PCB126 Induces Differential Changes in Androgen, Cortisol, and Aldosterone Biosynthesis in Human Adrenocortical H295R Cells
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《毒物学科学杂志》
Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaohsiung 807, Taiwan, Republic of China
Division of Metabolism and Endocrinology, Chang Gung Memorial Hospital, Kaohsiung 833, Taiwan, Republic of China
ABSTRACT
Dioxins and polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal glands when incorporated into the body. However, the impacts of exposure on adrenal steroidogenesis have not been thoroughly investigated. In this study, we demonstrated that dioxin-like PCB126 altered androgen, cortisol, and aldosterone biosynthesis differentially in human adrenocortical H295R cells. PCB126 diminished basal and cAMP-induced androstenedione production as well as CYP17 mRNA expression in a dose-dependent and time-dependent manner. The CYP17 repression was accompanied with decreases in the encoded 17-hydroxylase and 17,20-lyase activities, particularly the latter. In contrast, high concentrations of PCB126 stimulated basal cortisol and aldosterone biosynthesis, including induction of CYP21B, CYP11B1, and CYP11B2 mRNA expression and elevation of the conversion of cortisol from 17-OH-progesterone and aldosterone from progesterone. cAMP abolished the positive effect of PCB126 on cortisol synthesis, while it synergistically enhanced PCB126 stimulation on CYP11B2 expression and aldosterone production. It seemed likely that the downregulation of CYP21B caused by the combination of PCB126 and cAMP counteracted the CYP11B1 induction stimulated by the co-treatment. In addition, high concentrations of PCB126 might sensitize the regulation of adrenocorticotropin (ACTH) on the adrenocortical cells by increasing ACTH receptor levels. Because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, obesity, vascular function, and cardiac remodeling, this article also discusses the potential association of the detected adrenocortical alterations with increased diabetic and cardiovascular risk found among highly exposed people.
Key Words: coplanar PCB; adrenal steroidogenesis; cAMP induction; steroidogenic genes; diabetes; cardiovascular diseases.
INTRODUCTION
Accumulating epidemiological evidence reveals that people exposed to high levels of dioxins and dioxin-like compounds, such as coplanar polychlorinated biphenyls (PCBs), have increased risk of developing diabetes mellitus and cardiovascular diseases (Bertazzi et al., 2001; Cranmer et al., 2000; Fierens et al., 2003; Flesch-Janys, 1997; Gustavsson and Hogstedt, 1997; Hay and Tarrel, 1997; Henriksen et al., 1997; Longnecker et al., 2001; Michalek et al., 1998; Steenland et al., 1999; Vena et al., 1998). Abnormal secretion of steroid hormones from adrenal cortex has been suggested to constitute a pathophysiological basis for these diseases, because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, abdominal obesity, vascular function, and cardiac remodeling (details are given in the Discussion) (Bjorntorp, 1997; Delcayre and Swynghedauw, 2002; Grippo and Johnson, 2002; Rosmond and Bjorntorp, 2000; Suzuki et al., 2003). The adrenals, which contain massive blood flow and high lipid content, provide a major accumulation site for lipophilic PCBs and dioxins in the body (Brandt, 1977; Durham and Brouwer, 1990; Matthews and Dedrick, 1984; Pohjanvirta et al., 1990; Weber et al., 1993). Clearance of these organochlorine contaminants from the adrenals is considerably slower than clearance from other target organs like liver and kidney (Brandt, 1977; Durham and Brouwer, 1990). However, the exposure effects of PCBs and dioxins on adrenal steroidogenesis are not well characterized.
Three types of steroid hormones are synthesized in the human adrenal cortex. Dehydroepiandrosterone (DHEA) and androstenedione (A4) are the major androgens produced by the gland. Despite possessing only minimal androgenic activity, these two adrenal androgens can be converted to more potent androgens and estrogens in the peripheral tissues. In humans, particularly in postmenopausal women, a large proportion of active androgens and estrogens are derived from adrenal androgens locally (Labrie, 1991). Cortisol, involved in a variety of physiological processes including metabolism, stress response, immune response, vasoconstriction, growth, and development, is the primary glucocorticoid synthesized in the human adrenal cortex (Stewart, 2002). The third type of adrenal steroid is aldosterone, a mineralocorticoid essential for salt and water homeostasis and vascular tone (Osborn, 1991).
Despite functional differences, all three types of steroids are converted from cholesterol via series of reactions catalyzed by the same group of steroidogenic enzymes, except (1) androgen formation requires both 17-hydroxylase and 17,20-lyase activities of CYP17, whereas cortisol synthesis involves only the 17-hydroxylase activity and aldosterone synthesis needs neither; (2) 21-hydroxylation catalyzed by CYP21B is a step specific for cortisol and aldosterone biosynthesis; and (3) two similar but distinct 11-hydroxylases, CYP11B1 and CYP11B2, are responsible for the final rate-limiting conversion of cortisol and aldosterone, respectively. Adrenocorticotropin (ACTH) secreted from the anterior pituitary plays an important role in stimulating adrenal steroidogenesis. ACTH exercises its steroidogenic effects mainly by activating adenylyl cyclase to generate cAMP after binding to the G protein–coupled membrane receptor. Through the intracellular messenger cAMP, ACTH instantly speeds up the mobilization of cholesterol to the mitochondria, where steroidogenesis is initiated. ACTH/cAMP also raises a delayed increase in steroid yield by transcriptional activation of steroidogenic enzymes (Sewer and Waterman, 2003). In addition to ACTH, circulating potassium and angiotensin II (Ang II) have decisive effects on aldosterone synthesis. Potassium elevates aldosterone synthesis by activating the voltage-gated Ca2+ channel, whereas Ang II acts by inducing protein kinase C activation and Ca2+ release from the intracellular store via the phosphatidylinositol 4,5-bisphosphate signal transduction system (Foster, 2004).
In a previous study (Li et al., 2004b), we have employed the human adrenocortical H295R cell model to study the effects of the most toxic coplanar PCB congener PCB126 on adrenal aldosterone biosynthesis. We found that high levels of PCB126 concomitantly stimulated aldosterone production and steady-state mRNA expression of the key gene CYP11B2. When potassium or Ang II was added to induce aldosterone synthesis, the stimulatory effects of PCB126 were enhanced. However, PCB126 exhibited differential interactions with the potassium and Ang II signals. Although PCB126 and potassium synergistically upregulated CYP11B2 mRNA expression, transcriptional induction of the gene seemed less important in the presence of Ang II (Li et al., 2004b).
This study expands the PCB126 impact investigation to all three types of adrenal steroids. We assessed the production of A4, cortisol, aldosterone, and the biosynthetic intermediates progesterone (P4) and 17-OH-P4 under the influence of PCB126 alone and together with cAMP. We also compared the steroid conversion rates to gene expression levels. The results indicate that PCB126 has differential dose and time effects on basal and cAMP-induced androgen, cortisol, and aldosterone biosynthesis. The alterations in steroid production are closely related to the modulation of PCB126 on mRNA expression and enzyme activity of the pathway-specific steroidogenic genes.
MATERIALS AND METHODS
Cell culture and drug treatment.
