U.S. Environmental Protection Agency, ORD, NHEERL, ETD, Research Triangle Park, North Carolina 27711
Received October 9, 2000; accepted February 7, 2001
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ABSTRACT |
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Key Words: TCDD; dioxin; subchronic dosing; disposition; CYP1A1; CYP1A2; dosimetry; body burden.
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INTRODUCTION |
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Diet accounts for an estimated 90% of the daily exposure in humans (Safe, 1990). In the U.S., an estimated daily intake of TCDD from food is 34.4 pg and from nonfood sources an additional 0.424 pg (Travis and Huttemer-Fey, 1991). The daily intake of TCDD in the U.S. from dairy, meat, and fish as the principal sources has been estimated to be 15.9 pg (Henry et al., 1992
). Other investigators (Vanden Heuvel and Lucier, 1993
) have estimated the average daily intake from food in industrial countries with meat, fish, and dairy products being the principal sources as 13 pg toxic equivalency (TEQ)/kg. Using data from numerous studies in industrialized countries, daily intake of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) has been estimated to be 13 pg TEQ/kg body weight/day for a 60-kg adult (Galli and Marinovich, 1999
). This is equivalent to a body burden (BB) of 26 ng TEQ/kg bw (Galli and Marinovich, 1999
). The World Health Organization has set a tolerable daily intake (TDI) for TCDD at 14 pg/kg (WHO, 1998).
At high exposures, adverse human health effects to TCDD have been observed and are associated with activation of the Aryl hydrocarbon (Ah) receptor by TCDD (Birnbaum, 1994a). Although the general population is exposed to TCDD at low background exposures, the bio-accumulation of this chemical may pose a potential risk to human health. Because of this, an accurate assessment of human environmental exposure to TCDD and related compounds is important. Animal studies using repeated low exposures of TCDD over time to establish steady-state kinetics are important in assessing adverse and non-adverse biological endpoints for TCDD and other persistent bio-accumulative toxicants (PBTs). This type of dosing paradigm in experimental animals may mimic in humans the likely steady-state kinetics of TCDD resulting from daily exposure to low concentrations via food consumption. Hence, data from these subchronic animal studies may be more accurate to use in physiologically based pharmacokinetic (PBPK) models than studies using single acute doses resulting in models that will be more useful for the risk assessment of PBTs. A recent robust PBPK model for TCDD by Wang et al. (2000) is able to extrapolate across species, sex, dose, and route and will be useful for extrapolations to humans.
Studies in our laboratory have examined effects of subchronic exposure on dose-response relationships of various PHAHs in female B6C3F1 mice (DeVito et al., 1997, 1993
, 2000
, 1998
; van Birgelen et al., 1996
). In a series of subchronic studies with dioxin-like PHAHs, mice were dosed long enough for many of these compounds to reach or at least approach steady-state levels so that differences in pharmacokinetic factors did not play a major role in the biological response (Birnbaum, 1994b
; DeVito and Birnbaum, 1995
; DeVito et al., 1997
). As a result, relative potency (REP) values based on CYP1A enzymatic induction were proposed for these compounds (DeVito et al., 1997
, 2000
). The TEQ concept describes the relative potencies of these compounds by using TCDD as a reference compound (Ahlborg et al., 1994
; Birnbaum, 1999
; Birnbaum and DeVito, 1995
; Safe 1990
).
Presently, data is limited on the disposition of low, repeated doses of TCDD at steady state in the mouse (DeVito et al., 1997, 1993
, 2000
, 1998
; van Birgelen et al., 1996
; Vogel et al., 1997
). For that reason, the present study was conducted to examine the relationship of steady state and dose differences on disposition and CYP1A enzymatic activities of [3H]TCDD following subchronic oral exposure in B6C3F1 mice. One goal of repeated-dose studies is to achieve a steady-state concentration of the tested compound in the test animal. Steady-state concentration suggests a constant exposure of the test animal and its organs to the test compound throughout the study. Assuming complete bioavailability (amount of test compound that reaches the systemic circulation), steady state will be achieved when the rate of the compound's elimination equals the rate of the compound's administration. The present study is the first and most intensive pharmacokinetic study at low exposures to be reported with repeated dosing, multiple times, and multiple doses examining disposition of TCDD-derived radioactivity and CYP1A activities in mice.
The present study had several objectives. The first objective was to determine time-course effects on tissue dosimetry and enzymatic activities of TCDD approaching or at steady state. The second was to determine dose-response effects on tissue dosimetry and enzymatic activities of TCDD at or near steady state. The third was to study effects of a washout period (time with no dosing) subsequently to dosing at or near steady state on tissue dosimetry and enzymatic activities. The fourth was to examine disposition including metabolism and excretion of TCDD at or near steady state.
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MATERIALS AND METHODS |
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Animals and treatment.
