Introduction

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TCDD¹ is the most potent member of a family of PHAHs that is known to produce a spectrum of toxic effects in animals via a common receptor-mediated mechanism (DeVito and Birnbaum, 1994). This family of environmental contaminants includes polyhalogenated chlorinated and brominated dibenzo-p-dioxin (PCDD and PBDD), dibenzofuran (PCDF and PBDF), and biphenyl (PCB and PBB) isomers sharing a characteristic pattern of lateral substitution and relatively planar conformation. Among these compounds, toxic potency derives not only from the ability of a specific congener to bind the cytosolic Ah receptor in target tissues but also from its pharmacokinetic behavior (Safe, 1990).

All PHAHs are highly lipophilic and concentrate in biological systems on a purely physical basis by simple partitioning (Jackson et al., 1993). The extreme persistence of TCDD and related compounds in bodies of animals is largely dependent upon biotransformation to more polar metabolites. Specifically, PCDD/F congeners possess halogen substituents at preferred sites of enzymatic oxidationlateral 2, 3, 7, and 8 positions (Tulp and Hutzinger, 1978; Poiger et al., 1989). In vivo studies with TCDD and its analogues have identified essentially all of the congener-derived radioactivity present in tissues to be the parent compound. This suggests that metabolites of these compounds formed in liver are readily excreted, mainly in the bile, and are nontoxic. Hepatic metabolism has been understood to be rate-limiting for TCDD elimination andin effectdetoxification of TCDD and related PHAHs. In the case of TCDD, this transformation proceeds slowly in all species tested (Van den Berg et al., 1994). In rats, reported whole-body half-lives range from 17 to 31 days, depending on dose and strain (Van den Berg et al., 1994). Sex, age, and various physiological or pharmacological states may alter the metabolism and elimination of TCDD but have received little attention (Banks et al., 1990; Pegram et al., 1995).

The cytochrome P450-dependent monooxygenase system plays an important role in the metabolic disposition of many xenobiotics, transforming nonpolar, lipophilic compounds into more polar metabolites via hydroxylation. As such, it figures prominently in the deactivation and elimination of many PHAHs. In fact, the metabolite profiles reported so far for TCDD in the rat include products that suggest a characteristic P450 ring hydroxylation as an initial step (Poiger and Buser, 1984; Sawahata et al., 1982).

The hepatic metabolism of TCDD has generally been associated with the cytochrome P4501A forms that TCDD so potently induces. Using short-term biliary excretion of metabolites as an indirect measure of metabolism, autoinduction of metabolism (~2-fold) has been demonstrated in male rats for 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) (McKinley et al., 1993; Brewster and Birnbaum, 1987). In addition, TCDD induces rapid metabolism and biliary elimination of TCDF (McKinley et al., 1993). More dramatic autoinduction of metabolism (~20-fold) by TCDF has been reported in isolated rat hepatocytes (Olson et al., 1991). Other studies with liver microsomes from rats pretreated with TCDF and antibodies specific for CYP1A1 indicated that this was the primary enzyme responsible for the in vitro TCDF metabolism observed (Tai et al., 1993).

In contrast to these PCDF analogues, TCDD does not induce its own metabolism in short-term biliary excretion studies. Neither TCDD nor 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) induced its own metabolism in biliary excretion studies in male F344 rats, despite marked induction of CYP1A1 and CYP1A2 (Kedderis et al., 1991, 1993). Pretreatment with TCDF was no more effective at altering the 8-hr biliary elimination of a TCDD challenge dose (McKinley et al., 1993). However, bile collected for 72 hr from rats pretreated with 10 µg TCDD/kg 8 days before dosing with [³H]TCDD showed a significant increase in biliary excretion of TCDD-derived radioactivity, compared with that of controls (Poiger and Buser, 1984). Studies with isolated rat hepatocytes demonstrated that TCDD may induce its own metabolism in vitro but only at very high concentrations, which are not attained after in vivo exposures (Olson et al., 1991). Based on these findings, investigators have generally concluded that TCDD is a poor substrate for metabolism by CYP1A isoforms.

Studies of biliary elimination of TCDD in rats to date have not examined effects of aging, and little attention has been paid to the effect of sex and enzyme induction (Poiger and Buser, 1984; Ramsey et al., 1982). The objective of the present study was to investigate the effects of age, sex, and various pharmacologic pretreatments on TCDD metabolism and elimination in rats as characterized by the biliary elimination of TCDD-derived radioactivity.

