Acute Administration of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Pregnant Long Evans Rats: Association of Measured Tissue Concentrations with Developmental Effects

Christopher H. Hurst*,1, Michael J. DeVito{dagger}, R. Woodrow Setzer{dagger} and Linda S. Birnbaum{dagger}

* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599–7270; and {dagger} U. S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Experimental Toxicology Division, Research Triangle Park, North Carolina 27711

Received April 30, 1999; accepted August 30, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prenatal exposure to TCDD interferes with fetal development at doses lower than those causing overt toxicity in adult animals. Exposure to TCDD during development produces alterations in the reproductive system of the developing pups—delayed puberty and reduced sperm counts in males and malformations in the external genitalia of females. The objectives of this study were to determine maternal and fetal tissue concentrations of TCDD after acute exposure and whether these tissue concentrations can be used to estimate the intensity of the developmental abnormalities reported by other laboratories. Pregnant Long Evans rats received a single, oral dose of 0.05, 0.20, 0.80, or 1.0 µg [3H]-TCDD/kg on gestation day (GD) 15, and maternal and fetal tissue concentrations of TCDD were measured on GD16 and GD21. On GD16, maternal liver contained the greatest amount of TCDD (30–47% administered dose). One day after administration of 0.20 µg TCDD/kg on GD15, there were 13.2 pg TCDD/g present in an individual fetus. This concentration is associated with delayed puberty and decreased epididymal sperm counts in male pups as well as malformations in the external genitalia of females. For the responses studied, tissue concentration measured during a critical period of gestation adequately predicts the intensity of the response. In addition, there was a strong correlation between fetal body burden and maternal body burden on GD16. A dose of 0.05 µg TCDD/kg resulted in maternal body burdens of 30.6 ± 3.1 and 26.6 ± 3.1 ng TCDD/kg on GD16 and GD21, respectively. In conclusion, low-level TCDD exposure during the perinatal stage of life can produce adverse effects within the developing pups and that tissue concentration measured during a critical period is the appropriate dose metric to predict adverse reproductive and developmental effects.

Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; toxicokinetics; disposition; body burden; embryo; fetus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) belongs to a class of chemicals known as polyhalogenated aromatic hydrocarbons, which includes polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs). Exposure to these dioxinlike compounds results in a wide variety of effects, including a wasting syndrome, immunosuppression, thymic atrophy, chloracne, teratogenicity, and carcinogenicity, as well as other toxic and biochemical effects (Birnbaum, 1994bGo). Initiation of most, if not all, of these responses is due to binding of TCDD to the arylhydrocarbon (Ah) receptor (Birnbaum, 1994aGo).

TCDD and dioxinlike compounds adversely affect reproduction and development in part due to its ability to alter hormone and receptor levels in the endocrine system (Birnbaum, 1994bGo). Exposure to 0.064 µg TCDD/kg in female Holtzman rats on gestation day (GD) 15 decreased epididymal sperm reserves in male pups but produced no overt signs of toxicity in the pups or dams (Mably et al., 1992Go). Studies by Gray and coworkers examined the adverse effects in male and female Long Evans (LE) pups that were associated with exposure during critical periods of development. For example, a dose of 1.0 µg TCDD/kg on GD8 produced a broad spectrum of effects such as malformations in the external genitalia, premature reproductive senescence, enhanced incidence of constant estrous, and cystic endometrial hyperplasia in female LE rat pups (Gray and Ostby, 1995Go). In male offspring, this dose was associated with a persistent reduction in sperm counts (Gray et al., 1995Go). In addition, exposure to 1.0 µg TCDD/kg on GD15 produced a narrower spectrum of adverse effects, although the magnitude of the responses was much greater (Gray et al., 1995Go). For example, administration of 1.0 µg TCDD/kg on GD15 produced a greater decrease in cauda epididymal sperm numbers and ejaculated sperm numbers in males as compared to administration on GD8. In females, the GD15 group had a higher incidence of clefting of the phallus and a permanent thread of tissue across the vagina as compared to the GD8 group (Gray and Ostby, 1995Go). Results from Gray and coworkers show that administration of TCDD on GD15 is more detrimental to the offspring than an equivalent dose administered on GD8 with respect to male reproductive endpoints and in inducing malformations in the external genitalia of females (Gray et al., 1995Go; Gray and Ostby, 1995Go).