Female B6C3F1 mice (60-days-old, 20 g) were obtained from Charles River Breeding Laboratories (Raleigh, NC) and allowed 1 week to acclimate before dosing. By using this animal model, extensive comparisons to previous studies from our laboratory can be made (DeVito et al., 1995
, 1997
, 1994
, 2000
, 1998
; Diliberto et al., 1995
; van Birgelen et al., 1996
). Mice were randomly assigned to treatment groups and were housed in an AAALAC-approved animal facility at the U.S. EPA using the National Institutes of Health`s Guideline on the Care and Use of Laboratory Animals. Housing environment conditions included a 12-h light/dark cycle at ambient temperature of 22 ± 1°C and relative humidity of 55 ± 5%.
The present study consisted of 3 studies: Phase 1, time-course; Phase 2, dose-response; and Phase 3, disposition. Mice (Phases 1 and 2) were housed according to treatment groups (1 treatment group per cage; 5 animals/group). They were housed throughout the study in shoe box-type polycarbonate cages with bedding of hardwood shavings (Beta Chips, North Eastern Products Inc., Warrensburg, NY) and given Rodent Chow (Purina, St. Louis, MO) and tap water ad libitum.
Mice (Phase 3; 5 animals/treatment group) were housed individually 3 days prior to dosing for acclimation and throughout the study in Nalge metabolism cages (Nalgene, Rochester, NY). They were given dustless precision pellet feed (BioServe, Frenchtown, NJ) and tap water ad libitum.
The doses given to all animals (Phases 1, 2, and 3) were administered in a corn-oil vehicle by oral gavage at a dosing volume of 10 ml/kg body wt. Three days after the last dose, all animals were euthanized by CO2 asphyxiation followed by exanguination via cardiac puncture. At necropsy, tissues were collected and weighed. Residual radioactivity was determined in collected tissues (Phases 1, 2, and 3) and excreta (Phase 3). Enzymatic activities (Phases 1 and 2) were determined in liver, lung, and skin for CYP1A1 and in liver for CYP1A2.
Treatment (Phase 1, time-course study).
Time-points used were 4, 8, 13, and 17 weeks of daily dosing 5 days per week, Monday through Friday. For each time-point, mice were dosed with either 0.0 (corn oil), 1.5, or 150 ng [3H]TCDD/kg body weight/day (0.0, 0.14, or 1.4 µCi/kg/day, respectively). Another group of animals was dosed for 13 weeks (daily, MondayFriday) with either 0.0, 1.5, or 150 ng [3H]TCDD/kg body wt/day (0.0, 0.14, or 1.4 µCi/kg/day, respectively) followed by 4 weeks with no dosing to allow for elimination (washout) of the TCDD-derived radioactivity from the TCDD-treated animals. Tissues collected at the end of the study were blood, adipose tissue (subcutaneous and perirenal adipose tissue), liver, kidneys, lung, skin (whole body), muscle (right and left leg thigh muscle), spleen, thymus, adrenals, ovaries, and uterus.
Treatment (Phase 2, dose-response study).
Mice were dosed daily 5 days/week (MondayFriday) for 13 weeks at doses of 0.0 (corn oil), 0.15, 0.45, 1.5, 4.5, 15, 45, 150, or 450 ng [3H]TCDD/kg body weight (0.0, 0.014, 0.14, 0.42, 1.4, 1.4, 1.4, 1.4, or 1.4 µCi/kg, respectively). Doses were chosen based on previous studies in our laboratory (DeVito et al., 1997; 1993
). Animals dosed with 0, 1.5, or 150 ng [3H]TCDD/kg/day for 13 weeks were the same groups of animals used for the 13-week time-point in the Phase 1 time-course study; the time-course and dose-response studies were started at the same time. Tissues collected at the end of the study were similar to Phase 1 and were blood, adipose tissue (subcutaneous and perirenal fat), liver, kidneys, lung, skin (whole body), muscle (right and left leg thigh muscle), spleen, thymus, adrenals, ovaries, and uterus.
Treatment (Phase 3, disposition study).
Mice, housed individually in metabolism cages, were dosed 5 days/week (daily, MondayFriday) for 13 weeks with either a low or high dose of 1.5 or 150 ng [3H]TCDD/kg body weight (0.14 or 1.4 µCi/kg, respectively). Throughout the study, daily urine and feces were collected separately. Tissues collected at the end of the study were blood, liver, lung, kidneys, adrenals, thymus, skin (back of neck area), adipose tissue (subcutaneous and perirenal fat), muscle (right and left leg thigh muscle), brain, spleen, thyroid, heart, ovaries, uterus, stomach and contents, small intestines and contents, large intestines and contents, pancreas, bone (femur), and bone marrow (from the femur).
Tissue sample analysis (Phase 1, 2, and 3 studies).
