SETAC
PRESS
EFFECTS OF BROMINATED FLAME RETARDANTS AND BROMINATED DIOXINS ON STEROIDOGENESIS IN H295R HUMAN ADRENOCORTICAL CARCINOMA CELL LINE
LING DING, ; MARGARET B. MURPHY,# YUHE HE,# YAN XU,# LEO W.Y. YEUNG,# JINGXIAN WANG, } BINGSHENG ZHOU, *; PAUL K.S. LAM,¿ RUDOLF S.S. WU,# and JOHN P. GIESY§
+State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, People’s Republic of China #Center for Coastal Pollution and Conservation, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong Special Administrative Region, People’s Republic of China
§National Food Safety and Toxicology Center, Department of Zoology, and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824, USA
(Received 31 July 2006; Accepted 30 October 2006)
Abstract-Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo-p-dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genes- cytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3ß-hydroxysteroid dehydrogenase (3ßHSD2), 17ß-hydroxy- steroid dehydrogenase (17BHSD1 and 17ßHSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3BHSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo-p-dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption.
Keywords-Brominated flame retardants Bromodioxins
Steroidogenesis H295R Gene expression
INTRODUCTION
Brominated flame retardants (BFRs), such as polybromi- nated biphenyls (PBBs), bromophenols, tetrabromobisphenol A (TBBPA), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane have been used in large quantities to reduce fire risk in electrical equipment, cars, construction materials, plastics, foams, and textiles. These compounds are ubiquitous in sediments and biota, including marine mammals and humans [1]. Recently, BFRs have caused greater concern, because concentrations of some BFRs measured in biota have increased significantly [1,2].
Among these BFRs, bromophenols, such as 2,4-dibromo- phenol (DBP), 2,4,6-tribromophenol (TBP), and pentabromo- phenol (PBP), represent high-volume flame retardants [3]. Greater concentrations of 2,4,6-TBP have been detected in marine mussels and mammals [4] as well as in human milk and serum [5]. A commercial flame retardant containing PBBs accidentally added to animal feed in Michigan, USA, in 1975 resulted in loss of livestock and long-term impacts on the health of people who consumed the PBB-contaminated meat [6]. Although PBBs were phased out from the market during the 1970s in the United States and in the year 2000 in Europe, they are more resistant to degradation than polychlorinated biphenyls (PCBs) and have been measured at significant con-
centrations in the environment, including in birds [7], marine mammals [8], and humans [9].
The principal sources of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) are known to be combustion and thermal processes, but polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) are emitted into the atmosphere primarily from electronic waste recycling facilities and other thermal treatment of BFRs [10]. For example, significant amounts of these compounds are produced by combustion of television and circuit boards in electronic waste incinerators, accidental fires, municipal and industrial waste incineration, and burning of automotive fuels containing BFRs, such as PBDEs [11]. In addition, PBDD/Fs can be formed in the pro- cess of manufacturing bromophenols and TBBPA. Recent studies have shown that bromophenols can form great yields of PBDD/Fs from high-temperature oxidation [12]. Because bromine inhibits combustion, the incomplete combustion like- ly would produce a significant amount of PBDD/Fs. Indeed, PBDD/Fs were detected in flue gas, fly ash, and bottom ash of municipal solid waste incineration plants [13], and consid- erable levels of PBDD/Fs have been detected in the atmosphere [14], in blue mussels (Mytilus edulis) [15], and in the liver and eggs of common cormorants (Phalacrocorax carbo) [7]. Although the detected levels of PBDD/Fs were relatively less those of than PCDD/Fs, the rapid increase in the combustion of electrical waste may raise environmental concerns. The World Health Organization assessment of PBDD/Fs and mixed
* To whom correspondence may be addressed (bszhou@ihb.ac.cn).