Human adrenocortical H295R cells (ATCC, Manassas, VA, USA) were seeded at 1.2 x 104 cells/cm2 in phenol red–free DMEM/F12 medium (Sigma-Aldrich, St Louis, MO) plus 10% charcoal/dextran–treated fetal bovine serum (HyClone, Logan, UT) and cultured at 37°C in a humidified 5% CO2-in-air atmosphere. After attachment, the cells were treated with 3,3',4,4',5-pentachlorobiphenyl (PCB126; AccuStandard, Inc., New Haven, CT) or vehicle dimethyl sulfoxide (DMSO; Sigma-Aldrich) for 10 days. In the dose–response analysis, PCB126 was tested at a final concentration of 0 (0.1% DMSO), 10–9, 10–7, and 10–5 M. In the time effect study, the cells were first grown in the vehicle-containing medium and then switched to the 10–5 M PCB126-supplemented medium for 0, 1, 3, 6 or 10 days during a 10-day course. The medium was refreshed every 2–3 days during the treatment. To assess steroid production, the cells were incubated in serum-free medium for an additional 24 h after the treatment. The effects of ACTH on steroid production were mimicked by addition of 1 mM 8-Br-cAMP (Sigma-Aldrich) to the serum-free medium. Basal and cAMP-induced gene expression was examined using the cells harvested at the end of the 10-day treatment. 1 mM 8-Br-cAMP was added during the last 24 h of treatment.
Steroid measurement.
Cortisol, aldosterone, P4, and 17-OH-P4 secreted to the serum-free medium during the 24-h incubation were measured by radioimmunoassays (Diagnostic Products Corporation, Los Angeles, CA), whereas A4 was quantified with the LC-MS-MS method developed previously (Chang et al., 2003). The yield of each steroid was normalized to the cellular protein content determined by the Micro BCA protein assay (Pierce Biotechnology, Rockford, IL).
Gene expression assay.
The mRNA abundances of steroidogenic genes were measured in pair with the housekeeping gene -actin by reverse transcription-polymerase chain reaction (RT-PCR). RNA isolation, reverse transcription, real-time PCR, primer design, and PCR specificity verification were performed as described previously (Li et al., 2004a). Expression of a target gene in a sample was relatively quantified using the software RelQuant (Roche Diagnostics GmbH, Mannheim, German) by calibration against a target:-actin ratio curve, which was drawn based on the real-time PCR data of a series of diluted RT controls. The mathematical model used in this relative quantification is described by Pfaffl (2001).
Data analysis.
All the data are expressed as means ± S.E. The dose and time effects of PCB126 on steroid yield, gene expression, and product/precursor ratio were analyzed with one-way analysis of variance (ANOVA) in SPSS 10.0 (SPSS INC., Chicago, IL). When the p value of ANOVA was smaller than 0.05, Bonferroni's post-hoc test was performed to analyze the significance of difference between the treatment and control. The significance of 1 mM 8-Br-cAMP stimulation or 10–5 M PCB126 treatment to the product/precursor ratio was determined with Student's t-test in Excel 2000 (Microsoft Corporation, Redmond, WA).
RESULTS
Dose effects of PCB126 on basal and cAMP-induced steroid production
Because of lacking information on the adrenal PCB concentrations in exposed people, the PCB126 doses examined in this study were selected based on the blood and adipose tissue concentrations of "Yu-Cheng" patients who ingested contaminated cooking oil (Chen et al., 1985) and capacitor manufacture workers who had direct contact through their jobs (Wolff et al., 1982). The blood of Yu-Cheng patients sampled 9 to 18 months after the poisoning outbreak contained 10 to 720 ppb of PCBs (about 18 to 1300 ppb on a plasma basis) (Chen et al., 1985). The body burden of PCBs was even higher in industrially exposed workers. Over 2500 ppb was found in the plasma analyzed for ongoing exposure. The adipose tissue concentrations were hundreds to thousands-fold of the plasma levels (Wolff et al., 1982). Taking the lipid content into consideration, we assumed that the adrenal PCB concentrations should be between the blood and adipose tissue concentrations. Hence, we treated the human adrenocortical H295R cells with 10–9 to 10–5 M PCB126 (about 0.33 ppb to 3.3 ppm). Our cytotoxicity analysis showed that PCB126 at 10–6 M or lower concentrations caused no significant changes from the vehicle control; 10–5 M PCB126 slowed H295R cell growth, but it did not alter the cellular morphology (Li et al., 2004b).
The basal P4, 17-OH-P4, aldosterone, and cortisol productions of the H295R cells were gradually elevated in response to increasing concentrations of PCB126 supplemented to the medium in a 10-day treatment (p 0.036, one-way ANOVA). The amounts of P4, 17-OH-P4, aldosterone, and cortisol produced within the 24-h period after the 10-day 10–5 M PCB126 treatment were 1.57 ± 0.14, 1.40 ± 0.14, 3.31 ± 0.85, and 5.03 ± 1.27-fold of the vehicle control, respectively. In contrast, PCB126 suppressed basal A4 synthesis. The 24-h basal A4 yield was lowered by approximately 40–50% after 10 days of treatment with 10–7 M and 10–5 M PCB126 (Fig. 1).
Under stress or other physiological stimuli, the pituitary would secrete ACTH to increase adrenal steroid synthesis. To understand the interaction of PCB126 and ACTH in the regulation of steroidogenesis, 1 mM 8-Br-cAMP (an analog of the intracellular messenger cAMP) was added to the H295R cells during the 24-h incubation period to mimic the ACTH upsurge. The 8-Br-cAMP addition greatly increased the synthesis of all the steroids examined. However, while cAMP and PCB126 synergistically increased aldosterone synthesis, the cAMP treatment abolished the stimulation of PCB126 on P4, 17-OH-P4, and cortisol yields. Similar levels of P4, 17-OH-P4, and cortisol were produced under cAMP induction regardless of the PCB126 concentrations during the 10-day treatment. PCB126 diminished cAMP-induced A4 production in a dose-dependent manner (p = 0.037, one-way ANOVA), but the dose threshold and magnitude of the PCB126-induced inhibition was attenuated in the presence of cAMP as compared to the inhibition in basal synthesis (Fig. 1).
PCB126 effects on mRNA expression and enzyme activity of genes required for A4 biosynthesis
A4 formation involves three genes: CYP11A1, HSD3b2, and CYP17 (Fig. 2A). PCB126 had little effect on either basal or cAMP-stimulated mRNA expression of CYP11A1, the common first-step gene for all three steroidogenic pathways (data not shown). The steady-state mRNA abundance of HSD3b2 was dose-dependently increased by PCB126 under the basal condition (p < 0.001, one-way ANOVA). Basal HSD3b2 expression increased 1.86 ± 0.11-fold when the H295R cells were treated with 10–5 M PCB126 for 10 days. Stimulating the cells with 1 mM 8-Br-cAMP during the last 24 h of treatment blocked the PCB126 dose effect (Fig. 2B). This might explain why the PCB126-elicited P4 and 17-OH-P4 rises in basal production were absent under cAMP stimulation (Fig. 1). PCB126 exerted an obvious dose-dependent inhibition on CYP17 mRNA expression (p = 0.001, one-way ANOVA). Treating with 1 mM 8-Br-cAMP did not alter CYP17 expression. Similar levels of CYP17 mRNA were detected in the absence and presence of cAMP under each examined PCB126 concentration (Fig. 2C).