Residual radioactivity in tissues from Phase 1, 2, and 3 studies was determined by combustion (Packard 306B Biological Oxidizer; Packard Instrument Co., Downers Grove, IL) followed by liquid scintillation spectrometry (LSS; Beckman Scintillation Counter, Beckman Instruments, Fullerton, CA). Whole body tissues of lung, liver, and skin were homogenized (DeVito et al., 1993) and triplicate samples of 200 µl for each tissue homogenate (except for skin in Phase 3, triplicate samples of 200 mg/sample of intact skin taken from the dorsal neck area) were combusted. Triplicate samples of blood (200 mg/sample), muscle (100 mg/sample; right and left leg thigh muscles), and adipose tissue including triplicate sampling from each subcutaneous and perirenal fat (50 mg/sample) were combusted. All remaining tissues were combusted in their entirety. The form of radioactivity localized in hepatic and adipose tissue has been demonstrated to be > 95% unmetabolized TCDD (Kedderis et al., 1991
).
Analysis of urine (Phase 3 study).
TCDD-derived radioactivity was determined in urine for each time-point for the 2 doses (low and high) from the Phase 3 study. Triplicate urine samples of 200 µl each were delivered directly into scintillation cocktail (Ultima Gold; Packard Instrument Co., Downers Grove, IL) and analyzed by LSS.
Analysis of feces (Phase 3 study).
At all time points for the 2 doses (low and high), feces were analyzed for total TCDD-derived radioactivity. At selected time points, feces were extracted and analyzed for unmetabolized [3H]TCDD. Feces samples were air-dried and separated from any contaminating food particles, then weighed. Feces not prepared for extraction were combusted whole and entirely, as multiple samples (2 or 3) of 400 mg. Selected feces (including first and final weeks of both doses) for extraction were pulverized using an apparatus consisting of a stainless steel rod (pestle), a steel pipe sheath (to isolate crushing area), and a close-fitting bored aluminum base (mortar). Feces were manually pounded into a fine powder with minimal loss of sample. Single 100-mg samples of pulverized feces were combusted followed by LSS.
Fecal extraction (Phase 3 study).
A microsoxhlet method was developed using dichloromethane (DCM) as extracting solvent. Trials were conducted to test the method using 400 mg pulverized feces spiked with 0.2 µCi of [3H]TCDD in 50200 µl MeOH and dried for either 5 days or 3 weeks. Each sample was placed in a cellulose thimble and extracted by continuously refluxing with 30 ml DCM (plus 2 ml MeOH to prevent dryness) for
20 h. Total extract was collected and weighed. A weighed sample was analyzed for radioactivity by LSS. Residual feces and thimble were air-dried, and then analyzed for radioactivity by combustion followed by LSS. Results in the trials (5 days or 3 weeks) indicated that
84% of [3H]TCDD spike was extracted from feces; 83.8 ± 3.6% and 83.6 ± 0.46%, respectively, were quantifiable in extract, while 2.6 ± 0.6% and < 0.3%, respectively, were recovered by oxidizing the postextraction feces and extraction thimble. Minor modifications to the above method were used to extract selected experimental samples. During the first week of dosing at the low dose, daily fecal samples were extracted for Days 16. For each day's extraction, 400 mg of feces from each animal for that day were combined due to the low radioactivity. Similarly, during the first week of dosing (Days 16) at the high dose, feces (300 mg/animal) were combined from each day due to the low radioactivity and extracted. However, during the final week of dosing (Days 8490) at the high dose, feces from each animal were extracted individually using 500 mg/animal/day.
HPLC analysis of fecal extracts.
Duplicate injections of 100 µl concentrated fecal extract were analyzed by RP-HPLC as described previously. TCDD-derived radioactivity was detected by a Beckman 171 radioisotope detector with a 500 µl flow cell. Retention time for [3H]TCDD was 15 min.
CYP1A enzymatic assays.
Microsomal fractions were prepared from liver, skin, and lungs according to the method of DeVito et al. (1993). Microsomal protein concentrations were determined according to the method of Bradford (1976) using a Bio-Rad Protein Assay kit (Richmond, CA) with bovine serum albumin as standard. Activity of ethoxyresorufin O-deethylase (EROD), a marker for CYP1A1, was determined in liver, skin, and lungs spectrofluorimetrically based on the dynamic assay method of Pohl and Fouts (1981) as modified by DeVito et al. (1993). Activity of acetanilide-4-hydroxlyase (ACOH), a marker for CYP1A2, was determined in liver by the method of Liu et al. (1991) as modified by DeVito et al. (1993).
Data analysis.
For calculation of percent total dose, blood mass was assumed to be 8% of body weight (King et al., 1983). Total body skin, muscle, and fat percentages (of body weight) were 13 (13.3 ± 0.4%), 35 (34.9 ± 4.3%), and 8 (8.1 ± 1.2%), respectively, as determined by dissection of age-matched control animals (n = 5) for the time points of 30, 60, and 90 days. However, for the animals at the time point of 120 days, the total dissectable body fat percentages were 15.5 ± 5.5% as determined by dissection of body fat from each animal. For calculation of percent total dose at 120 days, the actual % body fat for each animal was used. Also, for the animals in the Phase 3 study, the total body fat was determined by dissection of body fat from each animal and the total dissectable body fat percentages were 12.9 ± 3.3%. The actual % body fat for each animal for these animals was used for calculation of percent total dose. Body burdens in Phases 1 and 2 were the terminal cumulative dose in the body as determined by % total dose in collected tissues.