	Materials and Methods

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Chemicals. Purity check of [³H]TCDD. The radiopurity was routinely assayed by reversed-phase high-performance liquid chromatography (System Gold; Beckman Instruments, Inc., Fullerton, CA) using a Zorbax 5-µm C₁₈ 4.6 × 250 mm analytical column (Mac-Mod Analytical, Inc., Chadds Ford, PA), an isocratic 95:5 MeOH:water mobile phase at a flow rate of 1 ml/min, and a Beckman 171 radiodetector using Flo-Scint II cocktail (Radiomatic Instruments, Tampa, FL) at a flow rate of 1 ml/min. The radiopurity of purified [³H]TCDD was additionally confirmed using an in vivo rat biliary excretion bioassay (Kedderis et al., 1993).

Other Compounds. Phenobarbital (PB) and DEX were purchased from Sigma (St. Louis, MO). The total P450 inhibitor ABT was purchased from Aldrich (Milwaukee, WI).

Animals. Fischer 344 rats were received from Charles River Laboratories (Raleigh, NC). Animals were maintained on a 12-hr light/dark cycle under conditions of constant temperature and humidity and were provided Purina 5001 Rodent Chow (Ralston-Purina, St. Louis, MO) and water ad libitum.

Pretreatments. Two groups (PB- and DEX-treated) of adult male rats were pretreated with specific P450-inducing compounds for 3 days prior to dosing with 1 nmol [³H]TCDD/kg. One group was treated with PB, 80 mg/kg ip daily in 1 ml saline/kg. The other group received DEX, 100 mg/kg ip twice daily in 0.5 ml corn oil/kg. Another group of adult rats was pretreated with the P450 suicide substrate inhibitor ABT (50 mg/kg ip in 2 ml saline/kg) 12-15 hr prior to treatment with 1 nmol [³H]TCDD/kg (Mico et al., 1988).

Surgical Procedures. The experimental procedures used for biliary excretion studies in adult rats have been previously described (McKinley et al., 1993; Kedderis et al., 1991, 1993). Throughout the surgical and biliary collection period, the physiological body temperature of each anesthetized rat was monitored and maintained using heat from a light; saline was given ip periodically to maintain hydration.

Anesthesia. In the present studies, groups of rats were anesthetized with either sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL; 50 mg/kg, ip) or urethane (1-1.25 g/kg, ip). Use of the anesthetic urethane was begun midway through these studies, after it was determined to have no effect on the short-term biliary elimination of TCDD-derived radioactivity in naive rats; only the volume of bile excreted was significantly reduced in the urethane anesthetized rats (data not shown). Urethane (Sigma Chemical Co., St. Louis, MO) was found to be a far more effective and humane anesthetic for this procedure. Male rats anesthetized with pentobarbital required supplemental 5-mg pentobarbital injections approximately every 2 hr, while pentobarbital females rarely required supplementation; urethane rats almost never required additional anesthetic.

Bile Duct Cannulation. The common bile duct of each animal was surgically exposed and then cannulated using either a ~20-cm length of PE10 tubing (0.011-in. i.d.; Clay Adams, Parsippany, NY) for adult rats, or a ~10-cm length of microteflon polytetrafluoroethylene tubing (0.008-in. i.d., 0.004-in. wall; Cole-Parmer, Niles, NY) for the juvenile rats. Anesthesia was maintained throughout the 6-hr collection period.

Administration of [³H]TCDD. Dose. Dosing solutions of [³H]TCDD were prepared using a vehicle of 3:1:1 water:ethanol:Emulphor^® (polyethoxylated castor oil; GAF Corporation, New York, NY). All animals received [³H]TCDD/kg (1 nmol/kg at 2 ml/kg) by injection into the femoral vein.

Bile Collection. Bile duct cannulation immediately preceded [³H]TCDD administration, and bile collection commenced immediately with the iv injection. Serial bile samples were collected for 6 hr in preweighed amber vials. At the end of bile collection, rats were killed by perforation of the diaphragm and exsanguination by cardiac puncture.

Sample Analysis of Radioactivity. Total biliary radioactivity was determined for each time interval. Duplicate aliquots of the preweighed fractions were added to the scintillant for analysis by liquid scintillation spectrometry. The bile was assumed to have a specific gravity = 1.

