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Evidence for Ah Receptor Mediation of Increased ACTH Concentrations in Primary Cultures of Rat Anterior Pituitary Cells Exposed to TCDD
TOXICOLOGICAL SCIENCES ARTICLE NO.
46, 294 –299 (1998)
TX982548
Evidence for Ah Receptor Mediation of Increased ACTH Concentrations in Primary Cultures of Rat Anterior Pituitary Cells Exposed to TCDD Lorelle L. Bestervelt,* Jeff A. Pitt,† and Walter N. Piper‡,1 *Toxicology Department, NSF International, Ann Arbor, Michigan 48105; †Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599; and ‡Toxicology Program, School of Public Health, Department of Pharmacology, Medical School and Reproductive Sciences Program, The University of Michigan, Ann Arbor, Michigan 48109-2029 Received December 30, 1997; accepted July 23, 1998
Evidence for Ah Receptor Mediation of Increased ACTH Concentrations in Primary Cultures of Rat Anterior Pituitary Cells Exposed to TCDD. Bestervelt, L. L., Pitt, J. A., and Piper, W. N. (1998). Toxicol. Sci. 46, 294 –299. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to increase plasma ACTH concentrations in male Sprague–Dawley rats and in male rat primary anterior pituitary cell cultures. The present study examined whether the anterior pituitary effects observed after TCDD exposure are mediated via the Ah receptor (AhR). Primary anterior pituitary cell cultures were prepared from normal 180- to 220-g male rats and the cultures treated with a -naphthoflavone (ANF), an antagonist; b -naphthoflavone (BNF), an agonist; BNF 1 TCDD; 3,3*,4,4*,5-pentachlorobiphenyl (PCB), which is known to bind to the AhR; and 2,2*,4,4*,5,5*hexachlorobiphenyl (HCB), which does not bind the AhR. Support for the TCDD–AhR-mediated increases in ACTH concentrations is provided by the following observations: (1) ANF inhibited both the 1.3- to 2-fold TCDD-induced increase in basal medium and intracellular ACTH concentrations and the 30% TCDD-induced decrease in medium ACTH levels and the 1.2-fold increase in intracellular ACTH levels in corticotropin-releasing hormone (CRH)-stimulated cells, (2) BNF increased basal medium (1.7fold) and intracellular (1.3-fold) ACTH concentrations, (3) BNF 1 TCDD demonstrated additivity by increasing basal medium (2.4fold) and intracellular (1.7-fold) ACTH concentrations, (4) PCB increased basal medium (1.8- to 2.1-fold) and intracellular (1.3- to 1.8-fold) ACTH concentrations and inhibited medium ACTH secretion in CRH stimulated cells by 24 – 43%, and (5) HCB did not effect basal or CRH stimulated medium and intracellular ACTH concentrations. From this study it appears that TCDD-induced changes in ACTH secretion and synthesis by cultured anterior pituitary cells is mediated through the Ah receptor. © 1998 Society of Toxicology.
1 To whom reprint requests should be addressed at Environmental & Industrial Health, M6108 SPH II, 1420 Washington Hts., Ann Arbor, MI 481092029.
1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.
Halogenated aromatic hydrocarbons (HAHs) represent a chemical family of environmental contaminants that include the polychlorinated and polybrominated biphenyls, the polychlorinated dibenzofurans, and the polychlorinated dibenzo-pdioxins. As these compounds are lipophilic and resistant to degradation, they have a propensity to persist in the environment and bioaccumulate in the food chain, posing a potential risk to human health (Kimbrough and Grandjean, 1989). Toxicological studies have shown that various HAHs produce similar adverse responses (wasting syndrome, immunological alterations, epidermal keratinization, and induction of several hepatic microsomal drug-metabolizing enzymes) and are believed to share a common mechanism of action (Poland and Glover, 1979; Safe, 1986; Whitlock, 1990; Denison, 1991). The ability of the prototypical HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related HAHs to produce a broad spectrum of effects suggests that these compounds may act to alter the expression of particular genes in a species-specific or tissue-specific manner (Poland and Knutson, 1982; Safe, 1986; Durrin et al., 1987; Whitlock, 1990; Denison, 1991). Despite many years of research, the precise mechanism by which HAHs cause toxic responses remains unknown. In the case of the paradigmatic HAH, TCDD, it is believed that a key step involves binding to the Ah receptor (AhR), a cytosolic protein which has been identified in a wide variety of species and tissues that specifically bind HAHs (Yamamoto, 1985). Once the TCDD binds to the AhR, the ligand–receptor complex accumulates in the cell’s nucleus and interacts with a specific DNA sequence, the dioxin-responsive element (DRE), resulting in modulation (increased or decreased activity) of a specific gene (Whitlock, 1990). It has previously been shown that the increase in transcription of the rat hepatic CYP4501A1 gene, observed after TCDD exposure, occurs via this mechanism (Poland and Knutson, 1982; Denison, 1991; Whitlock, 1990; Safe, 1986). However, one must be cognizant that other TCDD-induced toxic responses may occur via dissimilar mechanisms. Recent studies in this laboratory have shown that TCDD
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exposure results in increased ACTH concentrations in rat plasma in vivo (Bestervelt et al., 1993a) and in primary rat anterior pituitary cell cultures in vitro (Bestervelt et al., 1993b). The objective of the present study was to determine whether the AhR participates in altering basal and corticotropin-releasing hormone (CRH)-stimulated medium and intracellular (lysate) ACTH concentrations in primary cultures of rat anterior pituitary cells during TCDD exposure. ACTH secretion by primary rat anterior pituitary cell cultures treated with TCDD was evaluated after exposure to (1) the AhR antagonist a-naphthoflavone (ANF; Merchant et al., 1990), (2) the AhR agonist b-naphthoflavone (BNF; Oinonen et al., 1994; Zhang et al., 1997), (3) the coplanar HAH, 3,39,4,49,5-pentachlorobiphenyl (PCB), which binds to the AhR and has a TEF of 0.1 (Safe, 1990), and (4) the diortho coplanar HAH, 2,29,4,49,5,59hexachlorobiphenyl (HCB), which lacks binding affinity for the Ah receptor and has an assigned TEF of 0.00002 (Safe, 1990). Primary cultures of rat anterior