Intergroup comparisons were performed by a 1-way analysis of variance (ANOVA) followed by Scheffe's F-test. Differences between treatment groups were considered statistically significant when p < 0.05. Intergroup comparisons of the log transformation of enzyme activities, tissue concentrations of ng TCDD/g, and body burdens of ng TCDD/kg body weight were performed by ANOVA followed by Protected Fisher's Least Significant Difference test. All data are presented as mean ± standard deviation.
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RESULTS |
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Phase 1: Time Effects on Dosimetry
Tables 1 and 2 demonstrate effects of time on tissue dosimetry after dosing daily Monday through Friday with 1.5 or 150 ng [3H]TCDD/kg/day over 4, 8, 13, or 17 weeks or after dosing with 1.5 or 150 ng [3H]TCDD/kg/day (MondayFriday) over 13 weeks followed by 4 weeks washout with no dosing. At the low dose for all time points (Table 1
), concentration of ng TCDD/g tissue was less in liver than in adipose tissue (fat). Whereas, at the high dose for all time points (Table 2
), concentration of ng TCDD/g tissue was greater in liver than in adipose tissue. This dose difference was reflected by the induction of CYP1A2 enzymatic activity in liver (see below).
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Animals dosed for the 13-week period followed by 4 weeks with no dosing demonstrated dose- and tissue-dependent washout effects. At the low dose for all tissues, tissue concentrations following the washout period were approximately one-third compared to tissue concentrations following the dosing period. Whereas at the high dose, elimination of TCDD-derived radioactivity was faster in liver than in non-hepatic tissues with concentrations of approximately one-tenth and one-third, respectively. This faster elimination from the liver reflected the influence of the inducible hepatic binding protein, CYP1A2, and hepatic sequestration (see below). Differences in elimination were also reflected by the L/F concentration ratios.
Phase 1: Time Effects on CYP1A Enzymatic Activities
Table 3 demonstrates effects of time on CYP1A enzymatic activities after subchronic dosing with 0, 1.5, or 150 ng [3H]TCDD/kg (daily, MondayFriday) over 4, 8, 13, or 17 weeks or after dosing with 0, 1.5, or 150 ng [3H]TCDD/kg (daily, MondayFriday) over 13 weeks followed by 4 weeks washout with no dosing. Enzymatic activities were measured for EROD, marker for CYP1A1, in liver, lung, and skin and for ACOH, marker for CYP1A2, in liver.
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At the low dose for all time-points, hepatic ACOH activity was not induced. Whereas, the high-dose treated animals demonstrated induction of hepatic ACOH activity approximately five-fold greater than control treated animals. This increased ACOH activity was reflected by increased hepatic accumulation of TCDD (Tables 1 and 2).
Animals dosed for 13 weeks followed by 4 weeks with no dosing demonstrated dose- and tissue-dependent washout effects on enzymatic activities. With a half-life of approximately 10 days in mice (Birnbaum, 1986; Gasiewicz et al., 1983
), 4 weeks would be sufficient time for approximately 80% of the steady-state body burden to be eliminated as seen at the high dose and CYP1A induction partially reversed (Tables 1, 2, and 3
). This is very similar to studies in rats using higher doses than the present study (Li and Rozman, 1995
). However, at the low dose, only 50% of the steady-state body burden was eliminated (Tables 1 and 2
). Disposition kinetics have been shown to be nonlinear for TCDD and half-lives lengthen as tissue concentrations decrease with time (Carrier et al., 1995b
). In Table 3
, the greatest effects of washout were seen at the high dose for EROD enzymatic activities in liver and lung (10-fold reduction in activity) and for ACOH enzymatic activities in liver (3-fold reduction in activity). The effects of this residual induction of CYP1A2 on hepatic accumulation of TCDD was demonstrated by a L/F concentration ratio of 0.9 (Tables 1 and 2
). This L/F was very similar to the L/F concentration ratio at the dose of 15 ng/kg/day in the Phase 2 study (see below). In CYP1A2 KO mice, a L/F concentration ratio of
0.3 indicates no hepatic binding of TCDD to CYP1A2 (Diliberto et al., 1999
, 1997
).