Measurement of Total P450. Livers from ABT-treated and accompanying control animals were saved for measurement of total cytochrome P450. Whole livers were removed at the time of sacrifice, rinsed in cold 10 mM Na Hepes buffer (pH = 7.4), blotted, and sampled. Samples were immediately frozen in liquid N₂ and stored at 80 °C. On the day of the assay, microsomal fractions were prepared by centrifugation for 1 hr at 105,000g. The pellet was then homogenized in a wash buffer (1.15% KCl and 100 mM neutralized EDTA) and re-centrifuged for 1 hr at 105,000g. Total P450 concentration was measured by reduced CO-difference spectrum (Omura and Sato, 1964). Protein determinations were made using a modified Bradford method (Bio-Rad, Hercules, CA), with bovine serum albumin as the standard.

Data Analysis. All data are presented as means ± standard deviation (SD). Intergroup comparisons of cumulative biliary excretion of TCDD-derived radioactivity were performed by analysis of variance followed by Protected Fisher's Least Significant Difference test. Differences between treatment groups were considered statistically significant when p < 0.05.

	Results

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Biliary Elimination of TCDD-Derived Radioactivity. The effects of age, sex, and various pretreatments on metabolism of TCDD as characterized by the short-term cumulative biliary excretion of TCDD-derived radioactivity in F344 rats are presented in table 1 and fig. 1. The biliary elimination (table 1) is expressed as the cumulative percentage of administered dose of [³H]TCDD excreted in the bile for the time period of zero to 6 hr after dosing with [³H]TCDD. Biliary elimination of metabolites or parent-derived radioactivity represents an indirect measure of metabolism for TCDD and related compounds as determined by previous studies (McKinley et al., 1993; Brewster and Birnbaum, 1987; Kedderis et al., 1991, 1993).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Cumulative biliary elimination of TCDD-derived radioactivity from 0-6 hr of an iv dose of 1 nmol [3H]TCDD/kg for various age and sex groups of rats. Biliary elimination is expressed as cumulative percentage administered dose of [3H]TCDD excreted in the bile. Time is expressed as hours (0 to 6) of biliary collection. Data are presented as mean ± SD. Groups of naive rats include male and female rats of various ages (adult, juvenile, and senescent).

Effects of Age and Sex on Biliary Elimination of TCDD-Derived Radioactivity. Table 1 illustrates changes in biliary elimination of TCDD-derived radioactivity with sex and age. Gender-specific differences were noted in the adult rats, with males excreting a significantly greater (p < 0.05) amount of TCDD-derived radioactivity in the bile, compared with females. In contrast to the adult rats, the juvenile rats did not demonstrate gender-specific differences in the biliary excretion of TCDD-derived radioactivity. Moreover, biliary elimination was the same in juveniles, compared with adult males, but significantly greater (p < 0.05) compared with adult females. The senescent males demonstrated a significant decrease (p < 0.05) in biliary excretion of TCDD-derived radioactivity, compared with adult and juvenile males, and also to juvenile females. The biliary excretion of radioactivity by senescent males was the same as that seen in adult females. Vaginal smears performed on adult female rats indicated that the time of estrus did not affect the biliary elimination of TCDD-derived radioactivity (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Biliary excretion rates of TCDD-derived radioactivity from 0-6 hr of an iv dose of 1 nmol [3H]TCDD/kg for various age and sex groups of rats. Biliary excretion rate is expressed as percentage dose per min. The percentage dose was already corrected for body weight; since the dose to the animal was weight-normalized. Because the measured TCDD-derived radioactivity in the bile was a mixture of parent TCDD and its metabolites, percentage dose was the appropriate unit of measure to use. Data are presented as the means (error bars for standard deviations are omitted to prevent confusion).

Effects of Pretreatments on Biliary Elimination of TCDD-Derived Radioactivity. To determine the influence of cytochromes P450 (P450) on TCDD metabolism, various modulators of P450 function were used to pretreat animals. Age-matched adult male rats were pretreated with PB, DEX, or ABT as discussed in "Materials and Methods," and then dosed with [³H]TCDD. Serial bile samples were collected over the time period of zero thru 6 hours after dosing with [³H]TCDD. The results of these pretreatments on the cumulative biliary elimination of TCDD-derived radioactivity are presented in table 1.