Recent studies in our laboratory have shown that exposure to 1.15 µg TCDD/kg during a critical period of organogenesis (GD8) results in rat fetal tissue concentrations of 20–40 pg/g between GD9 and GD21 (Hurst et al., 1998Go). Although very little of this chemical reaches the developing fetus, it is still sufficient to produce adverse developmental effects. Therefore, our objective was to expand on these observations by conducting a dose-response study on GD15, which is the onset of sexual differentiation, in order to determine maternal and fetal tissue concentrations of TCDD. In this study, LE rats were dosed by gavage on GD15 with 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg, and tissue concentrations were measured on GD16 and GD21. This information is useful to determine how fetal tissue concentration relates to a response in light of the fact that dosing on different gestational days produces different responses as well as a varying magnitude of the response.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
[3H]-TCDD (specific activity 31.9 Ci/mmol) was obtained from Radian Corporation (Austin, TX) and was purified by reverse-phase high-pressure liquid chromatography to >=99% purity (Diliberto et al., 1995Go). Dosing solutions were prepared by adding [3H]-TCDD (0.91 mCi/ml) in toluene to corn oil. Volatile compounds were removed by evaporation using a Savant Speed-Vac (Savant Instruments Inc., Farmingdale, NY).

Animals and treatments.
Eight-week-old, time-pregnant Long Evans rats (200–250g) were obtained from Charles River Breeding Laboratories (Raleigh, NC) on GD9. The day following overnight mating, with evidence of a copulatory plug, is designated as GD0 by Charles River Laboratories. Animals were housed individually in clear plastic cages with hardwood bedding (Beta Chips, Northeastern Products, Warrensburg, NY). Animals were maintained during pregnancy on Laboratory Rodent Diet (#5001, PMI Feeds, Inc., St. Louis, MO) and unpurified tap water ad libitum in a room with a 12:12-h photoperiod and a temperature of 20–24°C with a relative humidity of 40–50%.

Experimental design and treatments.
Five time-pregnant rats were assigned to each group. Rats received a single oral dose of 0.05, 0.20, 0.80, or 1.0 µg [3H]-TCDD/kg in 5 ml corn oil/kg on GD15. The dams were anesthetized with CO2 on GD16 or GD21, and 5 ml of blood was removed via cardiac puncture. Animals were terminated by cervical dislocation while under anesthesia. The following maternal tissues were removed and weighed: liver, adipose, thymus, muscle, and skin (ears). On GD16 and GD21, four fetuses from each litter were randomly selected to determine the amount of TCDD reaching the entire fetus. The remaining fetuses were subdivided into urogenital tract, liver, head, remaining tissue (body), and placenta. All the fetal tissues within a dam were assayed individually except for the urogenital tract. This tissue was so small that all the urogenital tracts within an individual dam were pooled to determine TCDD concentrations. On GD21 only, whole fetuses were homogenized in a Waring blender with liquid nitrogen until a fine powder was formed. Three 250-mg samples of the resulting powder were prepared for sample oxidation to determine TCDD concentration.

Oxidation and quantitation of samples.
Tissues were oxidized using a Packard 307 Sample Oxidizer with an Oximate 80 Robotic Operator (Packard, Downers Grove, IL) and oxidized samples were analyzed in a Beckman Model LS6000 LL liquid scintillation spectrometer using Monophase S (Packard, Downers Grove, IL). Previous studies from our laboratory indicate that >95% [3H]-TCDD present in tissues is recovered as parent compound (Kedderis et al., 1991Go).

Data analysis.
Calculation of percentage total dose and tissue volumes for adipose, muscle, and skin were experimentally determined on GD16 and GD21 as described by Hurst et al. (1998). For both time points, blood mass was assumed to be 7.4% of body weight (International Life Sciences Institute, 1994Go). The data was analyzed in the following dose metric units: % dose/tissue, % dose/g tissue, ng TCDD/tissue, and ng TCDD/g tissue. Maternal body burdens were estimated based on analysis of dioxin in the following maternal tissues: liver, adipose, skin, and muscle. It was assumed that >90% of the body burden was due to the amount of TCDD present in these tissues.

