary considerably in their sensitivity to acute TCDD toxicity. Estimated EL25 values showed that the guinea pig was the most sensitive species to this effect, in accordance with the high sensitivity of this species to the acute toxic effects of TCDD. In addition, EL25 values in rats, mice, and hamsters for reduced hepatic vitamin A gain demonstrated altered vitamin A homeostasis to be a sensitive marker of TCDD exposure in all species examined in this study. The data highlighted a major species difference with respect to the effect of TCDD on renal vitamin A levels. The increased rat renal vitamin A content was in agreement with previous studies that have shown marked and persistent increases in kidney vitamin A levels following dosing with TCDD (Brouwer et al., 1989Go; Håkansson and Ahlborg, 1985Go; Håkansson et al., 1987Go; Pohjanvirta et al., 1990Go; Thunberg, 1983Go; Thunberg, 1984Go). Other studies in male Sprague-Dawley rats have shown the initial increase in renal vitamin A content to be attributable to increased levels of retinyl palmitate associated with enhanced lecithin:retinol acyltransferase (LRAT) activity, presumably a result of increased concentrations of retinoic acid (Nilsson et al., 1996Go; Nilsson et al., 2000Go). In contrast to the rat, there was no effect on renal vitamin A levels in guinea pigs, mice, or hamsters (Tables 1, 3, and 4GoGoGo). The reason for variations between renal vitamin A content between species is not readily apparent, but investigation of LRAT activity in these species may resolve this question, and provide further understanding of species differences both in endogenous retinoid homeostasis and retinoid effects following TCDD exposure.

CYP1A induction was measured enzymatically by the EROD assay in the four rodent species. In the guinea pig, EROD activity was increased about 5-fold, consistent with induction levels (3–5 fold) observed in previous studies with TCDD and PCBs (Huang and Gibson, 1991Go; Håkansson et al., 1994Go). A slightly lower ED50 value (0.03 µg/kg bw) has been reported for EROD activity in guinea pigs following TCDD treatment, probably a result of a shorter 2-week study duration (Holcomb et al., 1988Go). To our knowledge there were no previous reports of EROD dose-response relationships in hamsters after TCDD-dosing. The ED50 value for EROD activity in the hamsters (13.7 µg/kg bw) was greater than in other species. In rats and mice, EROD activities were generally comparable with previous studies with regard to magnitude of induction, though lower levels have been reported on occasion (Nilsson et al., 2000Go; Håkansson et al., 1994Go; Van Birglen et al., 1995Go; Weber et al., 1995Go; Iwasaki et al., 1986Go). In all species, induction maxima occurred at lower doses than the highest tested, consistent with previous results in mice and rats (Håkansson et al., 1994Go; Weber et al., 1995Go).

Tables 1–4GoGoGoGo indicate that CYP1A induction and decreased hepatic vitamin A gain were affected at similar doses. The shape of the dose-response curves (Figs. 1–4GoGoGoGo) suggested an inverse relationship between CYP1A induction and decreased hepatic vitamin A gain. Although current understanding of retinoic acid metabolism remains incomplete, a number of cytochrome P450 isoenzymes (including CYP1A1 and CYP1A2) have shown catalytic activity for the conversion of both retinol to retinal (Chen et al., 2000Go) and retinal to retinoic acid in vitro (Napoli, 2000Go; Raner et al., 1996Go; Roberts et al., 1992Go; Tomita et al., 1996Go; Zhang et al., 2000Go). Retinoic acid oxidation to more polar metabolites was also catalyzed in vitro by cytochrome P450 enzymes (Ahmad et al., 2000Go; Leo et al., 1984Go; Leo et al., 1989Go; Martini and Murray, 1993Go), but conflicting data exist for the role of the CYP1A2 isoform (Andreola et al., 1997Go; Roberts et al., 1992Go). Additionally, retinoic acid metabolism was increased in liver microsomes from TCDD-treated rats (Fiorella et al., 1995Go). One mechanism by which TCDD could decrease hepatic vitamin A storage is by induction of cytochrome P450 enzymes, resulting in increased retinoic acid synthesis and further conversion to more polar metabolites. As a result, hepatic retinoid mobilization may be increased and hepatic retinyl ester storage decreased. Although it remains likely that TCDD affects vitamin A homeostasis at multiple levels, the data from this study showing an apparent inverse relationship between hepatic vitamin A depletion and CYP1A induction across species further support a role for the AhR in dioxin-altered vitamin A homeostasis.