CYP17 is a bifunctional enzyme containing 17-hydroxylase and 17,20-lyase activities (Nakajin and Hall, 1981). Becausn CYP17 enzyme cannot effectively use 17-OH-P4 as a substrate like its rodent counterparts, A4 in humans is formed from 17-OH-pregnenolone via sequential reactions catalyzed by 17,20-lyase of CYP17 and 3-hydroxysteroid dehydrogenase of HSD3b2 (Auchus et al., 1998). Meanwhile, 3-hydroxysteroid dehydrogenase of HSD3b2 can convert 17-OH-pregnenolone into 17-OH-P4 (Fig. 2A). Therefore, the A4/17-OH-P4 ratio can reflect 17,20-lyase activity as well as the 17-OH-P4/P4 ratio represents 17-hydroxylase activity. Based on the product/precursor ratio estimation, cAMP and PCB126 each influenced the 17-hydroxylase and 17,20-lyase activities differentially. The 24-h treatment with 1 mM 8-Br-cAMP did not affect the conversion of 17-OH-P4 from P4 by 17-hydroxylase, but it reduced the 17,20-lyase activity by one fourth. PCB126 inhibited both 17-hydroxylase and 17,20-lyase activities, particularly the latter. The 10–5 M PCB126 treatment cut off less than 30% of the basal 17-OH-P4/P4 ratio but removed approximately half of the basal A4/17-OH-P4 ratio. cAMP softened the inhibition of PCB126 on 17,20-lyase activity. The relative A4/17-OH-P4 ratio was diminished from 0.76 ± 0.06 to 0.42 ± 0.04 by 10–5 M PCB126 under cAMP stimulation, less than the 50% reduction detected in basal synthesis (Fig. 2D).
The 10–5 M PCB126 treatment reduced CYP17 mRNA expression progressively with time despite the absence or presence of cAMP (p 0.007, one-way ANOVA) (Fig. 3A). Time-dependent inhibition was also observed in the relative 17-OH-P4/P4 and A4/17-OH-P4 ratios (Fig. 3B and 3C). The latter or 17,20-lyase activity appeared more sensitive to the 10–5 M PCB126 treatment. The A4/17-OH-P4 ratio declined significantly after one day of treatment, especially in combination with cAMP. However, the PCB126/cAMP-triggered A4/17-OH-P4 reduction recovered to a fair degree when the PCB126 treatment was extended to 10 days (Fig. 3C).
PCB126 effects on mRNA expression and enzyme activity of genes required for aldosterone and cortisol biosynthesis
CYP21B, together with CYP11B2, can further convert P4 to aldosterone while converting 17-OH-P4 to cortisol in conjunction with CYP11B1 (Fig. 4A). PCB126 displayed opposite effects on CYP21B mRNA expression in the absence and presence of cAMP. Basal CYP21B expression gradually rose 1.46 ± 0.06-fold when the PCB126 concentration was increased from 0 M to 10–5 M (p < 0.001, one-way ANOVA). Treating the H295R cells with 1 mM 8-Br-cAMP raised the steady-state CYP21B mRNA level to 2.26 ± 0.07-fold of the basal control, but it inverted the action of PCB126 from stimulation to suppression. The 10-day 10–5 M PCB126 treatment downregulated cAMP-induced CYP21B mRNA level by nearly 40% (Fig. 4B). CYP11B1 and CYP11B2 expression was extremely low unless cAMP was added. Both genes were insensitive to low concentrations of PCB126 under either the basal conditions or with cAMP stimulation. When the PCB126 concentration was raised to 10–5 M, the steady-state mRNA abundances of both genes went up. The stimulatory effect of 10–5 M PCB126 was particularly enormous on cAMP-induced CYP11B2 expression (Fig. 4C and 4D).
The conversion of cortisol from 17-OH-P4 and aldosterone from P4 was increased by the cAMP treatment (Fig. 4E), along with the transcriptional induction of CYP21B (Fig. 4B), CYP11B1 (Fig. 4C), and CYP11B2 (Fig. 4D). 10–5 M PCB126 elevated both basal and cAMP-stimulated aldosterone/P4 ratios. The rise in ratio evidenced that cAMP and PCB126 acted in synergy in promoting the formation of aldosterone from P4. In contrast, cAMP abolished the stimulation of 10–5 M PCB126 on the 17-OH-P4-to-cortisol conversion (Fig. 4E). This effect might be a result of the PCB126-induced CYP21B repression (Fig. 4B), which neutralized the mild upregulation of CYP11B1 in the presence of cAMP (Fig. 4C).
The time effect analysis showed that basal CYP21B expression had little change after 1 day of 10–5 M PCB126 treatment, but it exhibited an 1.5-fold induction when the treatment was prolonged to 3 days or longer. In contrast, cAMP-induced CYP21B expression was immediately downregulated after 1 day of PCB126 treatment, but no further decrease was detected in the prolonged treatments (Fig. 5A). Basal CYP11B1 expression was also significantly induced by a 3-day or longer treatment with 10–5 M PCB126. When stimulated with cAMP, CYP11B1 mRNA expression was above the vehicle control at the time points of 3 days and 6 days of PCB126 treatment, but fell back after a 10-day treatment (Fig. 5B). 10–5 M PCB126 exerted a time-dependent induction on the CYP11B2 gene under both basal and induced conditions (p < 0.001, one-way ANOVA). However, while 10–5 M PCB126 substantially elevated cAMP-induced CYP11B2 mRNA expression in 1 day, 10 days of PCB126 treatment were required for a significant increase in basal expression (Fig. 5C).
Consistent with basal expression of CYP21B (Fig. 5A) and CYP11B1 (Fig. 5B), the basal cortisol/17-OH-P4 ratio was significantly increased after 3 days of 10–5 M PCB126 treatment, and the elevated ratio was maintained to the end of the test course (Fig. 5D). When combined with cAMP, 10–5 M PCB126 showed similar patterns of time effect on CYP11B1 expression and cortisol/17-OH-P4 ratio, but the significance of the changes between different time points was reduced in the cortisol/17-OH-P4 ratio (Fig. 5B and 5D). The PCB126-elicited downregulation of inducible CYP21B expression shown in Figure 5A might contribute to this reduction. Likewise, the time effect of PCB126 on the aldosterone/P4 ratio echoed the time effect on CYP11B2 expression. The basal aldosterone/P4 ratio exhibited a time-dependent increase alongside basal CYP11B2 expression, and both showed a significant increase above the vehicle control after 10 days of treatment. The cAMP-induced aldosterone/P4 ratio was also elevated with duration of the PCB126 treatment as inducible CYP11B2 expression (Fig. 5C and 5E). However, the immediate downregulation of CYP21B expression in the presence of cAMP (Fig. 5A) seemed to hold back the P4-to-aldosterone conversion at the earlier time points (Fig. 5E).
Dose and time effects of PCB126 on ACTH receptor mRNA expression
Binding to the membrane receptor is a required step for ACTH to trigger cAMP formation and subsequent steroidogenic induction. The expression level of ACTH receptor on the adrenocortical cell surface is therefore an important factor determining ACTH responsiveness (Slawik et al., 2004). It is possible that PCB126 affects the steroidogenic regulation of ACTH by modulating expression of the ACTH receptor in addition to the steroidogenic enzymes examined above. To comprehensively understand the influence of PCB126 on ACTH-mediated regulation, this study has also examined ACTH receptor mRNA expression with and without cAMP stimulation. The dose–response assay demonstrated that PCB126 at 10–5 M significantly elevated both basal and cAMP-induced steady-state mRNA levels of ACTH receptor. The 10–5 M PCB126-elicited transcript increase appeared much greater in the absence of cAMP (3.32 ± 0.22-fold) than in the presence of cAMP (1.45 ± 0.13-fold) as compared to the vehicle control (Fig. 6A). A concentration of 10–5 M PCB126 raised ACTH receptor expression gradually under the basal condition, and plateau was reached after 3 days of treatment. In contrast, 10–5 M PCB126 stimulated inducible expression without delay. However, the inducible mRNA abundance started to decline when the treatment was extended to longer than 6 days (Fig. 6B).