StAR
HMGR
Cholesterol
CYP11A
CYP17
17a-OH-
CYP17
Pregnenolone
Pregnenolone
DHEA
3B-HSD
CYP17
3ß-HSD
CYP17
3฿ -HSD
Progesterone
17 a -OH-
Androstene
CYP21
Progesterone
-dione
11-Deoxy-
CYP21
17฿ -HSD
corticosterone
11-Deoxycortisol
Testosterone
CYP11B1
CYP19
CYP11B1
Corticosterone
Cortisol
17ß -estradiol
CYP11B2
Aldosterone
Zona fasciculata
Zona reticularis
Zona glomerulosa
brominated-chlorinated dioxins and dibenzofurans concluded that PBDD/Fs are persistent and toxic and that humans and the environment should be protected from these compounds. However, few studies on the toxicity of brominated dioxins have been undertaken.
The BFRs can cause toxicity through thyroidogenic, dioxin- like, and estrogenic mechanisms [16]. Classic 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD)-like responses, such as an- tiestrogenic activity, have been reported for some BFRs. Ev- idence exists that BFRs, particularly PBDEs, disrupt thyroid hormone function and interact with thyroid hormone receptor [16]. Some bromophenol congeners are suspected of disrupting endocrine function through thyroid hormone-like activity. For example, 2,4,6-TBP is suspected to have thyroid hormone- like activity by binding to transthyretin with greater affinity than that of thyroxine itself [16], whereas 2,4-DBP binds to the human estrogen receptor [17]. The PBBs also interact with the endocrine system, and exposure of rats and pigs has re- sulted in decreases in serum triiodothyronine and thyroxine [18]. In addition, several studies have demonstrated that some BFRs exert their effects on steroidogenic enzymes [19,20]. A recent study showed that exposure to PBDE 99 resulted in decreases in sex hormones (e.g., testosterone and 17ß-estra- diol) in male rates in vivo, leading to interference with sexual development and dimorphic behavior [21].
A novel bioassay based on the human adrenocortical car- cinoma cell line (H295R) has been used for screening endo- crine-disrupting chemicals. The H295R cells have physiolog- ical characteristics of zonally undifferentiated human fetal ad- renal cells and has been shown to have the ability to produce most of the steroid hormones of the three phenotypically dis- tinct zones found in the adult adrenal cortex [22] (Fig. 1). Heneweer et al. [23] showed that exposure of H295R cells to atrazine resulted in a threefold induction of aromatase activity, but no induction of this enzyme activity occurred in rat Leydig cell carcinoma cell line (R2C), suggesting that H295R cells are more sensitive than R2C cells. Although the mechanisms of disruption of corticosteroid production are currently still
unknown, Sanderson et al. [24] studied the induction of aro- matase (CYP19) activity by various classes of pesticides, sug- gesting that the mechanism of aromatase induction is mediated through inhibition of phosphodiesterase activity [24]. The H295R human adrenocortical carcinoma cell line has been suggested as a potential model for screening adrenocortical toxicity and steroidogenesis [25,26], and these cells have been used to examine the effects of PCBs on adrenal aldosterone biosynthesis [27], various pesticides and synthetic flavonoid compounds on CYP19 activity [28,29], and phthalate esters and alkylphenols [30] on steroid enzyme activities. Recently, a gene expression-based assay has been used for assessment of chemicals as well as environmental samples on steroido- genic gene expression using this cell line [25,31-33].
In the present study, we examined the effects of BFRs and related chemicals (e.g., PBDD/Fs) on the expression of key steroidogenic genes to determine whether these compounds could disrupt endocrine function through modulation of ste- roidogenesis. These genes include CYP11A (side-chain cleav- age enzyme), CYP11B2 (aldosterone synthetase), CYP17 (ste- roid 17a-hydroxylase/17,20-lyase), CYP19 (aromatase), CYP21 (steroid 1-hydroxylase), 3BHSD2 (3ß-hydroxysteroid dehydrogenase isomerase), 17ßHSD (17ß-hydroxysteroid de- hydrogenase), StAR (steroidogenic acute regulatory protein), and HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reduc- tase).