Figures 1–4GoGoGoGo show similarly shaped dose-response curves for body weight gain and hepatic vitamin A gain, while regression analyses between the two parameters indicated correlations of varying strength dependent upon species (r2 = 0.53–0.83; p < 0.01). Although these data do not necessarily imply a causative role for altered retinoid homeostasis in TCDD-induced weight loss, they were in accordance with the well-established importance of vitamin A status in body weight regulation, and the weight loss previously observed in vitamin A–deficient animals (Anzano et al., 1979Go; Orr and Richards, 1934Go; Patterson et al., 1942Go). Other evidence for a contributory involvement of altered retinoid homeostasis in TCDD-induced weight loss has been shown in studies in which vitamin A supplements were able to provide some protection to the weight loss suffered by rats exposed to PCBs (Innami et al., 1974Go) and TCDD (Håkansson et al., 1991bGo). Nevertheless, vitamin A supplements were not sufficient to allow normal growth in treated animals in either of these studies, and it is likely that decreased vitamin A content is in itself not sufficient to result in wasting.

More recently, it has been demonstrated that retinoic acid directly regulates transcription of the key rate-limiting enzyme of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) (Hall et al., 1992Go; Lucas et al., 1991Go; Pan et al., 1990Go; Shin et al., 1995Go), providing a molecular link between vitamin A status and intermediary metabolism. In addition, a response element for retinoic acid has been identified in the promotor region of another key gluconeogenic enzyme, fructose-1,6-biphosphatase (Fujisawa et al., 2000Go), and retinoids play a functional role in regulating insulin secretion (Chertow et al., 1987Go; Fernandez-Meija and Davidson, 1992Go).

TCDD-treatment has been shown to inhibit the retinoic acid-induced expression of a number of genes in vitro (Lorick et al., 1998Go; Rubin and Rice, 1998Go; Weston et al., 1995Go). While not implying positive or negative activation of genes, in vivo treatment of guinea pigs with TCDD leads to decreased binding of fractions of hepatic nuclear protein to a retinoic acid response element (Ashida and Matsumura, 1998Go). Together, these results suggest that TCDD downregulates the effects of retinoic acid in at least some cells, by as yet unknown mechanisms. A downregulation of the induction of retinoic acid–induced genes in the liver of TCDD-treated animals would be expected to have effects on gluconeogenic enzymes under retinoid control. Accordingly, the expression and/or activity of PEPCK has been shown to be decreased compared to controls in several species exposed to TCDD (Unkila et al., 1995Go; Viluksela et al., 1999Go; Weber et al., 1991aGo,bGo; Weber et al., 1995Go). PEPCK activity was also decreased in vitamin A–deficient rats, but inducible by administration of retinoic acid (Shin and McGrane, 1997Go). It may then be speculated that disturbance of vitamin A homeostasis at the level of signaling retinoids and/or receptor binding could contribute to the well-known depletion of glycogen in the livers of both vitamin A deficient rats and rats exposed to TCDD. In this way, altered retinoid homeostasis in TCDD-treated animals may play a role in the wasting syndrome by directly affecting transcription of genes important in intermediary metabolism.

In conclusion, dose-response relationships for depleted hepatic vitamin A levels established in guinea pigs, rats, mice, and hamsters showed that altered retinoid homeostasis was a sensitive marker of TCDD exposure in all of the examined species. Guinea pigs were the most sensitive of the species to depleted hepatic vitamin A gain, and effects were observed at doses 10-fold lower than in other species. The doses at which effects on hepatic vitamin A levels were observed were comparable with those of EROD induction, consistent with the hypothesis that TCDD-altered retinoid homeostasis is AhR mediated. In addition, the dose-response curves for body weight gain and reduced hepatic vitamin A gain showed some correlation which, when considered with the known importance of vitamin A in body weight regulation and intermediary metabolism, suggests that altered vitamin A homeostasis may contribute to the wasting syndrome observed in TCDD-treated animals.


    ACKNOWLEDGMENTS
 
The technical assistance of Ellu Manzoor, Laila Johansson, and Marina Nyborg is gratefully acknowledged. This study was supported by the Foundation for Strategic Environmental Research (grant 98657), the Karolinska Institute, the Swedish Environmental Protection Agency, and the Commission of the European Communities, specifically the RTD programme "Fair-Agriculture and Fisheries" (FAIR-CT 973220) "Retinoids (Vitamin A) interaction with organohalogen food residues: mechanisms, biomarkers and implications for developmental toxicity." It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area.


    NOTES
 
1 To whom correspondence should be addressed. Fax: + 46 8 34 38 49. E-mail: helen.hakansson{at}imm.ki.se. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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