DISCUSSION
Steroid hormones control a wide range of homeostatic processes. Adrenal cortex and gonads are the primary sources of circulating steroid hormones. Dysregulated steroidogenesis in these organs causes systematic pathophysiological changes in the body. There is an increasing amount of investigation indicating that PCBs and dioxins alter sex steroid biosynthesis in the gonads. In contrast, little is known about the effects of exposure on adrenal steroidogenesis, despite the fact that high levels of these chemicals accumulate in the adrenals after ingestion or inhalation (Brandt, 1977; Durham and Brouwer, 1990; Pohjanvirta et al., 1990; Weber et al., 1993). In this study, we demonstrated that coplanar PCB126 had diverse effects on the biosynthesis of all three types of steroids in the human adrenocortical H295R cell line.
High concentrations of PCB126 repressed basal and cAMP-induced CYP17 expression and diminished encoded 17-hydroxylase and 17,20-lyase activities, especially the latter, in the adrenocortical cells. As a consequence, the basal and cAMP-induced androgen production of the adrenocortical cells was greatly reduced. Similar inhibition was observed in the gonadal steroidogenic cells, where gonadotropin regulates steroidogenesis in a mechanism resembling ACTH in the adrenocortical cells (Richards, 2001). The incubation of rat testicular Leydig cells with a 5.2 μM PCB mixture significantly inhibited CYP17 activity and consequently decreased chorionic gonadotropin (hCG)-stimulated androgen production (Kovacevic et al., 1995). Exposure of human luteinized ovarian granulosa cells to 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) also reduced the hCG-stimulated production of estradiol (E2), a steroid downstream of androgen in the sex steroid biosynthetic pathway. As the action of PCB126 on inducible adrenal androgen synthesis, TCDD diminished inducible E2 production without altering the synthesis of intermediates P4 and 17-OH-P4 (Moran et al., 2000). Likewise, the reduction in E2 production was mainly due to the inhibitory effects of TCDD on CYP17 expression and its 17,20-lyase activity (Moran et al., 2003).
Our previous study demonstrated that PCB126 and potassium synergistically stimulated aldosterone synthesis. The synergistic action between PCB126 and potassium primarily involved upregulation of CYP11B2 (Li et al., 2004b). The present study found that high concentrations of PCB126 also activated CYP11B2 expression and elevated aldosterone yield in synergy with cAMP, although the rapid inhibition that PCB126 exerted on cAMP-induced CYP21B expression seemed to slow the conversion of P4 to aldosterone. The control of PCB126 over CYP21B had a more profound effect on the conversion of 17-OH-P4 to cortisol. The suppression of cAMP-induced CYP21B expression by PCB126 apparently neutralized the stimulation of PCB126 on CYP11B1. Hence, PCB126 elevated basal cortisol synthesis but exhibited little effect on cAMP-induced synthesis. Similar results were seen in animal studies. Daily exposure of male Fischer 344 rats to 0.1–25 mg/kg body weight of the PCB mixture Aroclor 1254 by gavage for 15 weeks elevated basal serum corticosterone (the cortisol counterpart in rodents) but did not affect stress-induced corticosterone rise (a result of stress activation of the pituitary-adrenal axis) (Miller et al., 1993). Male Sprague-Dawley rats treated with 125 μg/kg of TCDD also displayed elevated basal plasma corticosterone but showed no difference from the control rats in the stress-induced level (Gorski et al., 1988). At the same time, this study suggested that high concentrations of PCB126 might increase the sensitivity and responsiveness of the adrenocortical cells to ACTH by increasing receptor expression on the membrane. It has been reported that administration of 200 mg/kg of PCB77, a coplanar PCB congener like PCB126, induced similar morphological alterations in rat adrenocortical cells to those detected after ACTH administration (Durham and Brouwer, 1990).
Coplanar PCBs and dioxins can induce mRNA expression of several xenobiotic-metabolizing enzymes, including CYP1A1 and CYP1B1, through the aryl hydrocarbon receptor (AhR). AhR is a ligand-dependent transcription factor that interacts with specific upstream DNA elements and activates transcription of target genes after association with a ligand (Denison and Whitlock, 1995). However, we doubt that PCB126 alters steroidogenic gene expression directly through AhR. So far, no AhR-binding elements have been located in the promoter regions of steroidogenic genes. Moreover, PCB126 seems to have a more acute effect on the xenobiotic-metabolizing CYP1A1 gene than on the steroidogenic CYP genes (Hestermann et al., 2000). Even so, we cannot disregard the possibility that the steroidogenic changes we observed are the ripple effect of AhR-dependent gene activation. We are currently examining the role of AhR in the PCB126-modulated steroidogenesis using AhR antagonists.
The rise of cortisol and aldostereone and the fall of androgen after exposure may increase risk for diabetes mellitus and cardiovascular mortality in highly exposed people (Bertazzi et al., 2001; Cranmer et al., 2000; Fierens et al., 2003; Flesch-Janys, 1997; Gustavsson and Hogstedt, 1997; Hay and Tarrel, 1997; Henriksen et al., 1997; Longnecker et al., 2001; Michalek et al., 1998; Steenland et al., 1999; Vena et al., 1998). It has long been known that the illness of excess cortisol, such as Cushing's syndrome and metabolic syndrome, is associated with glucose intolerance and insulin resistance. Indeed, modulation of the cortisol levels of healthy people, even within the physiological range, can alter the actions of insulin on glucose metabolism (Dinneen et al., 1993; Nielsen et al., 2004). Aldosterone also diminishes insulin sensitivity. It has been shown that aldosterone downregulates insulin receptor expression and insulin binding in human U-937 promonocytic cells (Campion et al., 1999). Potassium depletion in consequence of prolonged aldosterone excess—e.g., primary aldosteronism—also impairs glucose tolerance and insulin secretion (Corry and Tuck, 2003). In contrast, dehydroepiandrosterone (DHEA) increases the binding of insulin to the insulin receptor (Buffington et al., 1991). The reduction of adrenal androgen synthesis owing to exposure likely minimizes such an interaction and lowers insulin sensitivity.
Diabetes mellitus is a well-recognized risk factor for cardiovascular diseases (Carr and Brunzell, 2004; Grundy, 2004; Nesto, 2004). In addition to the injuries secondary to diabetes, steroid hormones have direct effects on the cardiovascular system. Aldosterone regulates vascular electrolyte permeability, blood volume, and vasocontractility. High levels of aldosterone cause hypertension. Chronic animal studies further demonstrate that aldosterone together with high salt intake induces vascular inflammation and causes myocardial ischemia, necrosis, and fibrosis (Rocha and Funder, 2002). High levels of cortisol also provoke hypertension, although the underlying mechanism is not clear.
The association of adrenal steroids with diabetes and cardiovascular diseases also involves their effects on lipid metabolism and body fat distribution (Carr and Brunzell, 2004). Patients with excess cortisol have increased total cholesterol and low-density lipoprotein cholesterol (LDL cholesterol) and decreased high-density lipoprotein cholesterol (HDL cholesterol) (Colao et al., 1999; Tauchmanova et al., 2002). The general population also displays an inverse relationship between cortisol and HDL cholesterol (Fraser et al., 1999), implying that a small but chronic excess of cortisol would distort lipid metabolism. Furthermore, cortisol activates adipocyte differentiation and lipoprotein lipase expression, especially in the abdominal adipose tissue (Fried et al., 1993; Hauner et al., 1989). High lipoprotein lipase activity elevates the release of free fatty acids into circulation as well as fat accumulation in the adipocytes (Mead et al., 2002). Contrary to cortisol, plasma DHEA shows a negative correlation with abdominal fat deposition in men (Tchernof and Labrie, 2004).
Our results put forward plausibility that PCBs and dioxins may alter adrenal steroid synthesis and, in turn, damage metabolism and cardiovascular health. Although there are significant data gaps required to be addressed, our study suggests that adrenal endocrine toxicity is an important potential hazard and should not be ignored in the health effect assessment of PCBs and dioxins.