Statistical methods.
Intergroup comparisons were performed by a one-way analysis of variance (ANOVA) (Statview, Abacus Concepts, Inc., Berkeley, CA). When statistically significant effects were detected in the overall analysis of variance, means were compared using Scheffe's F test. When data were collected on more than one fetus per litter, the data were analyzed using litter means rather than individual fetus values. Differences between treatment groups were considered significantly different when p < 0.05. All data in the tables are presented as means ± standard deviations.

Model fitting.
This study was designed to evaluate the hypothesis that fetal tissue concentration of TCDD is sufficient to predict the intensity of developmental abnormalities. The approach involved incidences of various abnormalities for a range of maternal doses administered on GD15 and a single dose administered on GD8. In separate experiments, estimates of maternal and fetal TCDD concentrations were determined for the same administered dosages. A dose-response model was designed for each of four developmental abnormalities (ejaculated sperm counts and delayed puberty in males and urethra-phallus distance and incidence of vaginal thread in females), which related the incidence of the endpoint after GD15 dosing to fetal tissue concentration. These four endpoints were selected because they were consistently measured at the same postnatal time point in each of the papers by Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo). In addition, these responses were the most severely affected, which enabled a comparison of tissue concentration and adverse effects to be made. The question was then asked whether tissue concentration following GD8 exposure results in the incidence of developmental effects as predicted by the dose-response model.

The appropriate transformation for the dependent variable (developmental abnormality) was determined to make the variances homogeneous and unrelated to the group means and to make the population residuals within the groups approximately normally distributed. The dependent variable was first examined on its original scale, followed by log-square-root and three-quarters-power–transformed. Normality was checked by examining normal score plots of the residuals after subtracting off the group means, looking at graphs of within-group standard deviations versus group means, and boxplots of residuals for each group. As soon as a transformation was found that provided a satisfactory residual distribution and standard deviation to mean relationship, the search ended.

The fitted models took into account the two blocks in the study (group 1: GD15 doses 0–0.8 µg TCDD/kg; group 2: control and 1.0 µg TCDD/kg on GD8 and GD15). The full models always had the following form for the dependent variable yg in group g and independent variable t (where either y or t, or both, may be transformed): if g is 1, y1 = a1 + bt; if g is 2, and data are for GD15, y2 = a2 + bt; if g is 2 and the data are for GD8, y2 = a2 + {delta} + bt. This allows for the possibility that control values may differ between the two blocks, and fits a common slope to the values in both blocks. The parameter {delta} is an estimate of the difference between the mean GD8 value and the value predicted by the model for the GD15 data. Its value should be zero if tissue concentration is all that is needed to predict an outcome. The log-likelihood value for the various fitted models relative to that derived from the model in which each distinct dose and gestation group has a separate mean is used as a goodness-of-fit test.

All continuous endpoints were fit using data at the level of the individual pup or fetus. To accommodate the nested nature of the design (pups within litters), linear mixed effects models (Lindstrom and Bates, 1988Go) were fit by maximum likelihood using the function lme in Splus (version 3.4, Mathsoft Corporation, Seattle, Washington).

The variable vaginal thread is a dichotomous variable, so the above procedure was modified somewhat. To accommodate the nested design, the Rao-Scott transformation was used (Rao and Scott, 1992Go; Krewski and Zhu, 1995Go) and logistic models were fit using the Splus function glm. This has been shown to be an efficient procedure (Fung et al., 1998Go) and is simpler to implement than other approaches to nested design. Methods appropriate to measurement error models are formally appropriate for these data, as the mean tissue concentrations are measured with error. However, the proportion of the overall spread of tissue concentrations that is attributable to measurement error is so small that we have ignored this effect.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maternal TCDD Tissue Concentrations
GD16.
For all doses, the greatest amount (% dose/tissue) of radioactivity in the dam was found in the liver, followed by adipose tissue. The concentration in liver ranged from 369 to 7136 pg TCDD/g following exposure to 0.05–1.00 µg TCDD/kg (Table 1Go), representing 30–47% of the total dose. Adipose contained approximately 7–10% of the administered dose at all doses, which resulted in tissue concentrations of 65–1018 pg TCDD/g. There was a dose-dependent increase in liver to fat ratios following exposure to 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg, respectively (Table 1Go). It is interesting to note that 24 h after exposure, adipose TCDD concentrations have not reached their maximum due to its slow rate of perfusion (Wang et al., 1997Go). There was a dose-dependent decrease in the relative amount of TCDD (% dose/tissue) present in skin and muscle (p < 0.05), a pattern similar to that observed in adipose tissue. The concentration in blood ranged from 2 to 51 pg TCDD/g. Maternal body burdens were 30.6 ± 3.1, 97.4 ± 23.2, 522.8 ± 29.6, and 585.2 ± 98.3 ng TCDD/kg following exposure to 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg, respectively. One day after administration of 1.0 µg TCDD/kg on GD15, 23.8% of the administered dose was eliminated in the feces (73.8 ± 24.8 ng).