Phase 2: Dose Effects on Tissue Dosimetry and Body Burden
Table 4 illustrates effects of dose on tissue dosimetry and body burden following subchronic dosing with 0.15, 0.45, 1.5, 4.5, 15, 45, 150, or 450 ng [3H]TCDD/kg (daily, MondayFriday) over 13 weeks. The distribution of TCDD was dose-dependent in all tissues. Concentration expressed as ng/g tissue increased with dose in all tissues examined. However, there were differences between tissues. At the dose of 15 ng/kg/day, liver and adipose tissue concentrations were equal as demonstrated by a L/F concentration ratio of one. At doses below this exposure level, liver concentrations were less than adipose tissue with L/F concentration ratios < 1. At higher doses, liver concentrations were greater than adipose tissue with L/F concentration ratios > 1. No evidence of saturation of the adipose tissue depot was apparent. The hepatic sequestration appeared to be related to the induction of CYP1A2 (see below, Table 5
). The body burden of ng TCDD/kg body weight increased with increasing dose and ranged from
3 to 3000 ng TCDD/kg body weight, lowest to highest dose. Most of the body burden from 7190% (low to high dose) was distributed between adipose tissue and liver and this distribution was dose-dependent. The percentages of body burden in adipose tissue ranged from 6432%, low to high dose. In contrast, the body burden in liver ranged from 758%, low to high dose.
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Hepatic EROD activity was significantly increased (p < 0.05) starting at the lowest dose of 0.15 ng/kg/day and body burden of 2.8 ng TCDD/kg body weight (Table 4). At 15 ng/kg/day, the concentration in liver and adipose tissue are similar (L/F concentration ratio of 1 and body burden of 157 ng TCDD/kg body weight; Table 4
) and EROD activity started to increase at a much greater rate as seen by a 4-fold increase from control to a 140-fold increase from control at the highest dose.
Hepatic ACOH activity was significantly increased (p < 0.05) starting at 4.5 ng/kg/day and body burden of 54 ng TCDD/kg body weight (Table 4). The ACOH activity was maximally induced at 45 ng/kg/day and body burden of 300 ng TCDD/kg body weight. Although the enzymatic activity of CYP1A2 as measured by ACOH activity appeared to be maximal, the amount of TCDD sequestration in the liver continued to increase with dose (Table 4
). Previous studies have demonstrated that at the higher doses of TCDD with maximal ACOH activity the induction of CYP1A2 protein continues to increase (DeVito et al., 1996
).
The response of TCDD in the liver is not homogeneous. At low doses, induction of CYP1A1 and CYP1A2 occurs in the centrilobular region; and at high doses, areas of induction extend into the periportal regions (Bars and Elcombe, 1991; Tritscher et al., 1992
). A combined physiologically based PBPK and a multi-compartment liver model to account for the regional differences in the induction suggested that the hepatic CYP1A1/CYP1A2 induction by TCDD is highly nonlinear (Andersen et al., 1997a
,b
).
In lung, EROD activity was significantly increased (p < 0.05) starting at 1.5 ng/kg/day and body burden of 24 ng TCDD/kg body weight (Table 4). Whereas in skin, EROD activity was significantly increased (p < 0.05) starting at 15 ng/kg/day and body burden of 157 ng TCDD/kg body weight (Table 4
).
Phase 3: Effects on Tissue Dosimetry and Excreta
Table 6 illustrates the disposition of TCDD-derived radioactivity in tissue dosimetry and excreta after dosing with either 1.5 or 150 ng [3H]TCDD/kg (daily, MondayFriday) over 13 weeks. Adrenals and thyroid had higher concentrations of TCDD (pg/g tissue weight) than other non-fat extrahepatic tissues at both dose levels. Total recoveries of radioactivity were 85.67 and 81.14% (low and high doses, respectively; not significantly different).
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Figure 2A illustrates effects of the low dose 1.5 ng [3H]TCDD/kg (daily, MondayFriday) on daily fecal and urinary excretion of TCDD-derived radioactivity. Over the 13-week exposure period, fecal excretion was 34.9 ± 3.4% of administered dose and urinary excretion was 12.0 ± 1.0%, with an overall feces/urine excretion ratio of
3.
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In both dose groups, daily excretion of radioactivity varied with the dosing schedule and reflected the 5-day/week dosing regimen (rising during the week). The overall excretion profiles exhibited a saw tooth shape fluctuation with enhanced daily excretion except for weekends. During each interdose interval, the amount of TCDD-derived radioactivity in the excreta rose and then fell. These fluctuations appeared to be proportional to dosing interval and elimination half-time and blunted by slow absorption. Biliary excretion of TCDD-derived radioactivity in rats has been demonstrated to be less than 1% within the first 6 h of intravenous exposure to [3H]TCDD (Jackson et al., 1998). Although the radiopurity of [3H]TCDD in the TCDD dosing solution was
99%, the presence of minor impurities with more rapid clearance than TCDD and its metabolites may account for some of the fluctuation. Variations in the fecal excretion of radioactivity were more dramatic at the lower dose and appeared to be due primarily to amount of unabsorbed parent excreted (see results below on fecal extractions). In both dose groups, the overall feces/urine excretion ratio showed a similar weekly pattern, ranging from 2.53 on weekends, and peaking between 3.5 and 4 during the week.