MATERIALS AND METHODS
Chemicals
Bromophenols, 2-bromophenol (2-BP; purity, >99.0%), 2,4-DBP (purity, >99.5%), 2,6-DBP (purity, >98.0%), 2,4,6-TBP (purity, >98.6%), PBP (purity, >99.0%), and 3,3,4,4’,5,5’-hexabromobiphenyl (PBB 169; purity, >95.0%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The 3,3’,4,4’-tetrabromobiphenyl (PBB 77; purity, >99.7%), 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD; purity, >99.0%), and 2,3,7,8-tetrabromodibenzofuran (TBDF; purity, >99.0%) were purchased from AccuStandard (New Haven, CT, USA). Dimethyl sulfoxide (DMSO) was first added into all standards, and the original solvent (toluene, methanol, and iso-octane) was then blown out under a gentle stream of nitrogen before preparing their dilution series and stored at -20℃. The final solvent concentration in the culture medium did not exceed 0.5% (v/v).
Cell culture
The H295R human adrenocortical carcinoma cells (Amer- ican Type Culture Collection, Beltsville, VA, USA) were cul- tured in 1:1 (v/v) Dulbecco’s modified Eagle’s medium/F-12 Ham nutrient mixture. The culture medium contained 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer (Sigma-Aldrich), 1% insulin-transferring sodium sele- nite plus Premix (BD Bioscience, San Jose, CA, USA), 2.5% Nu-Serum (BD Bioscience), 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The H295R cells were cultured at 37℃ in 5% CO2. The culture medium was changed every 2 to 3 d.
Cell viability assay and exposure conditions
To test for cytotoxicity, the H295R cells were seeded in 96-well plates (Falcon, Franklin Lakes, NJ, USA) at a con- centration of 1 × 105 cells/well. When the cells reached ap- proximately 70% confluence, DMSO dissolved standards were diluted with culture medium to the desired concentrations and
16
I
I
1
12
0
8
4
0
DMSO
2-BP
2,4-DBP
2,6-DBP
2,4,6-TBP
PBP
PBB77
PBB169
TBDD
TBDF
added to each well. Three wells were used for each treatment and control as triplicates. After 48 h of exposure, the cell viability was assayed by using the Sulforhodamine B assay (Sigma-Aldrich). All plates included a negative control with no treatment (blank) and a solvent control treated with 0.5% DMSO. For gene expression examination, 1 × 106 cells were seeded in a six-well plate (Nalgene Nunc, Rochester, NY, USA) and cultured for 48 h. The culture medium was removed, fresh medium added, and the cells exposed to three concentrations of the test compounds for another 48 h. Each treatment was tested in triplicate. The cell condition was checked regularly under a microscope.
RNA isolation
Total RNA was isolated with the SV Total RNA Isolation system (Promega, Madison, WI, USA) following the manu- facturer’s introductions. Purity of the prepared RNA was ver- ified by agarose gel electrophoresis, and the quantity of the RNA sample was determined using ultraviolet photometry (Eppendorf, Hamburg, Germany). The purified RNA was used immediately for reverse transcription (RT) or stored at -80℃ until analysis. The housekeeping gene, ß-actin, which is ubiq- uitously expressed and the expression level of which does not vary under experimental conditions in the present study (Fig. 2), was used as an internal control to standardize the amount of sample RNA.
First-strand cDNA synthesis
First-strand cDNA synthesis was performed by use of the Superscript™ first-strand synthesis system (Invitrogen, Carls- bad, CA, USA). Total RNA (2 µg) was combined with 0.5 µg of biotinylated oligo (dT)12-18 and 0.5 mM deoxynucleotide triphosphate nucleotides, then RNase-free water was added to a final volume of 10 pl. Samples were denatured at 65℃ for 5 min and quickly chilled on ice for another 5 min. Reverse transcription was performed in a 9-ul reaction mixture con- taining the following buffer: 2 ul of 10X RT buffer, 4 ul of 25 mM MgCl2, 1ul of RNaseOUT (40 U/ul; Invitrogen), and 2 ul of RNase-free H2O. The mixtures were incubated at 42℃ for 2 min, then 50 U of SuperScript II RT (Invitrogen) were added. The reaction was incubated at 42℃ for 50 min and then inactivated by heating at 70°℃ for 15 min. To digest RNA, 1 pl of RNase H (2 U/pl) was added to each tube and incubated at 37°℃ for 20 min.