ACKNOWLEDGMENTS
This work is supported by grant EO-092-PP-07 from National Health Research Institutes, Taiwan, Republic of China.
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Division of Metabolism and Endocrinology, Chang Gung Memorial Hospital, Kaohsiung 833, Taiwan, Republic of China
ABSTRACT
Dioxins and polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal glands when incorporated into the body. However, the impacts of exposure on adrenal steroidogenesis have not been thoroughly investigated. In this study, we demonstrated that dioxin-like PCB126 altered androgen, cortisol, and aldosterone biosynthesis differentially in human adrenocortical H295R cells. PCB126 diminished basal and cAMP-induced androstenedione production as well as CYP17 mRNA expression in a dose-dependent and time-dependent manner. The CYP17 repression was accompanied with decreases in the encoded 17-hydroxylase and 17,20-lyase activities, particularly the latter. In contrast, high concentrations of PCB126 stimulated basal cortisol and aldosterone biosynthesis, including induction of CYP21B, CYP11B1, and CYP11B2 mRNA expression and elevation of the conversion of cortisol from 17-OH-progesterone and aldosterone from progesterone. cAMP abolished the positive effect of PCB126 on cortisol synthesis, while it synergistically enhanced PCB126 stimulation on CYP11B2 expression and aldosterone production. It seemed likely that the downregulation of CYP21B caused by the combination of PCB126 and cAMP counteracted the CYP11B1 induction stimulated by the co-treatment. In addition, high concentrations of PCB126 might sensitize the regulation of adrenocorticotropin (ACTH) on the adrenocortical cells by increasing ACTH receptor levels. Because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, obesity, vascular function, and cardiac remodeling, this article also discusses the potential association of the detected adrenocortical alterations with increased diabetic and cardiovascular risk found among highly exposed people.
Key Words: coplanar PCB; adrenal steroidogenesis; cAMP induction; steroidogenic genes; diabetes; cardiovascular diseases.
INTRODUCTION
Accumulating epidemiological evidence reveals that people exposed to high levels of dioxins and dioxin-like compounds, such as coplanar polychlorinated biphenyls (PCBs), have increased risk of developing diabetes mellitus and cardiovascular diseases (Bertazzi et al., 2001; Cranmer et al., 2000; Fierens et al., 2003; Flesch-Janys, 1997; Gustavsson and Hogstedt, 1997; Hay and Tarrel, 1997; Henriksen et al., 1997; Longnecker et al., 2001; Michalek et al., 1998; Steenland et al., 1999; Vena et al., 1998). Abnormal secretion of steroid hormones from adrenal cortex has been suggested to constitute a pathophysiological basis for these diseases, because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, abdominal obesity, vascular function, and cardiac remodeling (details are given in the Discussion) (Bjorntorp, 1997; Delcayre and Swynghedauw, 2002; Grippo and Johnson, 2002; Rosmond and Bjorntorp, 2000; Suzuki et al., 2003). The adrenals, which contain massive blood flow and high lipid content, provide a major accumulation site for lipophilic PCBs and dioxins in the body (Brandt, 1977; Durham and Brouwer, 1990; Matthews and Dedrick, 1984; Pohjanvirta et al., 1990; Weber et al., 1993). Clearance of these organochlorine contaminants from the adrenals is considerably slower than clearance from other target organs like liver and kidney (Brandt, 1977; Durham and Brouwer, 1990). However, the exposure effects of PCBs and dioxins on adrenal steroidogenesis are not well characterized.
Three types of steroid hormones are synthesized in the human adrenal cortex. Dehydroepiandrosterone (DHEA) and androstenedione (A4) are the major androgens produced by the gland. Despite possessing only minimal androgenic activity, these two adrenal androgens can be converted to more potent androgens and estrogens in the peripheral tissues. In humans, particularly in postmenopausal women, a large proportion of active androgens and estrogens are derived from adrenal androgens locally (Labrie, 1991). Cortisol, involved in a variety of physiological processes including metabolism, stress response, immune response, vasoconstriction, growth, and development, is the primary glucocorticoid synthesized in the human adrenal cortex (Stewart, 2002). The third type of adrenal steroid is aldosterone, a mineralocorticoid essential for salt and water homeostasis and vascular tone (Osborn, 1991).
Despite functional differences, all three types of steroids are converted from cholesterol via series of reactions catalyzed by the same group of steroidogenic enzymes, except (1) androgen formation requires both 17-hydroxylase and 17,20-lyase activities of CYP17, whereas cortisol synthesis involves only the 17-hydroxylase activity and aldosterone synthesis needs neither; (2) 21-hydroxylation catalyzed by CYP21B is a step specific for cortisol and aldosterone biosynthesis; and (3) two similar but distinct 11-hydroxylases, CYP11B1 and CYP11B2, are responsible for the final rate-limiting conversion of cortisol and aldosterone, respectively. Adrenocorticotropin (ACTH) secreted from the anterior pituitary plays an important role in stimulating adrenal steroidogenesis. ACTH exercises its steroidogenic effects mainly by activating adenylyl cyclase to generate cAMP after binding to the G protein–coupled membrane receptor. Through the intracellular messenger cAMP, ACTH instantly speeds up the mobilization of cholesterol to the mitochondria, where steroidogenesis is initiated. ACTH/cAMP also raises a delayed increase in steroid yield by transcriptional activation of steroidogenic enzymes (Sewer and Waterman, 2003). In addition to ACTH, circulating potassium and angiotensin II (Ang II) have decisive effects on aldosterone synthesis. Potassium elevates aldosterone synthesis by activating the voltage-gated Ca2+ channel, whereas Ang II acts by inducing protein kinase C activation and Ca2+ release from the intracellular store via the phosphatidylinositol 4,5-bisphosphate signal transduction system (Foster, 2004).
In a previous study (Li et al., 2004b), we have employed the human adrenocortical H295R cell model to study the effects of the most toxic coplanar PCB congener PCB126 on adrenal aldosterone biosynthesis. We found that high levels of PCB126 concomitantly stimulated aldosterone production and steady-state mRNA expression of the key gene CYP11B2. When potassium or Ang II was added to induce aldosterone synthesis, the stimulatory effects of PCB126 were enhanced. However, PCB126 exhibited differential interactions with the potassium and Ang II signals. Although PCB126 and potassium synergistically upregulated CYP11B2 mRNA expression, transcriptional induction of the gene seemed less important in the presence of Ang II (Li et al., 2004b).
This study expands the PCB126 impact investigation to all three types of adrenal steroids. We assessed the production of A4, cortisol, aldosterone, and the biosynthetic intermediates progesterone (P4) and 17-OH-P4 under the influence of PCB126 alone and together with cAMP. We also compared the steroid conversion rates to gene expression levels. The results indicate that PCB126 has differential dose and time effects on basal and cAMP-induced androgen, cortisol, and aldosterone biosynthesis. The alterations in steroid production are closely related to the modulation of PCB126 on mRNA expression and enzyme activity of the pathway-specific steroidogenic genes.
MATERIALS AND METHODS
Cell culture and drug treatment.