pituitary cells (an in vitro model) were used to exclude potential complications of circadian rhythm and feedback mechanisms within the hypothalamic–pituitary–adrenal axis. Both basal and CRH-stimulated pituitary cell cultures were tested since the ability of pituitary cells to produce and secrete ACTH in the presence or absence of a CRH stimulus may differ in the presence of TCDD. MATERIALS AND METHODS Primary Cultures of Anterior Pituitary Cells After decapitation, pituitary glands were aseptically removed from 20 –25 untreated Sprague–Dawley male rats (180 –220 g) obtained from The Reproductive Sciences Program of The University of Michigan. Posterior pituitary glands were separated from the anterior pituitary glands and discarded. Using a modification of the method of Vale et al. (1972), anterior pituitary glands were quartered, enzymatically dispersed in 20 ml Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL; Grand Island, NY) using 0.35% type I collagenase (Sigma, St. Louis, MO), and mechanically dissociated using a shaking water bath (37°C) for 60 min. After the resulting cell suspension settled (2 min), the collagenase was removed using a sterile flame-tipped Pasteur pipet. Complete DMEM (CDMEM: 2.2 g/liter sodium bicarbonate, 2.5% bovine calf serum, 10% horse serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, 0.1% bovine serum albumin, and 10 mM Hepes at pH 7.4) was added (5 ml) to the cell suspension (15 ml). The resulting mixture was passed through a sterile flame-tipped Pasteur pipet to facilitate dissociation of the anterior pituitary cells from the pituitary gland’s support membranes. After the membranous debris settled (2 min), the medium was removed and placed into a sterile 50-ml conical tube. This dissociation process was repeated three times to recover a maximal number of anterior pituitary cells. The resulting pooled cell suspension was brought to a final volume of 45 ml with CDMEM and centrifuged at 500g for 20 min at 5°C to pellet the cells. The pellet was gently resuspended in 5 ml of CDMEM using a sterile flame-tipped Pasteur pipet. The cell suspension was brought to 20 ml with CDMEM and centrifuged as previously described. The cell concentration was determined using a hemocytometer, diluted to 1 3 105 cells/ml, and plated in 24-well, 16-mm culture plates at 1 ml/well. The cells were maintained under standard culture conditions (95% air, 5% CO2, 37°C) for the duration of the experiment. Before dosing, cells were allowed to attach for 4 days and were rinsed twice with ice-cold phosphate buffered saline prior to treatment. For basal secretion studies, medium samples
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were collected following the treatment period and each well was rinsed twice (1 ml) with ice-cold 0.1 M sodium phosphate-buffered saline (pH 7.4). The cells were lysed with 150 ml lysis buffer (0.15 M NaCl, 1.5 mM MgCl2, 0.1% sodium dodecyl sulfate, and 20 mM Tris at pH 7.5; Anakwe and Payne, 1987). The medium and lysates were stored at 220°C until ACTH concentrations were measured by radioimmunoassay (RIA). For CRH stimulation studies, media were removed following the treatment period and cells were rinsed twice (1 ml) with fresh medium. Cells were then stimulated with 1027 M CRH for 5 h as this concentration of CRH has been shown to stimulate the synthesis and secretion of ACTH by cultured anterior pituitary cells (Vale et al., 1972 Rivier et al., 1984; Vale et al., 1983). Following CRH stimulation, medium and lysate samples were collected as described above, and stored at 220°C until ACTH concentrations were measured by RIA. Cell viability was assessed by trypan blue exclusion and was greater than 90% for all experiments. Experimental Design Primary cultures of rat anterior pituitary cells were initiated as described above. As only a finite number of cells can be obtained per pituitary gland, four experiments were required to analyze each chemical. Experiment 1: ANF. Anterior pituitary cells were treated for 2 h with 1 mM ANF, a dose that completely saturates the AhR (Gasiewicz and Rucci, 1991). Cells were rinsed twice with medium (1 ml) to remove free ANF and were then treated with TCDD (10213 M) for 6 h as a 6-h TCDD exposure has previously been shown to cause a significant increase in the secretion of ACTH by cultured anterior pituitary cells (Bestervelt et al., 1998). For the CRH stimulation study, anterior pituitary cells were treated with ANF and TCDD as described above followed by CRH stimulation. Experiment 2: BNF. Anterior pituitary cells were treated concurrently with 10 nM BNF and 10213 M TCDD for 6 h as a 6-h TCDD exposure has previously been shown to cause a significant increase in the secretion of ACTH by cultured anterior pituitary cells (Bestervelt et al., 1998). For the CRH stimulation study, anterior pituitary cells treated with 10 nM BNF and TCDD (10213 M) for 6 h were then stimulated with 1027 M CRH for 5 h. Experiment 3: PCB. Anterior pituitary cells were treated with 0, 1029, 10211, or 10213 M PCB for 24 h to allow for a maximal response as PCB’s affinity for the AHR is about 10-fold less than that of TCDD. For the CRH stimulation study, cells treated with PCB (1029, 10211, or 10213 M) for 24 h were then stimulated with 1027 M CRH for 5 h. Experiment 4: HCB. Anterior pituitary cells were treated with 0, 1029, 10 , or 10213 M HCB for 24 h to allow for a maximal response by the anterior pituitary cells and to duplicate the PCB treatment paradigm. For the CRH stimulation study, cells treated with HCB were stimulated with 1027 M CRH for 5 h. 211
Chemicals ANF and BNF were obtained from Sigma Chemical Co. (St. Louis, MO). 3,39,4,49,5-Pentachlorobiphenyl and 2,29,4,49,5,59-hexachlorobiphenyl were a generous gift from Dr. Mike Denison (University of California, Davis). CRH was obtained from Peninsula Laboratories (Belmont, CA). ACTH Measurement ACTH was quantified using a commercially available RIA kit (Diagnostic Products Corp., Los Angeles, CA). Intra- and interassay coefficients of variation for ACTH were 6 and 9%, respectively. Sensitivity of the ACTH RIA was 8 pg/ml. Statistics Differences between means were evaluated by analysis of variance. The level of statistical significance was set at a , 0.05 for all analyses.
