Pharmacokinetics of Ovarian Steroids in Sprague-Dawley Rats After Acute Exposure to 2,3,7,8-Tetrachlorodibenzo- P-Dioxin (TCDD)

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Vol. 3, No. 2 131

ORIGINAL PAPER

Pharmacokinetics of ovarian steroids in Sprague-Dawley rats after acute exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)

Brian K. Petroff 1,2,3 and Kemmy M. Mizinga4 2Department of Molecular and Integrative Physiology,Physiology, 3Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, KS 66160. 4Department of Pharmacology,Pharmacology, University of Health Sciences, Kansas City,City, MO 64106 Received: 3 June 2003; accepted: 28 June 2003

SUMMARY

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces abnormalities in steroid-dependent processes such as mammary cell proliferation, gonadotropin release and maintenance of pregnancy. In the current study, the effects of TCDD on the pharmacokinetics of 17ß-estradiol and progesterone were examined. Adult Sprague-Dawley rats were ovariectomized and pretreated with TCDD (15 µg/kg p.o.) or vehicle. A single bolus of 17ß-estradiol (E2,

0.3 µmol/kg i.v.) or progesterone (P4, 6 µmol/kg i.v.) was administered 24 hours after TCDD and blood was collected serially from 0-72 hours post- injection. Intravenous E2 and P4 in DMSO vehicle had elimination half-lives of approximately 10 and 11 hours, respectively. TCDD had no signifi cant effect on the pharmacokinetic parameters of P4. The elimination constant

1Corresponding author: Center for Reproductive Sciences, Department of Molecular and Integra- tive Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA; e-mail: [email protected]

and clearance of E2 were decreased by TCDD while the elimination half-life, volume of distribution and area under the time*concentration curve were not altered signifi cantly. Overall, these results indicate that diminished serum progesterone and estradiol concentrations following exposure to TCDD are due primarily to actions on steroid synthesis and release rather than any alterations in pharmacokinetics. Reproductive Biology 2003 3 (2): 131-141. Key words: TCDD, dioxin, ovary, progesterone, estradiol, pharmacokinetics

INTRODUCTION

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to block ovulation through alterations in preovulatory gonadotropin release [4, 5, 15, 17] and has direct actions on the ovary as well [3, 4, 8, 9, 13, 14, 15, 20]. This blockade of ovulation following exposure to TCDD is accompanied by aberrations in serum concentrations of ovarian steroids [17]. This is in agreement with other studies that have found decreases in serum estradiol and progesterone following acute exposure to TCDD [13, 14, 17]. Decreased steroid concentrations induced by TCDD may be due to alterations in gonadotropic support of ovarian steroidogenesis [17], direct impact on steroid synthesis [3, 8, 9, 13, 14] or altered distribution, metabolism and elimination [16]. In this study, we tested the effects of TCDD on the pharmacokinetics of intravenously administered 17β-estradiol

(E2) and progesterone (P4) in ovariectomized adult rats.

MATERIALS AND METHODS

Animals and Treatments

Adult female Sprague-Dawley rats (80-100 days old, 250-350 g, Charles River Laboratories, USA) were supplied by a commercial vendor and housed under scheduled lighting (12:12 hours light:dark) and provided with rodent chow (Purina, USA) and water ad libitum. Chemicals were supplied by Sigma (St. Louis, MO, USA) unless stated otherwise. TCDD was graciously donated by Dr. Karl K. Rozman (University of Kansas Medical Center). Petroff and Mizinga 133

Animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center. Rats were ovariectomized under a surgical plane of anesthesia (5 mg xy- lazine and 50 mg ketamine/kg i.p., Abbott Laboratories, North Chicago, IL, USA) via a paralumbar approach. A jugular cannula was placed to the level of the right atrium, fl ushed with heparinized saline and fi lled with a heparinized glycerol (50 IU/ml) lock solution to maintain cannula patency during the 3 day recovery period. At the end of this period rats (n = 4 per group) were pretreated with TCDD (15 µg/kg p.o.) or corn oil vehicle and 24 hours later received a single dose of E2 (0.3 µmol/kg i.v.) or P4 (6 µmol/kg i.v.) in DMSO vehicle. Steroids were delivered in 200 µl of DMSO (approximately 0.8 ml DMSO/kg i.v.), a dose below the commonly used therapeutic range for this compound [1]. These doses of E2 and P4 were chosen based on the administration of estradiol cypionate and P4 in previous studies [5, 15, 16] and were intended to provide steroid concentrations across the physiological range. The dose of TCDD chosen induced alterations in steroid concentrations in previous studies [15, 16, 17]. Blood (100-300 µl) was collected at 0, 0.25, 0.75, 2.5, 7.5, 24 and 72 hours post-injection. Blood removal was limited to less than 5% of estimated total blood volume and hematocrit was measured following each blood collection to insure that iatrogenic anemia was avoided. Serum was stored at –20oC until hormone measurement by previously validated radioimmunoassay.