Phase 3: Effects on Fecal Extractions
Table 7 illustrates the analysis of fecal excretion of TCDD-derived radioactivity during the first and final week of subchronic exposure to 1.5 ng [3H]TCDD/kg/day or 150 ng [3H]TCDD/kg/day. The chemical nature of TCDD-derived radioactivity in feces was investigated to determine relative amounts of parent compound (unabsorbed or absorbed yet nontransformed) and metabolites that were excreted on a given day following repeated oral exposure.
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Throughout the 13 weeks of dosing for both dose groups of animals, the weekly pattern of fecal excretion of TCDD-derived radioactivity appeared to be dependent on excretion of unabsorbed parent compound. This was clearly demonstrated by the low excretion of TCDD-derived radioactivity apparent on Sundays and Mondays. On these days, the TCDD-derived radioactivity in fecal excretion had to have been mostly absorbed and consisted of absorbed TCDD and metabolites.
During the first week of the low dose, TCDD-derived radioactivity excreted in the feces after the first day of dosing represented mainly unabsorbed/absorbed parent compound; fecal extraction confirmed 80% of radioactivity as TCDD. After 24 h of the first dose, 27 ± 5% of administered dose was excreted in the feces. Based on this, gastrointestinal absorption for the first day was
70%. This percentage of oral absorption is similar to previous disposition and absorption studies (Diliberto et al., 1996
, 1993
). In these rat studies, the intravenous (iv) and oral (po) routes of exposure for TCDD and related compounds were compared. For the first 24 h after dosing, most of the fecal excretion of derived-radioactivity from the parent compound was due to the unabsorbed parent compound (Diliberto et al., 1996
, 1993
). Days 2 and 3 showed decreases in both excretion and the portion attributable to TCDD, suggesting that absorption in the gut increased slightly with subsequent doses. By the end of the first week, 60% of the daily fecal excretion was attributable to TCDD, as absorbed with no metabolism and/or unabsorbed. During the final week of the low dose and at or near steady-state conditions, at least 70% of the daily fecal excretion of TCDD-derived radioactivity was attributable to parent compound, TCDD, as absorbed and not metabolized and/or unabsorbed.
During the first week of the high dose, excretion of TCDD-derived radioactivity in feces on the first day of dosing was mainly unabsorbed and/or absorbed parent compound; fecal extraction confirmed 70% of radioactivity as TCDD. After 24 h of the first dose, 19 ± 8% of administered dose was excreted in the feces. This suggested a better gastrointestinal absorption of the first dose at the high dose than at the low dose. Days 2 and 3 showed decreases in both excretion and the portion attributable to TCDD, suggesting an increased absorption, as also observed at the low dose. During the final week of the high dose, analysis of TCDD-derived radioactivity in feces demonstrated near steady-state conditions. On Monday (Day 83) and 3 days after the last dose, approximately 30% of the radioactivity in feces was assumed to be absorbed TCDD, while
70% in the unextracted was assumed to be metabolite(s). Between 28 and 54% of the daily TCDD-derived radioactivity in feces was attributable to parent compound on the final week. Variation in levels of unabsorbed parent excreted, an artifact of the dosing schedule, clearly accounted for daily variation in overall fecal excretion of TCDD-derived radioactivity.
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DISCUSSION |
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The disposition of TCDD is determined by its lipophilicity, binding affinity to the Ah receptor, binding affinity to CYP12 that is mediated by the Ah receptor, and metabolism. Because TCDD is highly lipophilic, TCDD concentrates in tissues by partitioning into a more lipid-rich environment. A majority of this compound in any body tissue is stored in the lipid compartment (van der Molen et al., 1996). However, at doses high enough to induce hepatic CYP1A2, TCDD sequesters in liver with less of the dose available for extrahepatic tissues. As seen clearly in the present study, disposition of TCDD was influenced not only by its lipophilicity but also by its binding to CYP1A2. Lipid solubility played a key role in the disposition of TCDD at low doses, but was overwhelmed by hepatic binding to CYP1A2 when it occurred (Diliberto et al., 1999
, 1997
). The dose in extra-hepatic tissues was modulated by dose-dependent hepatic sequestration as well as lipid solubility. Based on percent-administered dose, the present study demonstrated an inverse correlation between liver and extra-hepatic tissues. Clearly, TCDD accumulated or sequestered in liver at the expense of dosimetry in extra-hepatic tissues (Andersen et al., 1993
; Diliberto et al., 1999
, 1997
). Hepatic sequestration has also been reported in humans (Carrier et al., 1995a
; Grassman et al., 2000
).