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FIG. 1. The effect of TCDD on (A) basal media and lysate and (B) CRH-stimulated medium and lysate ACTH concentrations after pretreatment with ANF. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, anterior pituitary cells were treated with 1 mM ANF for 2 h. After pretreatment, ANF was removed. Basal cells (A) were treated with TCDD (10213 M) for 6 h. After ANF (2 h) and TCDD (10213 M, 6 h) treatment, CRH-stimulated cells (B) were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells. An asterisk denotes that the difference between TCDD or TCDD 1 ANF treatment and control values was statistically significant ( p , 0.05).
RESULTS
Experiment 1: ANF Pretreatment of anterior pituitary cells with a saturating concentration (1 mM) of the AhR antagonist ANF prevented the twofold TCDD-mediated increases of basal medium and the 1.3-fold increase of intracellular ACTH concentrations (Fig. 1A). ANF pretreatment of pituitary cells also prevented the 30% decrease in medium ACTH in cells exposed to TCDD and then stimulated with CRH (Fig. 1B) and the 1.2-fold increase in intracellular ACTH concentrations in cells stimulated with CRH that were exposed to TCDD (Fig. 1B). Experiment 2: BNF The AhR agonist BNF (10 nM) significantly increased both basal medium and intracellular (1.3-fold) ACTH concentrations comparably to 10213 M TCDD (Fig. 2A). Basal medium (2.4-fold) and intracellular (1.7-fold) ACTH concentrations showed a greater increase in the BNF–TCDD treatment group than either compound separately, suggesting additivity of response (Fig. 2A). Medium ACTH concentrations from cells stimulated with CRH were decreased by 35 and 5% in the 10 nM BNF and 10213 M TCDD–10 nM BNF treatment groups, respectively (Fig. 2B). In contrast, intracellular ACTH concentrations from cells stimulated with CRH were increased 1.3and 1.7-fold in the 10 nM BNF and the 10213 M TCDD–10 nM BNF treatment groups, respectively.
Experiment 3: PCB 3,39,4,49,5-Pentachlorobiphenyl (1029, 10211, and 10213 M PCB) caused dose-dependent 2.1-, 1.8-, and 1.3-fold increases in ACTH secreted into the medium. Intracellular ACTH concentrations were also increased in the PCB treatment groups (Fig. 3A). In PCB-treated cells stimulated with CRH, PCB treatment (1029, 10211, and 10213 M) caused a 24, 33, and 43% decrease in ACTH medium concentrations respectively, and 1.7-, 1.5-, and 1.4-fold increases in intracellular ACTH concentrations, respectively (Fig. 3B). Experiment 4: HCB 2,29,4,49,5,59-Hexachlorobiphenyl (HCB), a HAH analogue which lacks binding affinity for the Ah receptor, did not alter basal or CRH stimulated medium or intracellular ACTH concentrations (Fig. 4). DISCUSSION
Increased rat ACTH plasma concentrations have been found after TCDD exposure (Bestervelt et al., 1993a) and basal medium and intracellular (lysate) ACTH concentrations have been shown to be increased in rat anterior pituitary cell cultures after TCDD exposure (Bestervelt et al., 1993b). The data presented herein indicate that the AhR is responsible for mediating these phenomena. TCDD has been shown to increase the transcription of several genes through an AhR-mediated mechanism (Poland and Knutson, 1982; Whitlock, 1990) in-
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FIG. 2. The effects of TCDD and BNF on (A) basal medium and lysate and (B) CRH-stimulated medium and lysate ACTH concentrations. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, the anterior pituitary cells were treated with (A) 10 nM BNF and/or 10213 M TCDD for 6 h. After ANF (2 h) and TCDD (10213 M, 6 h) treatment, CRH-stimulated cells (B) were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells. An asterisk denotes that the difference between BNF, TCDD, or TCDD 1 BNF and control values was statistically significant ( p , 0.05).
cluding the cytochrome P4501A1 gene in mouse liver. It is possible that TCDD may increase ACTH concentrations by increasing the rate of transcription of the POMC gene through an AhR-mediated process. Several reasons exist for examining the participation of the AhR in increasing ACTH concentrations in the anterior pituitary gland after TCDD exposure. First, the AhR is found in the male rat pituitary gland (Bestervelt, Pitt, Denison, and Piper, 1992, unpublished data). Second, the POMC gene (Drouin et al., 1985) has a dioxin-responsive
element (Denison, 1991) located in the 59 flanking region upstream of the transcriptional start site. Therefore, the present study was designed to determine whether increased ACTH concentrations after TCDD exposure might be mediated through an AhR mechanism. An AhR antagonist (ANF) and an AhR agonist (BNF) were used to distinguish between Ah and non-AhR mechanisms. ANF prevented the increased ACTH concentrations in both basal media and cell lysates, blocked the ability of TCDD to
FIG. 3. The effect of PCB on (A) basal medium and lysate and (B) CRH-stimulated medium and lysate ACTH concentrations in rat anterior pituitary cells. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, anterior pituitary cells were treated with (A) PCB (0, 1029, 10211, or 10213 M) for 24 h. After PCB exposure, (B) CRH-stimulated cells were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells. An asterisk denotes that the difference between PCB and control values was statistically significant ( p , 0.05).