Radioimmunoassay

Serum concentrations of E2 and P4 were measured by specifi c radioimmunoassay following extraction with diethyl ether as described previously [16, 27]. Both intrassay and interassay coeffi cients of variation were less than 10% for progesterone and estradiol.

Data Analysis

Serum E2 and P4 concentration data were subjected to noncompartmental analysis for initial determination of pharmacokinetic parameters. Briefl y, 134 TCDD and ovarian steroid pharmacokinetics

area under the concentration vs. time curve (AUC0-inf) andand aarearea uundernder tthehe fi r srstt moment curve (AUMC0-inf) werewere calculatedcalculated usingusing thethe linearlinear trapezoidaltrapezoidal rulerule [19, 29] applied through the computer program “PK Functions for Microsoft Excel” (J.I. Usansky et al., Dept. of Pharmacokinetics and Drug metabolism,

Allergan, Irvine, CA). Half-life (t1⁄2) and elimination rate constants (Kelim) were calculated by regression of the semi-logarithmic concentration vs. time data [7] using the same computer program. Total systemic clearance (CL), calculated from Dose/AUC0-inf, meanmean residenceresidence timetime (MRT),(MRT), calculatedcalculated fromfrom

AUMC/AUC, and steady state volume of distribution (Vss), calculated from MRT*CL and other noncompartmental pharmacokinetic parameters [22] were evaluated but not reported because they yielded similar conclusions to those based on subsequent compartmental analysis. Serum E2 and P4 concentration data were then subjected to compartmental analysis to obtain micro-rate constants and volumes of distribution. Both E2 and P4 concentration vs. time curves exhibited bi-exponential decline best described by two- compartment models represented by the equation: C = Ae-αt + Be-βt (where

C = concentration of E2 or P4 in serum, t = time, A = ordinate intercept of distribution curve, α = hybrid rate constant (slope) of distribution curve, B = ordinate intercept of elimination curve, β = hybrid rate constant (slope) of elimination curve) [2, 11]. Volume of distribution (Vd) was calculated from Dose/(A+B) [30]. Serum steroid concentrations were analyzed by ANOVA for a repeated measures design. Effects tested included time and TCDD and their interaction. Comparisons yielding p≤0.05 were considered signifi cant. The elimination half-life, elimination constant, volume of distribution and clearance of P4 or E2 in vehicle and TCDD-treated rats following administration of E2 or P4 were compared using a student t –test.

RESULTS

17ß-estradiol

Concentrations of E2 in serum as measured by radioimmunoassay rose abruptly following intravenous administration of E2, reaching a measured peak of approximately 7000 pg/ml at 15 minutes post-administration Petroff and Mizinga 135

Fig. 1. Serum concentrations of 17β-estradiol (E2, log pg/ml) in ovariectomized rats receiving TCDD (15 µg/kg p.o.) or vehicle followed by E2, (n = 4).

(fi g. 1). Data from one rat in the control group were excluded due to cannula failure. E2 was undetectable in these animals by 72 hours post-administration and was eliminated with an elimination half-life of 8-13 hours for E2. While the calculated elimination constant and clearance of E2 were decreased in TCDD-treated animal, the dioxin had no signifi cant impact on the elimination half-life, area under the curve and volume of distribution of serum E2 following the intravenous administration of estradiol-17ß (tab. 1).

Progesterone

Serum P4 rose abruptly following intravenous administration in DMSO vehicle and reached a measured peak of approximately 400 ng/ml by 15 136 TCDD and ovarian steroid pharmacokinetics

Table 1. Pharmacokinetic parameters of serum estradiol following intravenous administration of 17ß-estradiol to ovariectomized female rats pretreated with TCDD or vehicle

Control TCDD (15 µg/kg p.o.) Mean SEM Mean SEM

t1/2 8.48 3.63 13.81 0.84 AUC (pg*h/ml) 17848 4197 22300 2106 CL(ml/h) 1613 574 986* 83 -1 Kelim (hr ) 0.11 0.04 0.051* 0.003

Vd (l/kg) 11.01 0.65 11.39 0.38 t1/2 = half life; AUC = area under curve; CL = clearance; Kelim = eliminationelimination cconstant;onstant;

TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; Vd = volume of distribution *a signifi cant (p<0.05) difference between controls and TCDD-treated animals

minutes post-administration (fi g. 2). The elimination half-life of intravenously administered P4 in this model was approximately 11 hours during the terminal phase. Pretreatment with TCDD was not associated with any signifi cant changes in the pharmacokinetic parameters of P4 (tab. 2) including terminal half-life, AUC, clearance, elimination constant and volume of distribution.