Human adrenocortical H295R cells (ATCC, Manassas, VA, USA) were seeded at 1.2 x 104 cells/cm2 in phenol red–free DMEM/F12 medium (Sigma-Aldrich, St Louis, MO) plus 10% charcoal/dextran–treated fetal bovine serum (HyClone, Logan, UT) and cultured at 37°C in a humidified 5% CO2-in-air atmosphere. After attachment, the cells were treated with 3,3',4,4',5-pentachlorobiphenyl (PCB126; AccuStandard, Inc., New Haven, CT) or vehicle dimethyl sulfoxide (DMSO; Sigma-Aldrich) for 10 days. In the dose–response analysis, PCB126 was tested at a final concentration of 0 (0.1% DMSO), 10–9, 10–7, and 10–5 M. In the time effect study, the cells were first grown in the vehicle-containing medium and then switched to the 10–5 M PCB126-supplemented medium for 0, 1, 3, 6 or 10 days during a 10-day course. The medium was refreshed every 2–3 days during the treatment. To assess steroid production, the cells were incubated in serum-free medium for an additional 24 h after the treatment. The effects of ACTH on steroid production were mimicked by addition of 1 mM 8-Br-cAMP (Sigma-Aldrich) to the serum-free medium. Basal and cAMP-induced gene expression was examined using the cells harvested at the end of the 10-day treatment. 1 mM 8-Br-cAMP was added during the last 24 h of treatment.
Steroid measurement.
Cortisol, aldosterone, P4, and 17-OH-P4 secreted to the serum-free medium during the 24-h incubation were measured by radioimmunoassays (Diagnostic Products Corporation, Los Angeles, CA), whereas A4 was quantified with the LC-MS-MS method developed previously (Chang et al., 2003). The yield of each steroid was normalized to the cellular protein content determined by the Micro BCA protein assay (Pierce Biotechnology, Rockford, IL).
Gene expression assay.
The mRNA abundances of steroidogenic genes were measured in pair with the housekeeping gene -actin by reverse transcription-polymerase chain reaction (RT-PCR). RNA isolation, reverse transcription, real-time PCR, primer design, and PCR specificity verification were performed as described previously (Li et al., 2004a). Expression of a target gene in a sample was relatively quantified using the software RelQuant (Roche Diagnostics GmbH, Mannheim, German) by calibration against a target:-actin ratio curve, which was drawn based on the real-time PCR data of a series of diluted RT controls. The mathematical model used in this relative quantification is described by Pfaffl (2001).
Data analysis.
All the data are expressed as means ± S.E. The dose and time effects of PCB126 on steroid yield, gene expression, and product/precursor ratio were analyzed with one-way analysis of variance (ANOVA) in SPSS 10.0 (SPSS INC., Chicago, IL). When the p value of ANOVA was smaller than 0.05, Bonferroni's post-hoc test was performed to analyze the significance of difference between the treatment and control. The significance of 1 mM 8-Br-cAMP stimulation or 10–5 M PCB126 treatment to the product/precursor ratio was determined with Student's t-test in Excel 2000 (Microsoft Corporation, Redmond, WA).
RESULTS
Dose effects of PCB126 on basal and cAMP-induced steroid production
Because of lacking information on the adrenal PCB concentrations in exposed people, the PCB126 doses examined in this study were selected based on the blood and adipose tissue concentrations of "Yu-Cheng" patients who ingested contaminated cooking oil (Chen et al., 1985) and capacitor manufacture workers who had direct contact through their jobs (Wolff et al., 1982). The blood of Yu-Cheng patients sampled 9 to 18 months after the poisoning outbreak contained 10 to 720 ppb of PCBs (about 18 to 1300 ppb on a plasma basis) (Chen et al., 1985). The body burden of PCBs was even higher in industrially exposed workers. Over 2500 ppb was found in the plasma analyzed for ongoing exposure. The adipose tissue concentrations were hundreds to thousands-fold of the plasma levels (Wolff et al., 1982). Taking the lipid content into consideration, we assumed that the adrenal PCB concentrations should be between the blood and adipose tissue concentrations. Hence, we treated the human adrenocortical H295R cells with 10–9 to 10–5 M PCB126 (about 0.33 ppb to 3.3 ppm). Our cytotoxicity analysis showed that PCB126 at 10–6 M or lower concentrations caused no significant changes from the vehicle control; 10–5 M PCB126 slowed H295R cell growth, but it did not alter the cellular morphology (Li et al., 2004b).
The basal P4, 17-OH-P4, aldosterone, and cortisol productions of the H295R cells were gradually elevated in response to increasing concentrations of PCB126 supplemented to the medium in a 10-day treatment (p 0.036, one-way ANOVA). The amounts of P4, 17-OH-P4, aldosterone, and cortisol produced within the 24-h period after the 10-day 10–5 M PCB126 treatment were 1.57 ± 0.14, 1.40 ± 0.14, 3.31 ± 0.85, and 5.03 ± 1.27-fold of the vehicle control, respectively. In contrast, PCB126 suppressed basal A4 synthesis. The 24-h basal A4 yield was lowered by approximately 40–50% after 10 days of treatment with 10–7 M and 10–5 M PCB126 (Fig. 1).
Under stress or other physiological stimuli, the pituitary would secrete ACTH to increase adrenal steroid synthesis. To understand the interaction of PCB126 and ACTH in the regulation of steroidogenesis, 1 mM 8-Br-cAMP (an analog of the intracellular messenger cAMP) was added to the H295R cells during the 24-h incubation period to mimic the ACTH upsurge. The 8-Br-cAMP addition greatly increased the synthesis of all the steroids examined. However, while cAMP and PCB126 synergistically increased aldosterone synthesis, the cAMP treatment abolished the stimulation of PCB126 on P4, 17-OH-P4, and cortisol yields. Similar levels of P4, 17-OH-P4, and cortisol were produced under cAMP induction regardless of the PCB126 concentrations during the 10-day treatment. PCB126 diminished cAMP-induced A4 production in a dose-dependent manner (p = 0.037, one-way ANOVA), but the dose threshold and magnitude of the PCB126-induced inhibition was attenuated in the presence of cAMP as compared to the inhibition in basal synthesis (Fig. 1).
PCB126 effects on mRNA expression and enzyme activity of genes required for A4 biosynthesis
A4 formation involves three genes: CYP11A1, HSD3b2, and CYP17 (Fig. 2A). PCB126 had little effect on either basal or cAMP-stimulated mRNA expression of CYP11A1, the common first-step gene for all three steroidogenic pathways (data not shown). The steady-state mRNA abundance of HSD3b2 was dose-dependently increased by PCB126 under the basal condition (p < 0.001, one-way ANOVA). Basal HSD3b2 expression increased 1.86 ± 0.11-fold when the H295R cells were treated with 10–5 M PCB126 for 10 days. Stimulating the cells with 1 mM 8-Br-cAMP during the last 24 h of treatment blocked the PCB126 dose effect (Fig. 2B). This might explain why the PCB126-elicited P4 and 17-OH-P4 rises in basal production were absent under cAMP stimulation (Fig. 1). PCB126 exerted an obvious dose-dependent inhibition on CYP17 mRNA expression (p = 0.001, one-way ANOVA). Treating with 1 mM 8-Br-cAMP did not alter CYP17 expression. Similar levels of CYP17 mRNA were detected in the absence and presence of cAMP under each examined PCB126 concentration (Fig. 2C).
CYP17 is a bifunctional enzyme containing 17-hydroxylase and 17,20-lyase activities (Nakajin and Hall, 1981). Becausn CYP17 enzyme cannot effectively use 17-OH-P4 as a substrate like its rodent counterparts, A4 in humans is formed from 17-OH-pregnenolone via sequential reactions catalyzed by 17,20-lyase of CYP17 and 3-hydroxysteroid dehydrogenase of HSD3b2 (Auchus et al., 1998). Meanwhile, 3-hydroxysteroid dehydrogenase of HSD3b2 can convert 17-OH-pregnenolone into 17-OH-P4 (Fig. 2A). Therefore, the A4/17-OH-P4 ratio can reflect 17,20-lyase activity as well as the 17-OH-P4/P4 ratio represents 17-hydroxylase activity. Based on the product/precursor ratio estimation, cAMP and PCB126 each influenced the 17-hydroxylase and 17,20-lyase activities differentially. The 24-h treatment with 1 mM 8-Br-cAMP did not affect the conversion of 17-OH-P4 from P4 by 17-hydroxylase, but it reduced the 17,20-lyase activity by one fourth. PCB126 inhibited both 17-hydroxylase and 17,20-lyase activities, particularly the latter. The 10–5 M PCB126 treatment cut off less than 30% of the basal 17-OH-P4/P4 ratio but removed approximately half of the basal A4/17-OH-P4 ratio. cAMP softened the inhibition of PCB126 on 17,20-lyase activity. The relative A4/17-OH-P4 ratio was diminished from 0.76 ± 0.06 to 0.42 ± 0.04 by 10–5 M PCB126 under cAMP stimulation, less than the 50% reduction detected in basal synthesis (Fig. 2D).