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FIG. 4. The effect of HCB on (A) basal medium and lysate (B) CRH-stimulated medium and lysate ACTH concentrations in rat anterior pituitary cells. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, anterior pituitary cells were treated with (A) HCB (0, 1029, 10211, or 10213 M) for 24 h. After HCB exposure, (B) CRH-stimulated cells were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells.
inhibit CRH stimulated ACTH secretion into the media and prevented the increased ACTH concentrations in cell lysates in anterior pituitary cells treated with TCDD. Previous results in anterior pituitary cell culture have shown that low concentrations of TCDD (1029 to 10213 M) decreased pituitary responsiveness to CRH (Bestervelt et al., 1998). A possible mechanism for decreased anterior pituitary responsiveness to CRH is that CRH may be homeostatically regulated by increased levels of ACTH. A multi-stage feedback system has been shown to participate in the regulation of ACTH by the anterior pituitary gland (Kovacs and Makara, 1988). ACTH feedback to the pituitary gland does occur (Ono et al., 1985; Suda et al., 1986), suggesting that ACTH is able to physiologically regulate its own secretion. Motto et al. (1967) have shown a short negative feedback mechanism linking synthesis and/or release of ACTH to preexisting blood levels of the hormone. Therefore, elevated levels of ACTH observed after TCDD exposure may feedback to the anterior pituitary via a short feedback loop to decrease pituitary responsiveness to CRH. The end result would be decreased secretion of ACTH. BNF increased both basal medium and cell lysate ACTH concentrations in anterior pituitary cell cultures. As BNF is an AhR agonist (Gasiewicz and Rucci, 1991), and increased ACTH concentrations were observed with BNF, it is possible that both BNF and TCDD increase medium and lysate ACTH concentrations through participation of the Ah receptor. BNF also inhibited CRH-stimulated ACTH concentrations in the medium and increased CRH-stimulated lysate ACTH concentrations similar to TCDD. Anterior pituitary cells appear to be able to synthesize ACTH as shown by a net increase in cellular lysate ACTH concentrations. The increased cellular ACTH concentrations and
decreased ACTH medium concentrations suggest that the secretory process of ACTH may be impaired by exposure to BNF. Decreased anterior pituitary responsiveness to CRH by BNF exposure may occur through a homeostatically regulated process in which increased levels of ACTH feedback to the anterior pituitary via a short feedback loop. As mentioned in the previous paragraph, the end result would be decreased pituitary responsiveness to CRH and depressed secretion of ACTH. Interestingly, these results parallel those observed in anterior pituitary cell culture after TCDD exposure (Bestervelt et al., 1998). The coplanar HAH 3,39,4,49,5-pentachlorobiphenyl (PCB) is believed to act similarly to TCDD based on closely related AhR binding properties and biochemical and toxic responses (Goldstein and Safe, 1989). As PCB and TCDD are believed to produce their toxic actions via the AhR and their binding affinities are within an order of magnitude (Safe, 1986, 1990; Goldstein and Safe, 1989; Whitlock, 1990), PCB should produce results in primary anterior pituitary cell culture similar to TCDD. Treatment with PCB increased basal media and lysate concentrations of ACTH in a similar manner as TCDD (Bestervelt et al., 1998), suggesting that it may stimulate the anterior pituitary gland to produce ACTH. PCB, like TCDD (Bestervelt et al., 1998), also decreased pituitary responsiveness to CRH as evidenced by decreased secretion of ACTH into the pituitary medium, although the anterior pituitary cells were able to synthesize ACTH demonstrated by a net increase in cellular lysate ACTH concentrations. The increased cellular lysate ACTH concentrations and decreased ACTH medium concentrations suggest that the secretory response is impaired, again similar to effects observed after TCDD exposure. As TCDD and PCB alter anterior pituitary secretion of ACTH in
AhR MEDIATION
a similar manner, it is possible that both do so by utilizing the same mechanism. Therefore, these findings indicate that increased ACTH concentrations in anterior pituitary cells treated with either PCB or TCDD may occur through an AhRmediated process. However, one would expect the effect of PCB treatment on ACTH synthesis/secretion to be about 10fold less than similar TCDD dosages as PCB has a binding affinity for the AhR an order of magnitude less than TCDD. This implies that another mechanism(s) may play a role in PCB’s altering ACTH synthesis/secretion in cultured anterior pituitary cells. 2,29,4,49,5,59-Hexachlorobiphenyl (HCB) is a diortho coplanar HAH analogue that has no affinity for the AhR (Goldstein and Safe, 1989). If increased ACTH concentrations are mediated through the Ah receptor, then HCB should not increase ACTH concentrations in anterior pituitary cell cultures. As HCB did not increase basal medium and lysate ACTH concentrations in anterior pituitary cells, these results suggest that chlorinated biphenyls interact with the AhR to be able to increase ACTH concentrations in anterior pituitary cells. In summary, the data from this study show support for the participation of the AhR in increasing ACTH concentrations in anterior pituitary cells exposed to TCDD. ACKNOWLEDGMENTS This research was supported in part by a predoctoral training grant fellowship to L.L.B. (T 32 ES-07062) and core facilities provided by P30-HD18258. The authors thank Dr. Mike Denison for the AhR agonists and antagonists as well as providing technical assistance and Dr. Paula A. Bank for consultation and input regarding the agonist and antagonist assays.