DISCUSSION

This is the fi rst study to directly address the impact of exposure to an aryl hydrocarbon receptor (AhR) agonist on the pharmacokinetics of ovarian steroids. This is of interest because TCDD and other toxicants thought to act through the AhR have profound effects on the female reproductive system including alterations in profi les of serum hormones [4, 5, 6, 12, 15, 16, 17, 20] and defects in steroid-dependent processes such as gonadotropin release and maintenance of pregnancy [6, 17]. The results of the current study found no signifi cant effect of pretreatment with TCDD on the pharmacokinetic profi le of progesterone following intravenous administration of the steroid Petroff and Mizinga 137

Fig. 2. Serum concentrations of progesterone (P4, log ng/ml) in ovariectomized rats receiving TCDD (15 µg/kg p.o.) or vehicle followed by P4, (n = 4). to ovariectomized rats. While the clearance and calculated elimination constant of estradiol were lower in TCDD-treated rats (indicating a potential prolongation of administered E2 in the serum) no other pharmacological parameters were altered signifi cantly for the estradiol. The AhR gene battery as characterized to date includes at least one enzyme (CYP1A1) involved in the hepatic metabolism of estradiol and progesterone [18]. Activation of this pathway by TCDD failed to signifi cantly alter serum estradiol in the pregnant rat [24]. Nevertheless, TCDD and re- lated compounds have profound effects on hepatic function and serum lipid composition [18, 25] and have been shown to alter peripheral metabolism of estradiol in target tissues such as the breast [26]. These effects in themselves can potentially alter the elimination of ovarian steroids but do not appear to signifi cantly hasten the metabolism of estradiol and progesterone based 138 TCDD and ovarian steroid pharmacokinetics

Table 2. Pharmacokinetic parameters of serum progesterone following intravenous administration of progesterone to ovariectomized female rats pretreated with TCDD or vehicle

Control TCDD (15 µg/kg p.o.) Mean SEM Mean SEM

t1/2 10.25 0.81 11.69 1.37 AUC (ng*h/ml) 1862 420 1750 201 CL (ml/h) 342.61 99.05 308.68 35.65 -1 Kelim (hr ) 0.069 0.005 0.062 0.008

Vd (l/kg) 5.62 1.36 5.01 0.45 t1/2 = half life; AUC = area under curve; CL = clearance; Kelim = eliminationelimination cconstant;onstant;

TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; Vd = volume of distribution There were no signifi cant effects of TCDD on calculated or observed endpoints.

on the results of the current study. TCDD did appear to actually prolong the persistence of E2 in the serum as indicated by a decreased clearance and elimination constant although further study is needed to establish a mechanism for this effect. One possibility is that the lipemia characteristic of

TCDD exposure [18, 25] resulted in increased binding of E2 to lipoproteins resulting in decreased availability to microsomal enzymes. The interaction between TCDD and estrogens, in particular, is complex. Estrogens have been shown to prolong the half-life of TCDD in the mouse, probably through retardation of biliary excretion [28]. Exogenous estrogens can promote the systemic toxicity (i.e. catabolic effects) of TCDD in the rat [15] while antagonizing the actions of the dioxin on estrogen-responsive tissues such as the uterus, mammary gland, ovary [17, 23], adenohypophysis and hypothalamus [5, 17]. TCDD, in turn, has well characterized estrogen modulatory effects for many estrogen responsive endpoints [10, 21, 23] and has been shown to decrease the synthesis of estradiol using both in vivo and in vitro approaches in several species [3, 8, 9, 13, 14, 21]. A single previous study addressed the impact of TCDD on hepatic microsomal enzyme activity in pregnant female rats and found that while catechol estrogen formation Petroff and Mizinga 139 was increased by exposure to TCDD, estradiol concentrations were not altered signifi cantly by this reaction [24]. In the current study, data suggested no effect or possibly a decrease in estradiol metabolism following acute exposure to TCDD. While one of these experimental groups (E2 controls) had decreased animal numbers due to cannula failure, data clearly argue against increased metabolism of E2 as a mechanism leading to decreased serum E2. Overall, the results of the current study fail to find a significant impact of exposure to TCDD on the distribution and excretion of progesterone administered intravenously to adult rats and TCDD may have been associated with diminished E2 elimination although further study is needed to verify this fi nding. Any decrease in serum concentrations of estradiol and progesterone following exposure to TCDD probably involve direct actions on ovarian steroidogenesis or modulation of pituitary gonadotropin release rather than alterations in the pharmacokinetics of these ovarian steroids.

ACKNOWLEDGEMENTS

This work was supported by grants from the National Institute of Health (NIH1F32ES05880, BKP), and the Mellon Foundation (BKP). The authors would also like to thank Drs. Xin Gao, Karl K. Rozman and Paul F. Ter- ranova for their assistance during this study.

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