Results from the present time-course study suggest that tissues levels of TCDD reached steady state by 8 and certainly by 13 weeks of TCDD exposure. For each of the 2 doses in the time-course study, liver and adipose tissue contained most of the body burden. Furthermore, the liver to adipose tissue (L/F) concentration ratios remained similar over time for each dose; and, suggested that TCDD had reached steady state (Krowke et al., 1989). Upon assessing changes in tissue distribution, the L/F concentration ratio provides a sensitive indicator (Krowke et al., 1989
). With an approximate half-life of 10 days for TCDD in mice (Birnbaum, 1986
; Gasiewicz et al., 1983
), time to reach 90% of steady state would be over 30 days and to reach 99%, over 60 days. In comparison, half-life estimates for TCDD in humans range from 7.2 to 8.7 years (Flesch-Janys et al., 1996
; Michalek and Tripathi, 1999
; Needham et al., 1994
).
The relative persistence of TCDD was tissue specific in the present study. Previous studies in mice (Birnbaum, 1986) and rats (Abraham et al., 1988
) have demonstrated a faster elimination half-life in liver than in adipose tissue. The elimination of TCDD from tissues appeared to be directly related to the amount of TCDD binding to CYP1A2, the inducible hepatic binding protein, and to the Ah receptor, the TCDD-activated transcriptional enhancer, that functions to regulate gene expression of CYP1A1 and CYP1A2. Because of TCDD's high affinity for the Ah receptor, this complex remained active for a relatively long time following exposure and the reversible biological responses of enzymatic activities for CYP1A1 and CYP1A2 continued to be induced, although at reduced levels. The importance of the Ah receptor is demonstrated in studies using knockout mice lacking expression of the Ah receptor (null mutant, AhR-/-). In these studies, constitutive expression of CYP1A2 was decreased by 90% and the Ah agonist TCDD did not induce CYP1A2 mRNA (Fernandez-Salguero et al., 1995
). Likewise, the importance of CYP1A2 is demonstrated in knockout mice lacking expression of the CYP1A2 (null mutant, CYP1A2-/-) and their parental strains with the normal (CYP1A2+/+) gene. These studies demonstrated that CYP1A2 was the inducible hepatic binding protein that sequesters TCDD (Diliberto et al., 1999
, 1997
). Accordingly, the amount of hepatic CYP1A2 appears to be a crucial determinant for the dosimetry of TCDD in the liver. The pharmacokinetic behavior of TCDD has been analyzed by a PBPK model developed by Andersen et al. (1993) that accurately describes the TCDD distribution in liver and adipose tissue and the CYP1A enzymatic induction. In addition, sensitivity analyses of the parameters of a PBPK model by Leung et al. (1990a) demonstrated that the liver concentrations of TCDD in TCDD-pretreated mice were influenced by the binding capacity of the hepatic microsomal protein.
A tissue-specific reversible biological response was associated with tissue dose and low body burden of TCDD in the present study. At the lowest dose of 0.15 ng/kg/day, a low body burden of 2.8 ng TCDD/kg body weight was associated with significant hepatic induction of CYP1A1. At this dose, most of the body burden was contained in the liver (5 pg TCDD/g) and adipose tissue (22 pg TCDD/g) with a L/F concentration ratio of 0.21. This low body burden of TCDD in mice with a concomitant reversible biological response was comparable to estimated background levels in humans (Galli and Marinovich, 1999). A recent study using a different subchronic dosing paradigm than the present study demonstrated similar induction at a low dose of 0.34 ng TCDD/kg/day and a liver concentration of 30 pg TCDD/g (Vogel et al., 1997
). Also, in the present study, a low body burden of 10.5 ng TCDD/kg body weight was associated with significant induction of CYP1A2. Again, most of the body burden was contained in the liver (20 pg TCDD/g) and adipose tissue (85 pg TCDD/g) with a L/F concentration ratio of 0.25. At the lower doses, greater concentrations of TCDD were found in adipose tissue than in liver with body burdens ranging from 2.8 to 54 ng TCDD/kg body weight and L/F concentration ratios ranging from 0.21 to 0.47. Similarly, humans exposed at low environmental levels have most of their body burden of TCDD in adipose tissue (Ryan et al., 1985
). An estimated 90% of the body burden was found in the adipose tissue of a human volunteer who ingested a dose of 1.14 ng [3H]TCDD/kg body weight (Poiger and Schlatter, 1986
). Human data on liver-to-fat concentration ratios are variable between individuals and dependent upon previous exposures (and CYP1A2 levels) as well as expression of the data on lipid or wet-weight basis (Leung et al., 1990b
; Thoma et al., 1990
).