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TABLE 1 Distribution of TCDD in Maternal Tissues after GD15 Exposure
 
GD21.
The largest amount of TCDD was still in the maternal liver on GD21 after exposure to 1.0 µg TCDD/kg on GD15 (27% of administered dose). Adipose also contained large amounts of TCDD, which ranged from 16% of the administered dose at an exposure of 1.0 µg TCDD/kg to 37% at the low dose (Table 1Go). These amounts resulted in adipose tissue concentrations of 177 and 1525 pg TCDD/g, respectively. Furthermore, adipose TCDD concentrations increased from GD16 to GD21 (Table 1Go). Another tissue containing relatively large amounts of TCDD was the skin. At all doses examined, the skin contained approximately 4% of the administered dose. At doses of 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg, the following concentrations were found: 34, 56, 318, and 348 pg TCDD/g, respectively. Maternal blood contained 1 pg TCDD/g at the lowest dose and 23 pg TCDD/g at the highest dose. The dose-dependent relative decrease in extrahepatic tissue concentration is still apparent. Maternal body burdens were 26.6 ± 2.7, 76.2 ± 16.7, 327.8 ± 59.3 and 431.1 ± 60.0 ng TCDD/kg following exposure to 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg, respectively. This indicates that some of the delivered material has been eliminated between GD16 and GD21.

Fetal TCDD Tissue Concentrations
GD16.
A dose of 0.05 µg TCDD/kg resulted in a concentration of 5 pg/g in a single fetus (0.02% of administered dose) (Table 2Go). This increased to 56 pg/g after exposure to 1.0 µg TCDD/kg. Individual fetuses were subdivided into liver, urogenital tract, head, and body to determine whether TCDD was preferentially sequestered within a fetus. A dose of 0.05 µg TCDD/kg produced a concentration of 4 pg/g in fetal liver, 5 pg/g in a head, 6 pg/g in a body, and 4 pg/g in the urogenital tract (Table 3Go). At a dose of 0.20, 0.80, and 1.0 µg TCDD/kg, there was approximately 14, 38, and 52 pg TCDD/g within each of the three fetal tissues (head, body, and urogenital tract), respectively. To determine the amount of TCDD reaching the entire fetal compartment, TCDD levels in all the fetuses and placentas were summed. At the low dose (0.05 µg/kg), the fetal compartment contained 0.5% of the administered dose, which resulted in a concentration of 7 pg TCDD/g (Table 2Go). At the higher doses (0.2, 0.8, and 1.0 µg/kg) the fetal compartment contained 0.3, 0.2, and 0.2% of the administered dose, respectively.


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TABLE 2 Distribution of TCDD to Fetus after GD15 Exposure
 

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TABLE 3 Distribution of TCDD in Different Fetal Tissues
 
GD21.
A dose of 0.05 µg TCDD/kg resulted in a concentration of 4 pg TCDD/g in a single fetus (0.03% of administered dose) (Table 2Go). At a dose of 0.05 µg TCDD/kg, total fetal liver contained 0.01% dose, which resulted in a tissue concentration of 4.8 pg TCDD/g (Table 3Go). Individual urogenital tracts contained only 0.5 pg (6.5 pg/g) in rats prenatally exposed to 0.05 µg TCDD/kg. An exposure of 0.20 µg TCDD/kg produced the following concentrations: 14, 14, 18, and 11 pg TCDD/g in head, liver, urogenital tract, and placenta, respectively. After a dose of 0.80 µg TCDD/kg, the urogenital tract contained 0.001% dose at a concentration of 50 pg TCDD/g. This concentration had increased to 58 pg TCDD/g after a single dose of 1.0 µg TCDD/kg. Exposure to 0.05 µg TCDD/kg resulted in 2.3% of the administered dose in the fetal compartment (Table 2Go). This resulted in a concentration of 4.3 pg TCDD/g. With increasing dose, there was a dose-dependent decrease in the relative amount (% dose/tissue) present in the fetal compartment. The concentration in the fetal compartment ranged from 4.3 to 37.4 pg TCDD/g following doses of 0.05–1.0 µg TCDD/kg on GD15 (Table 2Go).