Real-time polymerase chain reactions
Real-time polymerase chain reaction (PCR; quantitative PCR) was performed by using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in sterile, 96-well PCR plates (Applied Biosystems). The cDNA samples were then diluted five times with RNase-free H2O. The PCR reaction mixtures (20 pl) contained 1× SYBR Green™ PCR Master Mix (Applied Biosystems), 0.2 to 0.4 uM sense and antisense gene-specific primers (Table 1), and a 1-pg cDNA sample. Samples were denatured at 95°℃ for 10 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing with extension for 1 min at 60℃, and a final cycle of 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. Melting curve analyses were performed after the 60°℃ stage of the final cycle to differentiate between desired PCR products and prim- er-dimers or DNA contaminants.
The quantity of product amplicon was derived from the threshold cycle (C) number, which is determined as the lowest cycle at which significant signal over background noise (setup threshold) is observed. The C, number is proportional to the starting amount of the nucleic acid. The fold-change can be calculated using the following formulae:
AC, = Ct target - Ct B-actin A(AC) = ACt exp - ACt con Fold change = Xexp/Xcon = 2-A(AC)
| Gene | Product length (bp) | Concn. (MM) | Sense primer | Antisense primer |
|---|---|---|---|---|
| ß-Actin | 100 | 0.2 | 5'-CACCTTCCAGCCTTCCTTCC-3' | 5'-AGGTCTTTGCGGATGTCCAC-3' |
| CYP11A | 137 | 0.4 | 5'-GAGATGGCACGCAACCTGAAG-3' | 5'-CTTAGTGTCTCCTTGATGCTGGC-3' |
| CYP11B2 | 146 | 0.4 | 5'-TCCAGGTGTGTTCAGRAGTTCC-3' | 5'-GAAGCCATCTCTGAGGTCTGTG-3' |
| CYP17 | 134 | 0.4 | 5'-AGCCGCACACCAACTATCAG-3' | 5'-TCACCGATGCTGGAGTCAAC-3' |
| CYP19 | 128 | 0.2 | 5'-AGGTGCTATTGGTRCATCTTGCTC-3' | 5'-TGGTGGAATCGGGTCTTTATGG-3' |
| CYP21 | 108 | 0.4 | 5'-CGTGGTGCTGACCCGACTG-3' | 5'-GGCTGCATCTTGAGGATGACAC-3' |
| 3ßHSD2 | 95 | 0.4 | 5'-TGCCAGTCTTCATCTACACCAG-3' | 5'-TTCCCAGAGGCTCTTCTTCGTG-3' |
| 17@HSD1 | 136 | 0.4 | 5'-CTCCCTCTGACCAGCAACC-3' | 5'-TGTGTCTCCCACGCAATCTC-3' |
| 17ßHSD4 | 121 | 0.4 | 5'-TGCGGGATCACGGATGACTC-3' | 5'-GCCACCATTCTCCTCACAACTC-3' |
| StAR | 168 | 0.2 | 5'-GTCCCACCCTGCCTCTGAAG-3' | 5'-CATACTCTAAACACGAACCCCACC-3' |
| HMGR | 152 | 0.4 | 5'-TGCTTGCCGAGCCTAATGAAAG-3' | 5'-AGAGCGTTCGTGGGTCCATC-3' |
a bp = base pairs; CYP= cytochrome P450; 3ßHSD2 = 3ß-hydroxysteroid dehydrogenase isomerase; 17ßHSD = 17ß-hydroxysteroid dehydro- genase; StAR = steroidogenic acute regulatory protein; HMGR = 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
A (2-BP)
7
DMSO
0.17 μΜ
6:
1.74 μΜ
Fold
17.4 μΜ
N
1
C
CYP11A
CYP11B2
CYP17
CYP19
CYP21
3ßHSD2
17@HSD1
17ßHSD4
StAR
HMGR
B (2,4-DBP)
8
DMSO
0.12 µM
6
1.20 μΜ
Fold
12.0 μΜ
A
N
O
CYP11A
CYP11B2