	Discussion

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The objective of the present study was to determine the effects of age, sex, and various pretreatments on TCDD metabolism and elimination as characterized by the short-term biliary elimination of TCDD-derived radioactivity in F344 rats. Short-term biliary excretion of metabolites (e.g., TCDD-derived radioactivity) has been shown to be an indirect measure of metabolism for TCDD and related compounds (McKinley et al., 1993; Brewster and Birnbaum, 1987; Kedderis et al.,1991, 1993). Results of the biliary elimination studies presented here, in conjunction with findings from earlier investigations, permit several conclusions to be drawn regarding the short-term metabolism and elimination of TCDD in rats. Biliary elimination of TCDD-derived radioactivity following low-dose systemic exposure occurs slowly in F344 rats, with <1% of the dose being excreted in the bile in 6 hr when measured immediately after exposure. Collection of bile was limited to only 6 hr in these studies because previous research has shown that bile production and lipid secretion in anesthetized rats remain constant during the first 4-6 hr after bile duct cannulation and declines gradually thereafter (Kuipers et al., 1985). However, even within this admittedly small window of biotransformation, differences in the biliary elimination of TCDD related to age, sex, and various pretreatments were clearly and reproducibly measurable.

In the present study, the cumulative biliary elimination of TCDD-derived radioactivity in the naive adult males was significantly greater than that of naive adult females, although it was no greater than that of juvenile rats of either sex. The naive juvenile male and female rats demonstrated no sex differences with similar cumulative biliary elimination of TCDD-derived radioactivity. The decreased biliary elimination of TCDD-derived radioactivity in senescent males, compared with naive adult males, may suggest a substantial age-related compromise in metabolic activity toward TCDD.

Substantial evidence has accumulated which indicates that differences in expression of hepatic cytochrome P450 involved with age- and/or sex-related manners are governed by sex steroids and other hormones. In the rat, most of the constitutive P450 in livers of untreated adult rats belongs to the CYP2C subfamily; male specific forms include 2C11, 2C13, 2A2, and 3A2, and the major female-specific form is 2C12 (Ryan and Levin, 1993). The significantly higher rate of biliary elimination of TCDD-derived radioactivity in adult male rats, compared with adult females, may therefore indicate that one or more of the sex-specific hepatic cytochrome P450 forms is involved in TCDD metabolism. Levels of total hepatic microsomal P450 are 10%-30% lower in adult female than in adult male rats (Waxman et al., 1985). That the sex difference in biliary elimination of TCDD-derived radioactivity in the present study did not appear in juvenile rats may suggest that metabolism was linked to sexual maturation (Waxman et al., 1985; Marie et al., 1993). The juvenile male and female rats were 28 days of age and did not display secondary sexual dimorphism (e.g. same body size and weight). Depressed elimination in senescent male rats similar to that in adult females, as demonstrated in the present study, is consistent with involvement of hormonal regulation of sex-specific P450 forms and/or the general decline of metabolic activity in the aging liver (Kamataki et al., 1985; Birnbaum, 1991, 1993).

Pretreatment of adult rats with modulators of P450 activity provided additional insight into the metabolism of TCDD, although the results may be complicated by the broader effects of these compounds. Pretreatment of male rats with the three inhibitory compounds caused significant decreases in the metabolism of TCDD, compared with age-matched naive male rats, as characterized by the short-term cumulative biliary excretion of TCDD-derived radioactivity. The decreased cumulative biliary elimination of TCDD-derived radioactivity in males following PB pretreatment was similar to the cumulative biliary elimination for naive females. Interestingly, PB has been shown to decrease levels of the male constitutive forms 2A2, 2C11, and 2C13 (Funae and Imaoka, 1993). Results in the DEX rats may be confounded because these animals lost >10% of their weight during pretreatment, and the decrease in biliary elimination may have been due to toxicity. Starvation does repress expression of 2C11 and 2C13 (Imaoka et al., 1990). Also, decreased expression of CYP2C forms in male rat liver has been demonstrated 24 hr after treatment with DEX-type inducer pregnelone-16-carbonitrile (PCN) (Marie et al., 1993). Animals pretreated with ABT (P450 suicide substrate) demonstrated a significant decrease in biliary elimination of TCDD-derived radioactivity, compared with age- and TCDD dose-matched rats. As suggested by these results, biliary metabolism of TCDD in rats of both sexes may be only partially dependent upon hepatic P450-dependent monooxygenase activity.