Dose-Response Relationships
Although the administered dose is often associated with a response, the body burden may be a more appropriate dose metric for a persistent, poorly metabolized chemical such as TCDD. Gray and coworkers conducted several studies in which the reproductive and developmental effects of gestational TCDD-exposure were assessed. Specifically, Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go) examined the adverse effects in pups exposed to a single dose of 1.0 µg TCDD/kg on GD8 or GD15. In addition, they conducted a dose-response study in which the dams received 0, 0.05, 0.20, or 0.80 µg TCDD/kg on GD15 and the adverse effects in the pups were measured (Gray et al., 1997aGo; Gray et al., 1997bGo). Therefore, using response data from Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo), GD16 fetal tissue concentrations, which were measured in the present study, were plotted to relate tissue concentration to the incidence of developmental effect after similar exposures (Figs. 1–4GoGoGoGo). Note: All the statistical work was based on scaling the tissue concentrations following GD8 exposure by 1/1.15 (Hurst et al., 1998Go), as GD8 developmental results are based on 1.00 µg TCDD/kg (Gray et al., 1995Go; Gray and Ostby, 1995Go). On GD16, the model adequately predicts the response (sperm count and day of puberty in males; urethra-phallus distances and incidence of vaginal thread in females) associated with the concentration of TCDD in a single fetus after different acute exposures. For example, a dose of 1.15 µg TCDD/kg on GD8 produced a concentration of 16 pg/g on GD16 in a single fetus (Hurst et al., 1998Go). This tissue concentration (16 pg/g) predicts urethra-phallus distance in female pups based on GD16 tissue concentrations following GD15 administration (Fig. 3Go). In addition, fetal TCDD concentration predicts ejaculated sperm counts and day of puberty in males (Figs. 1 and 2GoGo). However, GD8 exposure to 1.15 µg TCDD/kg slightly underpredicts the incidence of vaginal thread formation (Fig. 4Go). This may be due to the large variability in the measurement of this response.



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FIG. 1. Percent decrease in ejaculated sperm count plotted versus estimated mean fetal TCDD concentration on GD16. ({blacksquare}) Time-pregnant LE rats administered 1.15 µg TCDD/kg on GD8. ({circ}) Time-pregnant LE rats administered 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg on GD15. There was no significant difference between the magnitude of response after GD8 exposure versus GD15 exposure based on fetal TCDD concentration (p = 0.80). Horizontal and vertical bars represent 95% confidence intervals. The equation describing the line was {surd}y = 10.88 – 0.0855(x). Response data following GD8 or GD15 exposure to TCDD are from the laboratory of Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo). Tissue concentrations after GD8 exposure to 1.15 µg TCDD/kg are from Hurst et al. (1998).

 


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FIG. 2. Puberty delay in males plotted versus estimated mean fetal concentration on GD16. ({blacksquare}) Time-pregnant LE rats administered 1.15 µg TCDD/kg on GD8. ({circ}) Time-pregnant LE rats administered 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg on GD15. Age at puberty modeled was adequately modeled as a linear function of tissue concentration. There was no significant difference between the magnitude of response after GD8 exposure versus GD15 exposure based on fetal TCDD concentration (p = 0.11). Horizontal and vertical bars represent 95% confidence intervals. The equation describing the line was y = 40.48 + 0.0697(x) and y = 40.48 – 0.563 + 0.0697(x). Response data following GD8 or GD15 exposure to TCDD are from the laboratory of Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo). Tissue concentrations after GD8 exposure to 1.15 µg TCDD/kg are from Hurst et al. (1998).