CYP17
CYP19
CYP21
3ßHSD2
17@HSD1
17@HSD4
StAR
HMGR
C (2,6-DBP)
10
DMSO
0.2 μΜ
2.0 μΜ
Fold
20.0 μΜ
P
2
0
CYP11A
CYP11B2
CYP17
CYP19
CYP21
3BHSD2
178HSD1
178HSD4
StAR
HMGR
D (2,4,6-TBP)
20
DMSO
0.09 μΜ
0.9 μΜ
Fold
9.0 μΜ
0
CYP11A
CYP11B2
CYP17
CYP19
CYP21
3ßHSD2
17BHSD1
17BHSD4
StAR
HMGR
E (PBP)
12
DMSO
10
0.001μΜ
0.14 µM *
20.0 μΜ
Fold
8
6
4
N
O
CYP11A
CYP11B2
CYP17
CYP19
CYP21
3BHSD2
17BHSD1
17BHSD4
StAR
HMGR
where AC, represents the value of each tested gene normalized by the C, for B-actin; A(AC) represents the difference between AC, values for the solvent control and chemically exposed cells; Xexp and Xcon represent the degree of expression in the exposed and control samples, respectively; and Xexp/Xcon rep- resents the fold-induction [31]. All data were reported and statistically analyzed as fold-induction between exposed and solvent control cells. Gene expression levels were measured in triplicate for each treatment and controls, and each exposure was repeated three times in the same experiment.
Statistical analysis
Statistical analysis of gene expression profiles was per- formed using SPSS (Ver 12.0; SPSS, Chicago, IL, USA). The gene expressions were checked for homogeneity of variances by Levene’s test. Because the data failed to pass the tests, a logarithmic transformation was performed on the data, and the data were checked again for homogeneity of variances. When the assumptions for homogeneity of variances were met, dif- ferences in gene expression were evaluated by one-way anal- ysis of variance followed by Tukey’s test. The criterion for statistical significance was p < 0.05.
RESULTS
The H295R cells were exposed to three concentrations of five brominated phenols, two PBBs, and TBDD/TBDF for 48 h, and relative responses of 10 genes involved in the steroido- genic pathway were evaluated. Cell viability was not affected by exposure to the tested concentrations. Expression profiles of 10 steroidogenic genes varied in response to these chemi- cals.
Effects of brominated phenols on adrenocortical steroidogenesis
Exposure to 0.17 u.M 2-BP significantly up-regulated CYP17, CYP19, 3BHSD2, and 17BHSD1 by 1.8-, 1.9-, 6.6-, and 1.7-fold, respectively, compared to the control (Fig. 3A). Treatment with greater concentrations (1.74 p.M) further re- sulted in a significant increase of 17BHSD4 induction by 2.3- fold (Fig. 3A). Exposure to the greatest tested concentration of 17.4 uM resulted in increased expression of CYP11A, 3BHSD2, and StAR by 1.9-, 6.7-, and 1.6-fold, respectively (Fig. 3A). Among the genes examined, 3ßHSD2 was the most markedly up-regulated (more than sixfold), whereas CYP11B2, CYP21, and HMGR mRNA levels remained unchanged after exposure to all concentrations.