The oral absorption of initial doses in corn oil in the present study are consistent with previously reported results from acute animal (Diliberto et al., 1996; Lakshmanan et al., 1986
; Rose et al., 1976
) and human (Poiger and Schlatter, 1986
; Schlummer et al., 1988) studies. In addition, the fraction of TCDD absorption appeared to increase with subsequent doses suggesting a potentiation of oral absorption as previously reported in mice (Curtis et al., 1990
). This increase in TCDD uptake in the induced mice is in agreement with previous in vitro studies by Shen and Olson (1987). These studies used hepatocytes from mice pretreated with TCDD and demonstrated an increased uptake of TCDD by induced hepatocytes in culture (Shen and Olson, 1987
). In addition, earlier studies by Poland et al. (1989a,b) and Leung et al. (1990a) appear consistent with differences in kinetic behavior of the daily absorption seen at low and high doses and over time in the present study. In those earlier studies, the iodinated analog of TCDD was used to determine dose dependencies in mice for liver accumulation. Naïve mice were treated with a noninducing dose and had a greater proportion of the dose in the adipose tissue. Whereas, animals pretreated to induce the hepatic binding protein had a greater proportion of the dose in the liver and a more rapid absorption of the iodinated analog than naïve mice (Leung et al., 1990a
; Poland et al., 1989a
,b
). Also, PBPK model simulations demonstrated similar increase absorption in the induced mice (Leung et al., 1990a
).
TCDD is slowly metabolized in both animals and humans, and metabolism is required for urinary and biliary elimination. The limited excretion in urine, based on earlier findings in experimental animals, is attributed solely to metabolites of TCDD (Birnbaum, 1986; Gasiewicz et al., 1983
). The excretion of metabolites from kidneys may be dependent on the metabolic rate in the liver and/or the membrane transfer process. Metabolism of TCDD in the liver is very limited. Biliary excretion of TCDD-derived radioactivity in rats has been demonstrated to be less than 1% within the first 6 h of exposure to TCDD (Jackson et al., 1998
). TCDD is eliminated primarily in feces (Diliberto et al. 1996
; Gasiewicz et al., 1983
) as parent compound (TCDD) and its metabolites (Gasiewicz et al., 1983
; Neal et al., 1982
; Olson, 1986
; Olson et al., 1980
; Van den Berg et al., 1994
). Excretion as parent compound may result from the transluminal excretion of nonabsorbed and/or absorbed TCDD. Data in humans indicate that TCDD is partially excreted in feces as metabolites (Wendling et al., 1990
). Similar to previous studies (Kedderis et al., 1991
), fecal elimination of TCDD-derived radioactivity was dose-dependent with greater elimination at the high exposure than at the low. This significant difference in fecal excretion may be related to greater accumulation of TCDD in adipose tissue at the low dose than at the high dose and to slower elimination of TCDD from adipose tissue than liver. Elimination half-life of TCDD is longer in adipose tissue than liver (Abraham et al., 1988
; Birnbaum, 1986
). There was, also, a dose difference in fecal excretion of parent compound and its metabolites. At the lower dose,
70% of the radioactivity in the feces was present as parent compound; whereas at the higher dose, it was 3050%. The rest of the radioactivity at both doses was attributable to metabolites. At the higher dose, induction of Ah receptor-regulated proteins could affect the metabolism and elimination of TCDD. The variations in daily excretion of TCDD-derived radioactivity at both doses were associated with the dosing regimen. These patterns at the high dose appeared 12 days sooner in the feces than in the urine because daily fecal excretion varied primarily with amount of unabsorbed parent.
In summary, the present study provides direct evidence of low dose effects at steady-state levels on induction of CYP1A1 and CYP1A2 enzymatic activities; and, CYP1A2 was shown as the crucial determinant of hepatic sequestration. Induction of these enzymes occurs through binding and activation of the Ah receptor. Similar to the present study, exposure of dioxin to the general population involves continuously low-level exposures. A greater proportion of dioxin is available to adipose tissue and other extrahepatic tissues at low doses than at higher doses where more goes to the liver (present study; Diliberto et al., 1997; 1999). Consequently, relatively more body burden of dioxin is available to the immune and reproductive systemspotential targets for adverse health effects and toxicity. Body burdens in animals associated with adverse effects or biochemical alterations that may or may not be adverse have previously been reported at < 50 ng/kg body weight and even as low as 10 ng/kg body weight (DeVito et al., 1995). This is very similar to average body burdens based on TEQs in the human population (DeVito et al., 1995
). However, this study confirmed for the first time that at steady-state conditions a measured response (enzyme induction) was determined at a body burden of 2.8 ng/kg body weight. This body burden found in the present study is within background levels of the general human population (Galli and Marinovich, 1999
). After a period of time (30 days) with no dosing, induction of CYP1A1 and 1A2 continued. Whether persisent induction of these enzymes leads to adverse health effects remains to be determined. As seen by the present study, the elimination of TCDD in excreta was faster at the higher dose with a smaller percentage of the dose (absorbed and/or unabsorbed TCDD) eliminated than at the lower dose. In addition, the present study supports the argument that body burden or tissue dose for a persistent, bioaccumulative chemical such as TCDD is more important to consider than daily dose.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the U.S. Environmental Protection Agency (USEPA), Experimental Toxicology Division (ETD), Pharmacokinetics Branch, Mail Drop-74, Research Triangle Park, NC 27711. Fax: (919) 541-5394. E-mail: diliberto.janet{at}epa.gov.
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