 


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FIG. 3. Female urethra-phallus distance plotted versus log fetal tissue concentration on GD16. ({blacksquare}) Time-pregnant LE rats administered 1.15 µg TCDD/kg on GD8. ({circ}) Time-pregnant LE rats administered 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg on GD15. Urethra-phallus distance was modeled as a quadratic function in log-tissue concentration. There was no significant difference between the magnitude of response after GD8 exposure versus GD15 exposure based on fetal TCDD concentration (p = 0.81). Horizontal and vertical bars represent 95% confidence intervals. The equation describing the line was log(y) = –0.2689 – (0.005163 * log(x)) + (0.07668 * log(x2)). Response data following GD8 or GD15 exposure to TCDD are from the laboratory of Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo). Tissue concentrations after GD8 exposure to 1.15 µg TCDD/kg are from Hurst et al. (1998).

 


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FIG. 4. Incidence of vaginal thread plotted versus log tissue concentration on GD16. ({blacksquare}) Time-pregnant LE rats administered 1.15 µg TCDD/kg on GD8. ({circ}) Time-pregnant LE rats administered 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg on GD15. The incidence of vaginal thread was modeled as a logistic function of the log of the tissue concentration. There was a significant difference between the magnitude of response after GD8 exposure versus GD15 exposure based on fetal TCDD concentration (p = 0.03). Horizontal and vertical bars represent 95% confidence intervals. The equation describing the line was

Response data following GD8 or GD15 exposure to TCDD are from the laboratory of Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo). Tissue concentrations after GD8 exposure to 1.15 µg TCDD/kg are from Hurst et al. (1998).

 
Dose-response relationships were also plotted using GD21 urogenital tract TCDD concentrations. Again, the response data was obtained from work done by Gray and coworkers under similar conditions (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo). Figure 5Go illustrates that concentrations of TCDD in urogenital tract adequately predict TCDD`s affect on puberty delay in male pups. Because GD21 is already past the peak window of sensitivity for this effect, tissue concentrations on this day may not accurately predict the responses studied. It appears that tissue concentration measured during a period of peak sensitivity is an appropriate dose metric to predict the magnitude of a response. Obviously, tissue concentration after the window of sensitivity (e.g., GD21) would not be the appropriate dose metric, as exposure occurred after the critical period.



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FIG. 5. Mean puberty delay in males plotted versus mean urogenital tract TCDD concentrations on GD21. ({blacksquare}) Time-pregnant LE rats administered 1.15 µg TCDD/kg on GD8. ({circ}) Time-pregnant LE rats administered 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg on GD15. Urogenital tract concentration was modeled as a linear function of tissue concentration. There was no significant difference between the magnitude of response after GD8 exposure versus GD15 exposure based on fetal TCDD concentration (p = 0.17). Horizontal and vertical bars represent 95% confidence intervals. The equation describing the line was y = 40.51 + 0.0605(x). Response data following GD8 or GD15 exposure to TCDD are from the laboratory of Gray and coworkers (Gray et al., 1995Go; Gray and Ostby, 1995Go; Gray et al., 1997aGo; Gray et al., 1997bGo). Tissue concentrations after GD8 exposure to 1.15 µg TCDD/kg are from Hurst et al. (1998).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Developmental and reproductive studies are useful in assessing toxicity; however, one drawback is that many do not provide information on the concentration of the chemical found in target tissues. In the current study, tissue concentrations of TCDD in maternal and fetal LE rat tissues were examined after low-dose, acute exposure and related to developmental effects reported previously by Gray and coworkers. Concentrations of TCDD in fetal urogenital tract on GD21 were 3.8, 17.5, 50.0, and 58.3 pg/g after GD15 administration of 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg, respectively. A concentration of 17.5 pg TCDD/g after exposure to 0.20 µg TCDD/kg in this study was associated with a 27% incidence of a vaginal thread in females, as well as delayed puberty and reduction in sperm counts of males (Gray et al., 1997aGo; Gray et al., 1997bGo). A dose as low as 0.05 µg TCDD/kg produced fetal tissue concentrations of 3.8 pg/g, and this was associated with premature eye opening and reduction in ejaculated sperm counts (Gray et al., 1997aGo; Gray et al., 1997bGo). Several studies have examined fetal tissue concentration following different exposure paradigms; however, less is known about the effect of tissue concentration and the intensity of the response. Li et al. (1995) reported that a dose of 5.6 µg TCDD/kg on GD18 resulted in fetal liver concentrations of 0.01% dose/g on GD19 and GD20. Although developmental responses were not measured, this concentration is similar to those obtained in our studies (Table 3Go). In addition, Faqi and coworkers (1998) examined maternal and fetal TCDD levels following low-dose exposure. However, fetal TCDD levels were below their limit of detection, which makes comparisons between the studies difficult.