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Evidence for Ah Receptor Mediation of Increased ACTH Concentrations in Primary Cultures of Rat Anterior Pituitary Cells Exposed to TCDD Lorelle L. Bestervelt,* Jeff A. Pitt,† and Walter N. Piper‡,1 *Toxicology Department, NSF International, Ann Arbor, Michigan 48105; †Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599; and ‡Toxicology Program, School of Public Health, Department of Pharmacology, Medical School and Reproductive Sciences Program, The University of Michigan, Ann Arbor, Michigan 48109-2029 Received December 30, 1997; accepted July 23, 1998
Evidence for Ah Receptor Mediation of Increased ACTH Concentrations in Primary Cultures of Rat Anterior Pituitary Cells Exposed to TCDD. Bestervelt, L. L., Pitt, J. A., and Piper, W. N. (1998). Toxicol. Sci. 46, 294 –299. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to increase plasma ACTH concentrations in male Sprague–Dawley rats and in male rat primary anterior pituitary cell cultures. The present study examined whether the anterior pituitary effects observed after TCDD exposure are mediated via the Ah receptor (AhR). Primary anterior pituitary cell cultures were prepared from normal 180- to 220-g male rats and the cultures treated with a -naphthoflavone (ANF), an antagonist; b -naphthoflavone (BNF), an agonist; BNF 1 TCDD; 3,3*,4,4*,5-pentachlorobiphenyl (PCB), which is known to bind to the AhR; and 2,2*,4,4*,5,5*hexachlorobiphenyl (HCB), which does not bind the AhR. Support for the TCDD–AhR-mediated increases in ACTH concentrations is provided by the following observations: (1) ANF inhibited both the 1.3- to 2-fold TCDD-induced increase in basal medium and intracellular ACTH concentrations and the 30% TCDD-induced decrease in medium ACTH levels and the 1.2-fold increase in intracellular ACTH levels in corticotropin-releasing hormone (CRH)-stimulated cells, (2) BNF increased basal medium (1.7fold) and intracellular (1.3-fold) ACTH concentrations, (3) BNF 1 TCDD demonstrated additivity by increasing basal medium (2.4fold) and intracellular (1.7-fold) ACTH concentrations, (4) PCB increased basal medium (1.8- to 2.1-fold) and intracellular (1.3- to 1.8-fold) ACTH concentrations and inhibited medium ACTH secretion in CRH stimulated cells by 24 – 43%, and (5) HCB did not effect basal or CRH stimulated medium and intracellular ACTH concentrations. From this study it appears that TCDD-induced changes in ACTH secretion and synthesis by cultured anterior pituitary cells is mediated through the Ah receptor. © 1998 Society of Toxicology.
1 To whom reprint requests should be addressed at Environmental & Industrial Health, M6108 SPH II, 1420 Washington Hts., Ann Arbor, MI 481092029.
1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.
Halogenated aromatic hydrocarbons (HAHs) represent a chemical family of environmental contaminants that include the polychlorinated and polybrominated biphenyls, the polychlorinated dibenzofurans, and the polychlorinated dibenzo-pdioxins. As these compounds are lipophilic and resistant to degradation, they have a propensity to persist in the environment and bioaccumulate in the food chain, posing a potential risk to human health (Kimbrough and Grandjean, 1989). Toxicological studies have shown that various HAHs produce similar adverse responses (wasting syndrome, immunological alterations, epidermal keratinization, and induction of several hepatic microsomal drug-metabolizing enzymes) and are believed to share a common mechanism of action (Poland and Glover, 1979; Safe, 1986; Whitlock, 1990; Denison, 1991). The ability of the prototypical HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related HAHs to produce a broad spectrum of effects suggests that these compounds may act to alter the expression of particular genes in a species-specific or tissue-specific manner (Poland and Knutson, 1982; Safe, 1986; Durrin et al., 1987; Whitlock, 1990; Denison, 1991). Despite many years of research, the precise mechanism by which HAHs cause toxic responses remains unknown. In the case of the paradigmatic HAH, TCDD, it is believed that a key step involves binding to the Ah receptor (AhR), a cytosolic protein which has been identified in a wide variety of species and tissues that specifically bind HAHs (Yamamoto, 1985). Once the TCDD binds to the AhR, the ligand–receptor complex accumulates in the cell’s nucleus and interacts with a specific DNA sequence, the dioxin-responsive element (DRE), resulting in modulation (increased or decreased activity) of a specific gene (Whitlock, 1990). It has previously been shown that the increase in transcription of the rat hepatic CYP4501A1 gene, observed after TCDD exposure, occurs via this mechanism (Poland and Knutson, 1982; Denison, 1991; Whitlock, 1990; Safe, 1986). However, one must be cognizant that other TCDD-induced toxic responses may occur via dissimilar mechanisms. Recent studies in this laboratory have shown that TCDD
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exposure results in increased ACTH concentrations in rat plasma in vivo (Bestervelt et al., 1993a) and in primary rat anterior pituitary cell cultures in vitro (Bestervelt et al., 1993b). The objective of the present study was to determine whether the AhR participates in altering basal and corticotropin-releasing hormone (CRH)-stimulated medium and intracellular (lysate) ACTH concentrations in primary cultures of rat anterior pituitary cells during TCDD exposure. ACTH secretion by primary rat anterior pituitary cell cultures treated with TCDD was evaluated after exposure to (1) the AhR antagonist a-naphthoflavone (ANF; Merchant et al., 1990), (2) the AhR agonist b-naphthoflavone (BNF; Oinonen et al., 1994; Zhang et al., 1997), (3) the coplanar HAH, 3,39,4,49,5-pentachlorobiphenyl (PCB), which binds to the AhR and has a TEF of 0.1 (Safe, 1990), and (4) the diortho coplanar HAH, 2,29,4,49,5,59hexachlorobiphenyl (HCB), which lacks binding affinity for the Ah receptor and has an assigned TEF of 0.00002 (Safe, 1990). Primary cultures of rat anterior pituitary cells (an in vitro model) were used to exclude potential complications of circadian rhythm