While the present studies examined the effects of sex and age on the short-term biliary elimination at a dose of 1 nmol (0.322 µg) TCDD/kg, sex-specific differences in biliary elimination were strikingly similar to reported sex-specific susceptibilities to toxic effects after exposure to relatively high doses of TCDD. For example, the male/female ratio for 6-hr excretion of TCDD-derived radioactivity was ~1.5. Approximate male/female ratios of acute LD50 estimates reported for various rat strains are as follows: 1.7 for Long-Evans (Fan and Rozman, 1995), 1.8 for Finnish Long-Evans (Pohjanvirta et al., 1993), and 2.4 for Sprague-Dawley (Beatty et al., 1978). An earlier study (Pohjanvirta et al., 1993) also reported TCDD LD50 values for weanling male (25.2 ± 1.4 µg/kg), castrated adult male (39.1 ± 2.1 µg/kg), and testosterone-treated adult female (44.5 ± 1.5 µg/kg) Sprague-Dawley rats; changes in susceptibility were well-correlated with hepatic microsomal aniline hydroxylase activity. When purified from rat liver, male-specific CYP2C11 has more than twice the aniline hydroxylase activity of the female-specific CYP2C12 or the non-sex-specific CYP2C7 (Funae and Imaoka, 1993). These reported effects of sex hormone modulation on both lethality and microsomal enzyme activity are consistent with the suggested male-specific role of CYP450 in metabolism of TCDD in rats.

Recent studies have elucidated the importance of hepatic proteins that transport endogenous and xenobiotic substances across hepatocyte canalicular membranes into the bile and the ensuing physio- and toxicological responses. The multispecific organic anion transporter and multi-drug-resistant P-glycoproteins (mdr1a and mdr1b genes) have been identified as hepatic canalicular transport proteins involved with this transfer process (Keppler and Arias, 1997; Keppler and König, 1997; Silverman and Schrenk, 1997; Suchy et al., 1997). Although TCDD has been shown not to induce mdr mRNA in rat and mouse hepatocytes, indicating that the Ah receptor pathway is distinct from the mdr regulatory pathway (Silverman and Schrenk, 1997), transporter proteins may facilitate the hepatobiliary elimination of TCDD and its metabolites. For instance, glucuronide conjugates of TCDD (Poiger and Buser, 1984) may act as substrates for the canalicular multispecific organic anion transporter carrier. Since TCDD is metabolized very slowly compared with PB, the decreased biliary excretion of TCDD in the PB-treated animals in the present study may be related to competition of the PB and TCDD conjugates for the hepatic transporters. Interestingly, DEX has been shown to decrease mdr expression (Silverman and Schrenk, 1997); in the present study, this decrease in transporter protein could inhibit biliary excretion of TCDD in the DEX-treated rats. Further studies are needed in order for the effects of the various hepatic transporter proteins and genes on the hepatic disposition of TCDD to be fully understood.

Despite the extreme resistance of TCDD to metabolism and elimination in all animals examined thus far, the present studies have demonstrated clear sex- and age-related differences in short-term biliary elimination of TCDD-derived radioactivity within a single strain of rats. Modulation of hepatic enzyme activities with various pharmacologic agents have enabled further insights into the specific metabolic pathways involved in these important detoxifying processes. TCDD metabolism has been shown to be dose- and time-dependent in rats, involving both P450- and non-P450-dependent mechanisms. Even though metabolism occurs very slowly, commensurate with its overall rate of elimination, the effects of sex hormone modulation may in fact support a role for gender-specific P450 in TCDD metabolism for highly exposed subgroups of the general population.

	Note Added in Proof

A recent report suggested a sex difference in a TCDD-exposed population, with higher TCDD levels in women than in men, and that this difference may be explained by variations in metabolism or elimination. Also, the study suggested a lack of gender differences at older ages (Landi et al., 1998).

	Dedication

The authors wish to dedicate this work to the memory of Renu Batra, a Ph.D. candidate in our laboratory who helped with some of the early experiments, who died in an automobile accident on July 5, 1994, at the young age of 33. Her beauty, talent, and inspiration are sorely missed.