Another interesting finding is the nonlinear distribution of TCDD within maternal and fetal tissues. Table 1Go illustrates a dose-dependent increase in the maternal liver/adipose ratio, indicating hepatic sequestration. This is due to the induction of a hepatic binding protein, CYP1A2, by TCDD (Diliberto et al., 1997Go; Santostefano et al., 1996Go). A dose-dependent increase in the liver to fat ratio implies that at lower doses there is relatively more of the chemical available for extrahepatic tissues. For example, a dose of 0.05 µg TCDD/kg results in 0.51% of the administered dose present in the fetal compartment, as opposed to 0.17% administered dose after exposure to 1.0 µg TCDD/kg (Table 2Go). Therefore, it is important to consider that many high-dose animal studies may underpredict extrahepatic responses at low doses.

It is important to understand the disposition of a chemical to target tissues, as this information is crucial in understanding the relationship between tissue concentration and the response. The present study investigated whether tissue concentration was an appropriate dose metric to predict adverse reproductive and developmental effects. In addition, other dose metrics should be examined for use in species extrapolation. For example, lifetime area under the curve (AUC), body burden, tissue concentration, average blood concentration, or daily dose provide a means to estimate the risk of human exposure to these compounds. For chemicals with a mechanism of action that depends on maintaining a critical tissue or blood concentration for a specific duration to elicit a toxic response, AUC is probably the appropriate dose metric (Benet et al., 1996Go). However, for adverse effects due to prenatal exposure, it is important to consider the critical time at which exposure occurs. Birnbaum and coworkers (1985) demonstrated that the peak period for TCDD-induced effects on palate formation in C57BL/6N mice occurs between GD11 and GD12. In addition, a single dose of 24 µg TCDD/kg administered to pregnant C57BL/6N mice increased the incidence of prenatal mortality on GD6 but did not increase mortality on GD8, 10, 12, or 14 (Couture et al., 1990Go). This suggests that the sensitive window for fetal lethality in mice occurs on or before GD6. For this reason, tissue concentration during this critical period may be a better indicator of embryo toxicity.

Results from two different exposures during gestation (GD8 and GD15) show that fetal tissue concentration better predicts the intensity of the developmental abnormality as compared to administered dose. For example, Figs. 1–4GoGoGoGo clearly show that the model predicts the change in ejaculated sperm counts and puberty delay in males and urethra-phallus distance and vaginal thread incidence in females based on fetal TCDD concentration on GD16 following exposure on GD8 or GD15. Although fetal TCDD tissue concentrations on GD16 following GD8 exposure slightly underpredict the incidence of vaginal thread, the discrepancy may be attributed to a block effect in the design of the reproductive studies or to the fact that GD16 is past the critical window of sensitivity for this effect. It is important to understand that tissue concentration at a critical period during gestation is the appropriate dose metric. In contrast, administered dose fails to predict the severity of the response. For example, administration of 1.0 µg TCDD/kg on GD8 produces a 14% incidence of vaginal thread in female LE pups (Gray and Ostby, 1995Go) (Table 4Go). However, the same dose administered on GD15 produces 79% incidence of vaginal thread (Gray and Ostby, 1995Go). Therefore, administered dose poorly predicts these reproductive and developmental responses. Although other dose metrics (AUC, body burden, and blood concentration) may also be used to predict risk, results from this study clearly show that fetal tissue concentration measured during a critical period is a better measure than administered dose.


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TABLE 4 Effects of GD8 or GD15 Exposure to 1.0 µg TCDD/kg on LE Rat Offspring
 
For most of the responses examined, the critical window appears to be around GD16. However, the model prediction for urogenital tract concentration and puberty delay in males was better on GD21 than on GD16 (data not shown). This indicates that the maximum period of sensitivity for this effect may be closer to GD21. For other responses, the critical window may occur prior to TCDD administration. For this reason, TCDD tissue concentrations measured after the critical time point would be inappropriate to predict the response. Furthermore, tissue concentrations measured after the peak window of sensitivity may underpredict the response.