and feedback mechanisms within the hypothalamic–pituitary–adrenal axis. Both basal and CRH-stimulated pituitary cell cultures were tested since the ability of pituitary cells to produce and secrete ACTH in the presence or absence of a CRH stimulus may differ in the presence of TCDD. MATERIALS AND METHODS Primary Cultures of Anterior Pituitary Cells After decapitation, pituitary glands were aseptically removed from 20 –25 untreated Sprague–Dawley male rats (180 –220 g) obtained from The Reproductive Sciences Program of The University of Michigan. Posterior pituitary glands were separated from the anterior pituitary glands and discarded. Using a modification of the method of Vale et al. (1972), anterior pituitary glands were quartered, enzymatically dispersed in 20 ml Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL; Grand Island, NY) using 0.35% type I collagenase (Sigma, St. Louis, MO), and mechanically dissociated using a shaking water bath (37°C) for 60 min. After the resulting cell suspension settled (2 min), the collagenase was removed using a sterile flame-tipped Pasteur pipet. Complete DMEM (CDMEM: 2.2 g/liter sodium bicarbonate, 2.5% bovine calf serum, 10% horse serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, 0.1% bovine serum albumin, and 10 mM Hepes at pH 7.4) was added (5 ml) to the cell suspension (15 ml). The resulting mixture was passed through a sterile flame-tipped Pasteur pipet to facilitate dissociation of the anterior pituitary cells from the pituitary gland’s support membranes. After the membranous debris settled (2 min), the medium was removed and placed into a sterile 50-ml conical tube. This dissociation process was repeated three times to recover a maximal number of anterior pituitary cells. The resulting pooled cell suspension was brought to a final volume of 45 ml with CDMEM and centrifuged at 500g for 20 min at 5°C to pellet the cells. The pellet was gently resuspended in 5 ml of CDMEM using a sterile flame-tipped Pasteur pipet. The cell suspension was brought to 20 ml with CDMEM and centrifuged as previously described. The cell concentration was determined using a hemocytometer, diluted to 1 3 105 cells/ml, and plated in 24-well, 16-mm culture plates at 1 ml/well. The cells were maintained under standard culture conditions (95% air, 5% CO2, 37°C) for the duration of the experiment. Before dosing, cells were allowed to attach for 4 days and were rinsed twice with ice-cold phosphate buffered saline prior to treatment. For basal secretion studies, medium samples
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were collected following the treatment period and each well was rinsed twice (1 ml) with ice-cold 0.1 M sodium phosphate-buffered saline (pH 7.4). The cells were lysed with 150 ml lysis buffer (0.15 M NaCl, 1.5 mM MgCl2, 0.1% sodium dodecyl sulfate, and 20 mM Tris at pH 7.5; Anakwe and Payne, 1987). The medium and lysates were stored at 220°C until ACTH concentrations were measured by radioimmunoassay (RIA). For CRH stimulation studies, media were removed following the treatment period and cells were rinsed twice (1 ml) with fresh medium. Cells were then stimulated with 1027 M CRH for 5 h as this concentration of CRH has been shown to stimulate the synthesis and secretion of ACTH by cultured anterior pituitary cells (Vale et al., 1972 Rivier et al., 1984; Vale et al., 1983). Following CRH stimulation, medium and lysate samples were collected as described above, and stored at 220°C until ACTH concentrations were measured by RIA. Cell viability was assessed by trypan blue exclusion and was greater than 90% for all experiments. Experimental Design Primary cultures of rat anterior pituitary cells were initiated as described above. As only a finite number of cells can be obtained per pituitary gland, four experiments were required to analyze each chemical. Experiment 1: ANF. Anterior pituitary cells were treated for 2 h with 1 mM ANF, a dose that completely saturates the AhR (Gasiewicz and Rucci, 1991). Cells were rinsed twice with medium (1 ml) to remove free ANF and were then treated with TCDD (10213 M) for 6 h as a 6-h TCDD exposure has previously been shown to cause a significant increase in the secretion of ACTH by cultured anterior pituitary cells (Bestervelt et al., 1998). For the CRH stimulation study, anterior pituitary cells were treated with ANF and TCDD as described above followed by CRH stimulation. Experiment 2: BNF. Anterior pituitary cells were treated concurrently with 10 nM BNF and 10213 M TCDD for 6 h as a 6-h TCDD exposure has previously been shown to cause a significant increase in the secretion of ACTH by cultured anterior pituitary cells (Bestervelt et al., 1998). For the CRH stimulation study, anterior pituitary cells treated with 10 nM BNF and TCDD (10213 M) for 6 h were then stimulated with 1027 M CRH for 5 h. Experiment 3: PCB. Anterior pituitary cells were treated with 0, 1029, 10211, or 10213 M PCB for 24 h to allow for a maximal response as PCB’s affinity for the AHR is about 10-fold less than that of TCDD. For the CRH stimulation study, cells treated with PCB (1029, 10211, or 10213 M) for 24 h were then stimulated with 1027 M CRH for 5 h. Experiment 4: HCB. Anterior pituitary cells were treated with 0, 1029, 10 , or 10213 M HCB for 24 h to allow for a maximal response by the anterior pituitary cells and to duplicate the PCB treatment paradigm. For the CRH stimulation study, cells treated with HCB were stimulated with 1027 M CRH for 5 h. 211
Chemicals ANF and BNF were obtained from Sigma Chemical Co. (St. Louis, MO). 3,39,4,49,5-Pentachlorobiphenyl and 2,29,4,49,5,59-hexachlorobiphenyl were a generous gift from Dr. Mike Denison (University of California, Davis). CRH was obtained from Peninsula Laboratories (Belmont, CA). ACTH Measurement ACTH was quantified using a commercially available RIA kit (Diagnostic Products Corp., Los Angeles, CA). Intra- and interassay coefficients of variation for ACTH were 6 and 9%, respectively. Sensitivity of the ACTH RIA was 8 pg/ml. Statistics Differences between means were evaluated by analysis of variance. The level of statistical significance was set at a , 0.05 for all analyses.