The 10–5 M PCB126 treatment reduced CYP17 mRNA expression progressively with time despite the absence or presence of cAMP (p 0.007, one-way ANOVA) (Fig. 3A). Time-dependent inhibition was also observed in the relative 17-OH-P4/P4 and A4/17-OH-P4 ratios (Fig. 3B and 3C). The latter or 17,20-lyase activity appeared more sensitive to the 10–5 M PCB126 treatment. The A4/17-OH-P4 ratio declined significantly after one day of treatment, especially in combination with cAMP. However, the PCB126/cAMP-triggered A4/17-OH-P4 reduction recovered to a fair degree when the PCB126 treatment was extended to 10 days (Fig. 3C).
PCB126 effects on mRNA expression and enzyme activity of genes required for aldosterone and cortisol biosynthesis
CYP21B, together with CYP11B2, can further convert P4 to aldosterone while converting 17-OH-P4 to cortisol in conjunction with CYP11B1 (Fig. 4A). PCB126 displayed opposite effects on CYP21B mRNA expression in the absence and presence of cAMP. Basal CYP21B expression gradually rose 1.46 ± 0.06-fold when the PCB126 concentration was increased from 0 M to 10–5 M (p < 0.001, one-way ANOVA). Treating the H295R cells with 1 mM 8-Br-cAMP raised the steady-state CYP21B mRNA level to 2.26 ± 0.07-fold of the basal control, but it inverted the action of PCB126 from stimulation to suppression. The 10-day 10–5 M PCB126 treatment downregulated cAMP-induced CYP21B mRNA level by nearly 40% (Fig. 4B). CYP11B1 and CYP11B2 expression was extremely low unless cAMP was added. Both genes were insensitive to low concentrations of PCB126 under either the basal conditions or with cAMP stimulation. When the PCB126 concentration was raised to 10–5 M, the steady-state mRNA abundances of both genes went up. The stimulatory effect of 10–5 M PCB126 was particularly enormous on cAMP-induced CYP11B2 expression (Fig. 4C and 4D).
The conversion of cortisol from 17-OH-P4 and aldosterone from P4 was increased by the cAMP treatment (Fig. 4E), along with the transcriptional induction of CYP21B (Fig. 4B), CYP11B1 (Fig. 4C), and CYP11B2 (Fig. 4D). 10–5 M PCB126 elevated both basal and cAMP-stimulated aldosterone/P4 ratios. The rise in ratio evidenced that cAMP and PCB126 acted in synergy in promoting the formation of aldosterone from P4. In contrast, cAMP abolished the stimulation of 10–5 M PCB126 on the 17-OH-P4-to-cortisol conversion (Fig. 4E). This effect might be a result of the PCB126-induced CYP21B repression (Fig. 4B), which neutralized the mild upregulation of CYP11B1 in the presence of cAMP (Fig. 4C).
The time effect analysis showed that basal CYP21B expression had little change after 1 day of 10–5 M PCB126 treatment, but it exhibited an 1.5-fold induction when the treatment was prolonged to 3 days or longer. In contrast, cAMP-induced CYP21B expression was immediately downregulated after 1 day of PCB126 treatment, but no further decrease was detected in the prolonged treatments (Fig. 5A). Basal CYP11B1 expression was also significantly induced by a 3-day or longer treatment with 10–5 M PCB126. When stimulated with cAMP, CYP11B1 mRNA expression was above the vehicle control at the time points of 3 days and 6 days of PCB126 treatment, but fell back after a 10-day treatment (Fig. 5B). 10–5 M PCB126 exerted a time-dependent induction on the CYP11B2 gene under both basal and induced conditions (p < 0.001, one-way ANOVA). However, while 10–5 M PCB126 substantially elevated cAMP-induced CYP11B2 mRNA expression in 1 day, 10 days of PCB126 treatment were required for a significant increase in basal expression (Fig. 5C).
Consistent with basal expression of CYP21B (Fig. 5A) and CYP11B1 (Fig. 5B), the basal cortisol/17-OH-P4 ratio was significantly increased after 3 days of 10–5 M PCB126 treatment, and the elevated ratio was maintained to the end of the test course (Fig. 5D). When combined with cAMP, 10–5 M PCB126 showed similar patterns of time effect on CYP11B1 expression and cortisol/17-OH-P4 ratio, but the significance of the changes between different time points was reduced in the cortisol/17-OH-P4 ratio (Fig. 5B and 5D). The PCB126-elicited downregulation of inducible CYP21B expression shown in Figure 5A might contribute to this reduction. Likewise, the time effect of PCB126 on the aldosterone/P4 ratio echoed the time effect on CYP11B2 expression. The basal aldosterone/P4 ratio exhibited a time-dependent increase alongside basal CYP11B2 expression, and both showed a significant increase above the vehicle control after 10 days of treatment. The cAMP-induced aldosterone/P4 ratio was also elevated with duration of the PCB126 treatment as inducible CYP11B2 expression (Fig. 5C and 5E). However, the immediate downregulation of CYP21B expression in the presence of cAMP (Fig. 5A) seemed to hold back the P4-to-aldosterone conversion at the earlier time points (Fig. 5E).
Dose and time effects of PCB126 on ACTH receptor mRNA expression
Binding to the membrane receptor is a required step for ACTH to trigger cAMP formation and subsequent steroidogenic induction. The expression level of ACTH receptor on the adrenocortical cell surface is therefore an important factor determining ACTH responsiveness (Slawik et al., 2004). It is possible that PCB126 affects the steroidogenic regulation of ACTH by modulating expression of the ACTH receptor in addition to the steroidogenic enzymes examined above. To comprehensively understand the influence of PCB126 on ACTH-mediated regulation, this study has also examined ACTH receptor mRNA expression with and without cAMP stimulation. The dose–response assay demonstrated that PCB126 at 10–5 M significantly elevated both basal and cAMP-induced steady-state mRNA levels of ACTH receptor. The 10–5 M PCB126-elicited transcript increase appeared much greater in the absence of cAMP (3.32 ± 0.22-fold) than in the presence of cAMP (1.45 ± 0.13-fold) as compared to the vehicle control (Fig. 6A). A concentration of 10–5 M PCB126 raised ACTH receptor expression gradually under the basal condition, and plateau was reached after 3 days of treatment. In contrast, 10–5 M PCB126 stimulated inducible expression without delay. However, the inducible mRNA abundance started to decline when the treatment was extended to longer than 6 days (Fig. 6B).
DISCUSSION
Steroid hormones control a wide range of homeostatic processes. Adrenal cortex and gonads are the primary sources of circulating steroid hormones. Dysregulated steroidogenesis in these organs causes systematic pathophysiological changes in the body. There is an increasing amount of investigation indicating that PCBs and dioxins alter sex steroid biosynthesis in the gonads. In contrast, little is known about the effects of exposure on adrenal steroidogenesis, despite the fact that high levels of these chemicals accumulate in the adrenals after ingestion or inhalation (Brandt, 1977; Durham and Brouwer, 1990; Pohjanvirta et al., 1990; Weber et al., 1993). In this study, we demonstrated that coplanar PCB126 had diverse effects on the biosynthesis of all three types of steroids in the human adrenocortical H295R cell line.