For the reproductive and developmental effects examined in this study, fetal TCDD concentrations accurately predict the overall severity of the response. However, the usefulness of analyzing fetal concentrations is questionable in terms of assessing human health risk. Therefore, a more realistic approach to assess exposure is to determine concentrations of dioxin and dioxinlike compounds in maternal tissues or serum. On GD21, maternal body burdens were 27–431 ng TCDD/kg following exposure to 0.05–1.0 µg TCDD/kg on GD15. These calculations are based on the assumption that 90% of the residue is located in liver, adipose, muscle, and skin. Although these types of calculations can be done in human populations, a more realistic approach would be to analyze serum concentrations of dioxinlike chemicals. For this reason, we explored the relationship between maternal and fetal TCDD tissue concentrations. On GD16 there was a significant correlation between fetal body burden and maternal body burden (1:9; r2 = 0.91, p < 0.0001). In addition, there was a strong correlation between fetal body burden and maternal blood levels (1.8:1; r2 = 0.932, p < 0.0001). These data suggest that a measurement of maternal blood levels at a critical time provides a means to estimate concentrations of dioxin within the developing fetus.

Most people are exposed to low levels of dioxins and dioxinlike compounds, which are found in the food supply. The background body burden of PCDD/PCDF/PCB in humans is 9–13 ng TEQ/kg body weight (DeVito et al., 1995Go). However, dioxin body burdens in humans are log-normally distributed and for this reason, it is likely than certain individuals contain greater concentrations (DeVito et al., 1995Go; Sielkin, 1977). Several epidemiologic studies suggest a correlation between TCDD exposure and adverse outcomes. For example, a body burden of 109-7000 ng TCDD/kg is associated with increased risk of cancer (Bertazzi et al., 1993Go; Fingerhut et al., 1991Go). In addition, decreased birth weights and delayed developmental milestones are associated with a maternal body burden of 2130 ng TEQ/kg (Chen et al., 1992Go; DeVito et al., 1995Go; Lucier, 1991Go; Rogan et al., 1988Go). Maternal body burdens slightly above background (16 ng TEQ/kg) are associated with lower birth weight, lower psychomotor scores, and poorer neurologic condition (Huisman et al., 1995aGo; Huisman et al., 1995bGo; Koopman-Esseboom et al., 1994Go; Koopman-Esseboom et al., 1996Go; Patandin et al., 1998Go). The lowest dose used in this study (0.05 µg TCDD/kg) resulted in a maternal body burden of 27 ng TCDD/kg on GD21, which is associated with accelerated eye opening and a reduction in sperm counts of male offspring (Gray et al., 1997aGo). This body burden is only 2–3 times higher than the average background TEQ in U. S. populations.

In conclusion, TCDD induces a wide range of adverse effects in experimental animals. Epidemiologic studies indicate that humans are similar to animals in their response to dioxins, such as diminished immune function and growth and development, and carcinogenesis (Birnbaum, 1994bGo). Results from our study indicate that fetal tissue concentrations at a critical period of sensitivity provide a means to assess the potential for the development of adverse effects. Furthermore, concentrations of TCDD in the developing fetus are highly correlated with concentrations found in maternal blood. This indicates that maternal concentrations of TCDD would provide a means to determine fetal exposure to dioxin and the potential effects associated with this exposure. A better understanding of the relationship between tissue concentration of TCDD and the development of adverse outcomes may facilitate the ability to predict whether human populations are at risk for effects associated with low-level exposure to TCDD.


    ACKNOWLEDGMENTS
 
The authors thank David Ross, Vicki Richardson, and Frances McQuaid for their excellent technical assistance in this project and Drs. Mike Hughes and Barbara Abbott (USEPA) for their helpful review of the manuscript.


    NOTES
 
1 To whom correspondence should be addressed at U. S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Experimental Toxicology Division, Pharmacokinetics Branch, Mail-Drop 74, Research Triangle Park, NC 27711. Fax: (919) 541-5394. E-mail: hurst.christopher{at}epamail.epa.gov. Back

The research described in this article has been funded in part by the U. S. Environmental Protection Agency Cooperative Training Agreement (ES07126) with the University of North Carolina at Chapel Hill, NC 27599–7270. The manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or use recommendation.


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