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FIG. 1. The effect of TCDD on (A) basal media and lysate and (B) CRH-stimulated medium and lysate ACTH concentrations after pretreatment with ANF. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, anterior pituitary cells were treated with 1 mM ANF for 2 h. After pretreatment, ANF was removed. Basal cells (A) were treated with TCDD (10213 M) for 6 h. After ANF (2 h) and TCDD (10213 M, 6 h) treatment, CRH-stimulated cells (B) were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells. An asterisk denotes that the difference between TCDD or TCDD 1 ANF treatment and control values was statistically significant ( p , 0.05).
RESULTS
Experiment 1: ANF Pretreatment of anterior pituitary cells with a saturating concentration (1 mM) of the AhR antagonist ANF prevented the twofold TCDD-mediated increases of basal medium and the 1.3-fold increase of intracellular ACTH concentrations (Fig. 1A). ANF pretreatment of pituitary cells also prevented the 30% decrease in medium ACTH in cells exposed to TCDD and then stimulated with CRH (Fig. 1B) and the 1.2-fold increase in intracellular ACTH concentrations in cells stimulated with CRH that were exposed to TCDD (Fig. 1B). Experiment 2: BNF The AhR agonist BNF (10 nM) significantly increased both basal medium and intracellular (1.3-fold) ACTH concentrations comparably to 10213 M TCDD (Fig. 2A). Basal medium (2.4-fold) and intracellular (1.7-fold) ACTH concentrations showed a greater increase in the BNF–TCDD treatment group than either compound separately, suggesting additivity of response (Fig. 2A). Medium ACTH concentrations from cells stimulated with CRH were decreased by 35 and 5% in the 10 nM BNF and 10213 M TCDD–10 nM BNF treatment groups, respectively (Fig. 2B). In contrast, intracellular ACTH concentrations from cells stimulated with CRH were increased 1.3and 1.7-fold in the 10 nM BNF and the 10213 M TCDD–10 nM BNF treatment groups, respectively.
Experiment 3: PCB 3,39,4,49,5-Pentachlorobiphenyl (1029, 10211, and 10213 M PCB) caused dose-dependent 2.1-, 1.8-, and 1.3-fold increases in ACTH secreted into the medium. Intracellular ACTH concentrations were also increased in the PCB treatment groups (Fig. 3A). In PCB-treated cells stimulated with CRH, PCB treatment (1029, 10211, and 10213 M) caused a 24, 33, and 43% decrease in ACTH medium concentrations respectively, and 1.7-, 1.5-, and 1.4-fold increases in intracellular ACTH concentrations, respectively (Fig. 3B). Experiment 4: HCB 2,29,4,49,5,59-Hexachlorobiphenyl (HCB), a HAH analogue which lacks binding affinity for the Ah receptor, did not alter basal or CRH stimulated medium or intracellular ACTH concentrations (Fig. 4). DISCUSSION
Increased rat ACTH plasma concentrations have been found after TCDD exposure (Bestervelt et al., 1993a) and basal medium and intracellular (lysate) ACTH concentrations have been shown to be increased in rat anterior pituitary cell cultures after TCDD exposure (Bestervelt et al., 1993b). The data presented herein indicate that the AhR is responsible for mediating these phenomena. TCDD has been shown to increase the transcription of several genes through an AhR-mediated mechanism (Poland and Knutson, 1982; Whitlock, 1990) in-
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FIG. 2. The effects of TCDD and BNF on (A) basal medium and lysate and (B) CRH-stimulated medium and lysate ACTH concentrations. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, the anterior pituitary cells were treated with (A) 10 nM BNF and/or 10213 M TCDD for 6 h. After ANF (2 h) and TCDD (10213 M, 6 h) treatment, CRH-stimulated cells (B) were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells. An asterisk denotes that the difference between BNF, TCDD, or TCDD 1 BNF and control values was statistically significant ( p , 0.05).
cluding the cytochrome P4501A1 gene in mouse liver. It is possible that TCDD may increase ACTH concentrations by increasing the rate of transcription of the POMC gene through an AhR-mediated process. Several reasons exist for examining the participation of the AhR in increasing ACTH concentrations in the anterior pituitary gland after TCDD exposure. First, the AhR is found in the male rat pituitary gland (Bestervelt, Pitt, Denison, and Piper, 1992, unpublished data). Second, the POMC gene (Drouin et al., 1985) has a dioxin-responsive
element (Denison, 1991) located in the 59 flanking region upstream of the transcriptional start site. Therefore, the present study was designed to determine whether increased ACTH concentrations after TCDD exposure might be mediated through an AhR mechanism. An AhR antagonist (ANF) and an AhR agonist (BNF) were used to distinguish between Ah and non-AhR mechanisms. ANF prevented the increased ACTH concentrations in both basal media and cell lysates, blocked the ability of TCDD to
FIG. 3. The effect of PCB on (A) basal medium and lysate and (B) CRH-stimulated medium and lysate ACTH concentrations in rat anterior pituitary cells. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, anterior pituitary cells were treated with (A) PCB (0, 1029, 10211, or 10213 M) for 24 h. After PCB exposure, (B) CRH-stimulated cells were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells. An asterisk denotes that the difference between PCB and control values was statistically significant ( p , 0.05).