Treatment with 2,4-DBP resulted in up-regulation of most of the steroidogenic genes measured and showed a good con- centration-dependent response (Fig. 3B). The 3BHSD2, 17BHSD1, and 17ßHSD4 genes were induced after exposure
← Fig. 3. Effects of bromophenols on steroidogenic gene expression in the H295R cells. (A) 2-Bromophenol (2-BP). (B) 2,4-dibromophenyl (2,4-BDP). (C) 2,6-dibromophenyl (2,6-BDP). (D) 2,4,6-Tribromo- phenol (2,4,6-TBP). (E) Pentabromophenol (PBP). Relative mRNA expression represents the fold-change with exposure to 0.5% dimethyl sulfoxide (DMSO). Expression of steroidogenic genes was normalized to the expression of ß-actin. Fold-induction represents the increase in expression compared to the relevant solvent control. Values are the means of three determinations on each of three replicate exposures (mean ± standard deviation). An asterisk indicates a statistically sig- nificant (p < 0.05) difference from the DMSO control. See Figures 1 and 2 for definitions of the other abbreviations.
to all concentrations (0.12, 1.2, and 12 (M), and CYP11A, CYP11B2, and CYP17 expression increased moderately with exposure to 1.2 and 12 µM 2,4-DBP. Significantly up-regulated expression of CYP19, StAR, and HMGR of 3.2, 1.8-, and 1.5- fold, respectively, was observed only at the greatest concen- tration (12 µM). As with 2-BP, 3BHSD2 was the most sig- nificantly up-regulated gene (more than fivefold). Expression of CYP21 was not significantly altered by any concentration of 2,4-DBP.
In the 2,6-DBP exposure, suppressed expression of most of the tested genes generally was observed after treatment with the lowest concentration (0.2 p.M) (Fig. 3C), but no significant difference in expression was seen after exposure to greater concentrations (2 and 20 µM), with the exception of 3BHSD2, which was the only gene that was significantly up-regulated at all tested concentrations (1.9- to 8.4-fold). Expression of the CYP19 gene was not significantly altered.
In the exposure to 2,4,6-TBP, 3BHSD2 was the most mark- edly induced gene, with greater than 15-fold induction at all test concentrations (0.09, 0.9, and 9 p.M) (Fig. 3D). The CYP11A, CYP19, and StAR genes also were significantly up- regulated by approximately 1.5-fold at the greatest concentra- tion (9 p.M). In contrast, suppression of CYP11B2 and CYP17 (1.6-fold) was observed at all three concentrations, and CYP21 was suppressed (1.6-fold) after treatment at concentrations of 0.9 and 9 p.M. None of the other genes was significantly af- fected.
Treatment with PBP resulted in a complicated gene ex- pression profile (Fig. 3E). Up-regulation of gene expression generally was observed. The 3BHSD2 gene showed the great- est PBP-induced activation (10.3-fold) among all the genes examined. Expression of the CYP11A, CYP11B2, CYP17, CYP19, CYP21, and 17BHSD1 genes was less induced de- pending on the exposure concentrations. The CYP21 and 17BHSD1 genes were less induced (1.5- to 1.7-fold), whereas the CYP11B2 gene was moderately induced by 5.7- to 7.3- fold. For HMGR, a significant induction of gene expression (1.5-fold) was observed at 0.14 µM, and a slight but significant suppression of the gene (1.4-fold) was observed at 20 p.M. No effect of PBP on 17BHSD4 and StAR was observed.
Effects of PBBs on adrenocortical steroidogenesis
In the exposure to PBB 77, up-regulation of gene expression generally was observed in most of the genes examined (Fig. 4A). Expression of the CYP11A, CYP11B2, CYP19, CYP21, 3BHSD2, and 17ßHSD1 genes was elevated by more than 1.6- fold compared to the solvent control. The StAR mRNA levels increased by 2.4-fold, and CYP11B2 was the most induced, by more than 20-fold at the greatest concentration (3.75 p.M). No significant induction of 17BHSD4 and HMGR gene ex- pression was observed on exposure to the lowest concentration (0.06 µM), but significantly greater gene expression was ob- served at the median concentration (0.5 M).