High concentrations of PCB126 repressed basal and cAMP-induced CYP17 expression and diminished encoded 17-hydroxylase and 17,20-lyase activities, especially the latter, in the adrenocortical cells. As a consequence, the basal and cAMP-induced androgen production of the adrenocortical cells was greatly reduced. Similar inhibition was observed in the gonadal steroidogenic cells, where gonadotropin regulates steroidogenesis in a mechanism resembling ACTH in the adrenocortical cells (Richards, 2001). The incubation of rat testicular Leydig cells with a 5.2 μM PCB mixture significantly inhibited CYP17 activity and consequently decreased chorionic gonadotropin (hCG)-stimulated androgen production (Kovacevic et al., 1995). Exposure of human luteinized ovarian granulosa cells to 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) also reduced the hCG-stimulated production of estradiol (E2), a steroid downstream of androgen in the sex steroid biosynthetic pathway. As the action of PCB126 on inducible adrenal androgen synthesis, TCDD diminished inducible E2 production without altering the synthesis of intermediates P4 and 17-OH-P4 (Moran et al., 2000). Likewise, the reduction in E2 production was mainly due to the inhibitory effects of TCDD on CYP17 expression and its 17,20-lyase activity (Moran et al., 2003).
Our previous study demonstrated that PCB126 and potassium synergistically stimulated aldosterone synthesis. The synergistic action between PCB126 and potassium primarily involved upregulation of CYP11B2 (Li et al., 2004b). The present study found that high concentrations of PCB126 also activated CYP11B2 expression and elevated aldosterone yield in synergy with cAMP, although the rapid inhibition that PCB126 exerted on cAMP-induced CYP21B expression seemed to slow the conversion of P4 to aldosterone. The control of PCB126 over CYP21B had a more profound effect on the conversion of 17-OH-P4 to cortisol. The suppression of cAMP-induced CYP21B expression by PCB126 apparently neutralized the stimulation of PCB126 on CYP11B1. Hence, PCB126 elevated basal cortisol synthesis but exhibited little effect on cAMP-induced synthesis. Similar results were seen in animal studies. Daily exposure of male Fischer 344 rats to 0.1–25 mg/kg body weight of the PCB mixture Aroclor 1254 by gavage for 15 weeks elevated basal serum corticosterone (the cortisol counterpart in rodents) but did not affect stress-induced corticosterone rise (a result of stress activation of the pituitary-adrenal axis) (Miller et al., 1993). Male Sprague-Dawley rats treated with 125 μg/kg of TCDD also displayed elevated basal plasma corticosterone but showed no difference from the control rats in the stress-induced level (Gorski et al., 1988). At the same time, this study suggested that high concentrations of PCB126 might increase the sensitivity and responsiveness of the adrenocortical cells to ACTH by increasing receptor expression on the membrane. It has been reported that administration of 200 mg/kg of PCB77, a coplanar PCB congener like PCB126, induced similar morphological alterations in rat adrenocortical cells to those detected after ACTH administration (Durham and Brouwer, 1990).
Coplanar PCBs and dioxins can induce mRNA expression of several xenobiotic-metabolizing enzymes, including CYP1A1 and CYP1B1, through the aryl hydrocarbon receptor (AhR). AhR is a ligand-dependent transcription factor that interacts with specific upstream DNA elements and activates transcription of target genes after association with a ligand (Denison and Whitlock, 1995). However, we doubt that PCB126 alters steroidogenic gene expression directly through AhR. So far, no AhR-binding elements have been located in the promoter regions of steroidogenic genes. Moreover, PCB126 seems to have a more acute effect on the xenobiotic-metabolizing CYP1A1 gene than on the steroidogenic CYP genes (Hestermann et al., 2000). Even so, we cannot disregard the possibility that the steroidogenic changes we observed are the ripple effect of AhR-dependent gene activation. We are currently examining the role of AhR in the PCB126-modulated steroidogenesis using AhR antagonists.
The rise of cortisol and aldostereone and the fall of androgen after exposure may increase risk for diabetes mellitus and cardiovascular mortality in highly exposed people (Bertazzi et al., 2001; Cranmer et al., 2000; Fierens et al., 2003; Flesch-Janys, 1997; Gustavsson and Hogstedt, 1997; Hay and Tarrel, 1997; Henriksen et al., 1997; Longnecker et al., 2001; Michalek et al., 1998; Steenland et al., 1999; Vena et al., 1998). It has long been known that the illness of excess cortisol, such as Cushing's syndrome and metabolic syndrome, is associated with glucose intolerance and insulin resistance. Indeed, modulation of the cortisol levels of healthy people, even within the physiological range, can alter the actions of insulin on glucose metabolism (Dinneen et al., 1993; Nielsen et al., 2004). Aldosterone also diminishes insulin sensitivity. It has been shown that aldosterone downregulates insulin receptor expression and insulin binding in human U-937 promonocytic cells (Campion et al., 1999). Potassium depletion in consequence of prolonged aldosterone excess—e.g., primary aldosteronism—also impairs glucose tolerance and insulin secretion (Corry and Tuck, 2003). In contrast, dehydroepiandrosterone (DHEA) increases the binding of insulin to the insulin receptor (Buffington et al., 1991). The reduction of adrenal androgen synthesis owing to exposure likely minimizes such an interaction and lowers insulin sensitivity.
Diabetes mellitus is a well-recognized risk factor for cardiovascular diseases (Carr and Brunzell, 2004; Grundy, 2004; Nesto, 2004). In addition to the injuries secondary to diabetes, steroid hormones have direct effects on the cardiovascular system. Aldosterone regulates vascular electrolyte permeability, blood volume, and vasocontractility. High levels of aldosterone cause hypertension. Chronic animal studies further demonstrate that aldosterone together with high salt intake induces vascular inflammation and causes myocardial ischemia, necrosis, and fibrosis (Rocha and Funder, 2002). High levels of cortisol also provoke hypertension, although the underlying mechanism is not clear.
The association of adrenal steroids with diabetes and cardiovascular diseases also involves their effects on lipid metabolism and body fat distribution (Carr and Brunzell, 2004). Patients with excess cortisol have increased total cholesterol and low-density lipoprotein cholesterol (LDL cholesterol) and decreased high-density lipoprotein cholesterol (HDL cholesterol) (Colao et al., 1999; Tauchmanova et al., 2002). The general population also displays an inverse relationship between cortisol and HDL cholesterol (Fraser et al., 1999), implying that a small but chronic excess of cortisol would distort lipid metabolism. Furthermore, cortisol activates adipocyte differentiation and lipoprotein lipase expression, especially in the abdominal adipose tissue (Fried et al., 1993; Hauner et al., 1989). High lipoprotein lipase activity elevates the release of free fatty acids into circulation as well as fat accumulation in the adipocytes (Mead et al., 2002). Contrary to cortisol, plasma DHEA shows a negative correlation with abdominal fat deposition in men (Tchernof and Labrie, 2004).
Our results put forward plausibility that PCBs and dioxins may alter adrenal steroid synthesis and, in turn, damage metabolism and cardiovascular health. Although there are significant data gaps required to be addressed, our study suggests that adrenal endocrine toxicity is an important potential hazard and should not be ignored in the health effect assessment of PCBs and dioxins.
ACKNOWLEDGMENTS
This work is supported by grant EO-092-PP-07 from National Health Research Institutes, Taiwan, Republic of China.
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