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FIG. 4. The effect of HCB on (A) basal medium and lysate (B) CRH-stimulated medium and lysate ACTH concentrations in rat anterior pituitary cells. Primary cultures of rat anterior pituitary cells were initiated as described under Materials and Methods. Following a 72-h attachment time, anterior pituitary cells were treated with (A) HCB (0, 1029, 10211, or 10213 M) for 24 h. After HCB exposure, (B) CRH-stimulated cells were treated with 1027 M CRH for 5 h. Medium and lysate samples were collected following TCDD exposure and ACTH concentrations were measured by RIA. Values represent means 6 SEM for nine wells.
inhibit CRH stimulated ACTH secretion into the media and prevented the increased ACTH concentrations in cell lysates in anterior pituitary cells treated with TCDD. Previous results in anterior pituitary cell culture have shown that low concentrations of TCDD (1029 to 10213 M) decreased pituitary responsiveness to CRH (Bestervelt et al., 1998). A possible mechanism for decreased anterior pituitary responsiveness to CRH is that CRH may be homeostatically regulated by increased levels of ACTH. A multi-stage feedback system has been shown to participate in the regulation of ACTH by the anterior pituitary gland (Kovacs and Makara, 1988). ACTH feedback to the pituitary gland does occur (Ono et al., 1985; Suda et al., 1986), suggesting that ACTH is able to physiologically regulate its own secretion. Motto et al. (1967) have shown a short negative feedback mechanism linking synthesis and/or release of ACTH to preexisting blood levels of the hormone. Therefore, elevated levels of ACTH observed after TCDD exposure may feedback to the anterior pituitary via a short feedback loop to decrease pituitary responsiveness to CRH. The end result would be decreased secretion of ACTH. BNF increased both basal medium and cell lysate ACTH concentrations in anterior pituitary cell cultures. As BNF is an AhR agonist (Gasiewicz and Rucci, 1991), and increased ACTH concentrations were observed with BNF, it is possible that both BNF and TCDD increase medium and lysate ACTH concentrations through participation of the Ah receptor. BNF also inhibited CRH-stimulated ACTH concentrations in the medium and increased CRH-stimulated lysate ACTH concentrations similar to TCDD. Anterior pituitary cells appear to be able to synthesize ACTH as shown by a net increase in cellular lysate ACTH concentrations. The increased cellular ACTH concentrations and
decreased ACTH medium concentrations suggest that the secretory process of ACTH may be impaired by exposure to BNF. Decreased anterior pituitary responsiveness to CRH by BNF exposure may occur through a homeostatically regulated process in which increased levels of ACTH feedback to the anterior pituitary via a short feedback loop. As mentioned in the previous paragraph, the end result would be decreased pituitary responsiveness to CRH and depressed secretion of ACTH. Interestingly, these results parallel those observed in anterior pituitary cell culture after TCDD exposure (Bestervelt et al., 1998). The coplanar HAH 3,39,4,49,5-pentachlorobiphenyl (PCB) is believed to act similarly to TCDD based on closely related AhR binding properties and biochemical and toxic responses (Goldstein and Safe, 1989). As PCB and TCDD are believed to produce their toxic actions via the AhR and their binding affinities are within an order of magnitude (Safe, 1986, 1990; Goldstein and Safe, 1989; Whitlock, 1990), PCB should produce results in primary anterior pituitary cell culture similar to TCDD. Treatment with PCB increased basal media and lysate concentrations of ACTH in a similar manner as TCDD (Bestervelt et al., 1998), suggesting that it may stimulate the anterior pituitary gland to produce ACTH. PCB, like TCDD (Bestervelt et al., 1998), also decreased pituitary responsiveness to CRH as evidenced by decreased secretion of ACTH into the pituitary medium, although the anterior pituitary cells were able to synthesize ACTH demonstrated by a net increase in cellular lysate ACTH concentrations. The increased cellular lysate ACTH concentrations and decreased ACTH medium concentrations suggest that the secretory response is impaired, again similar to effects observed after TCDD exposure. As TCDD and PCB alter anterior pituitary secretion of ACTH in
AhR MEDIATION
a similar manner, it is possible that both do so by utilizing the same mechanism. Therefore, these findings indicate that increased ACTH concentrations in anterior pituitary cells treated with either PCB or TCDD may occur through an AhRmediated process. However, one would expect the effect of PCB treatment on ACTH synthesis/secretion to be about 10fold less than similar TCDD dosages as PCB has a binding affinity for the AhR an order of magnitude less than TCDD. This implies that another mechanism(s) may play a role in PCB’s altering ACTH synthesis/secretion in cultured anterior pituitary cells. 2,29,4,49,5,59-Hexachlorobiphenyl (HCB) is a diortho coplanar HAH analogue that has no affinity for the AhR (Goldstein and Safe, 1989). If increased ACTH concentrations are mediated through the Ah receptor, then HCB should not increase ACTH concentrations in anterior pituitary cell cultures. As HCB did not increase basal medium and lysate ACTH concentrations in anterior pituitary cells, these results suggest that chlorinated biphenyls interact with the AhR to be able to increase ACTH concentrations in anterior pituitary cells. In summary, the data from this study show support for the participation of the AhR in increasing ACTH concentrations in anterior pituitary cells exposed to TCDD. ACKNOWLEDGMENTS This research was supported in part by a predoctoral training grant fellowship to L.L.B. (T 32 ES-07062) and core facilities provided by P30-HD18258. The authors thank Dr. Mike Denison for the AhR agonists and antagonists as well as providing technical assistance and Dr. Paula A. Bank for consultation and input regarding the agonist and antagonist assays.
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