After PBB 169 exposure, 3BHSD2 was the most stimulated among the steroidogenic genes examined, with a 16-fold in- duction at the greatest concentration (4.0 p.M) (Fig. 4B). The CYP11A, CYP11B2, CYP17, CYP19, and 17@HSD1 genes also were up-regulated depending on the concentration (Fig. 4B). However, complex gene expression profiles were ob- served for 17BHSD1, StAR, and HMGR, in which both up- regulation and suppression of gene expression were observed depending on the exposure concentrations.
A (PBB77)
22
DMSO
*
0.06 μΜ
0.5 μΜ
*
Fold
4
3.7 μΜ
*
*
*
N
0
CYP11A
CYP11B2
CYP17
CYP19
CYP21
3ßHSD2
17@HSD1
17@HSD4
StAR
HMGR
₭
B (PBB169)
16
*
6
DMSO
0.001 μΜ
0.06 µM
Fold
4
4.0 μΜ
N
C
CYP11A
CYP11B2
CYP17
CYP19
CYP21
38HSD2
178HSD1
17BHSD4
StAR
HMGR
Effects of TBDD/TBDF on adrenocortical steroidogenesis
Only CYP11B2 and 3BHSD2 were up-regulated after ex- posure to TBDD (>1.5-fold) (Fig. 5A). Expression of the CYP11A, CYP17, CYP21, and 17ßHSD4 genes was signifi- cantly suppressed by 1.6- to 2.2-fold. None of the other genes was induced compared to the control.
The TBDF exposure induced 3BHSD2 expression by 15- to 20-fold (Fig. 5B). A lesser concentration (0.005 µM) in- duced greater mRNA expression in CYP11A, CYP11B2, CYP19, 17BHSD1, 17BHSD4, StAR, and HMGR. The CYP17 gene was not affected, and a 1.8-fold suppression of CYP21 was observed with exposure to all concentrations (0.005, 0.05, and 0.5 p.M) (Fig. 5B). Moreover, a good concentration-de- pendent response was observed for CYP19, 17ßHSD4, and HMGR.
DISCUSSION
In the present study, we used the H295R cells to demon- strate the effects of BPs, PBBs, and TBDD/TBDF on mRNA expression of steroidogenic genes. Previous studies have shown that these toxicants can affect thyroid hormones, the aryl hydrocarbon receptor, and the estrogen receptor and, hence, have endocrine-disruptive activities [16,34]. Therefore, the present study further supports the mechanism of endocrine disruption via a non-receptor mediated pathway as indirect or direct stimulators or inhibitors of the key enzymes involved in the metabolism of steroid hormones [24]. By using H295R cells, Sanderson et al. [24] studied the induction of CYP19 activity by various classes of pesticides, suggesting that the
A (TBDD)
*
2.5
*
DMSO
0.004 μΜ
*
2
0.04 µM
0.4 μΜ
Fold
1.5
1
0.5
0
CYP11A
CYP1182
CYP17
CYP19
CYP21
38HSD2
17BHSD1
17BHSD4
StAR
HMGR
*
B (TBDF)
25
DMSO
0.005 μΜ
0.05 μΜ
20
Fold
0.5 µM
*
*
*
*
5
*
*
*
*
*
*
*
*
**
0
CYP11A
CYP11B2
CYP17
CYP19
CYP21
3ßHSD2
17@HSD1
17@HSD4
StAR
HMGR
mechanism of aromatase induction is mediated through inhi- bition of phosphodiesterase activity.
Among the steroidogenic genes examined, the 3BHSD2 gene was up-regulated the most by almost all the tested chem- icals. The gene is responsible for oxidation and isomerization of 5-ene-3ß-hydroxy steroids to 4-ene-3-ketosteroids, an ob- ligate step in the biosynthesis of not only androgens and es- trogens but also mineralocorticoids and glucocorticoids [35]. In humans, two types of 3BHSD (3BHSD1 and 3(HSD2) have been identified, of which 3BHSD2 is exclusively expressed in the adrenal cortex and gonads [36]. The present results dem- onstrated that bromophenols, in addition to showing thyroid hormone-like activity [16] and weakly binding to human es- trogen receptor (e.g., 2,4-DBP) [17], exert endocrine-disrup- tive potential by affecting steroidogenesis in vitro. It should be noted that the gene expression profiles varied depending on the number and position of substituted bromines as well as on the dosing concentrations.
Bromophenols also may affect neuroendocrine function through other mechanisms. For example, a recent study using a neuroendocrine cell line (PC12) showed that bromophenols (e.g., 2,4-DP and 2,4,6-TBP) disrupt the endocrine system by affecting cell calcium homeostasis [37]. In the present study, PBB 169 and TBDF induced greater gene expression (>15- fold and >25-fold, respectively), whereas PBB 77 and TBDD induced moderate gene expression (>4-fold and >2.5-fold, respectively), suggesting greater inductive potency of the two compounds. In contrast, exposure of the H295R cells to PCB 149 resulted in a moderate induction of 3@HSD1 (greater than
twofold), whereas 3BHSD2 was comparably up-regulated by PCBs (PCBs 149, 110, and 101) [32]. The discrepancy of the magnitude in gene expression may suggest that the inductive potencies of PBBs are stronger than those of PCBs. Clearly, exposure respective structure of chlorinated and brominated biphenyls would compare the inductive potency of these com- pounds. In addition to up-regulation of 3ßHSD2, suppression of 3BHSD2 expression also was observed in the H295R cells after exposure to sediment extracts containing mainly PAHs and persistent chlorinated chemicals [33]. It should be noted that differential expression of 3HSD2 and 3@HSD1 also was observed in the present study, suggesting distinct functional roles of the two isogenes in steroidogenesis. Nevertheless, the up-regulation of 3BHSD2 in the present study suggested that the tested compounds could alter sex steroid hormone syn- thesis.
In addition to the 3ßHSD2 enzyme, many others catalyze the biosynthetic pathway from cholesterol to cortisol, includ- ing CYP11A, CYP11B, CYP17, and CYP21 [36]. The CYP11A, also known as CYP450scc, is responsible for cat- alyzing the side-chain cleavage of cholesterol to pregnenolone and also is the first step controlling the conversion of choles- terol to steroid hormones. In the present study, most of the tested chemicals significantly induced the expression of this gene, whereas the significant inhibition of this gene was ob- served after TBDD exposure.
The CYP11B2 enzyme is responsible for the rate-limiting final conversion in the aldosterone biosynthetic process. In the present study, PBB 169 caused significant dose-dependent in- duction of the CYP11B2 gene. Most of the tested chemicals altered the expression of the CYP11B2 gene at different con- centrations. Because CYP11B enzymes are important in ad- renal steroid synthesis, their modulation could significantly alter various physiological processes controlled by cortisol and aldosterone. Using H295R cells, Li et al. [27] showed that CYP11B2 is sensitive to coplanar PCB 126 and that signifi- cantly increased expression was accompanied by elevated pro- duction of aldosterone. Likewise, a recent study by Xu et al. [32] showed that the greatest transcriptional activation of this gene (29.9-fold) was caused by PCB 110 in H295R cells. These results suggested that PCB congeners may exert toxic effects by increasing aldosterone production. In the present study, PBBs 77 and 169 also greatly induced CYP11B2 gene ex- pression (>20-fold and 7-fold, respectively), which suggests that some PBB congeners may have similar toxic mechanism to PCBs.
The CYP17 enzyme catalyzes the conversion of aldosterone to corticosteroid substrates, and for cortisol synthesis in ad- renocortical cells. In the present study, gene expression pat- terns varied depending on the toxicants and concentrations. For example, 2,4,6-TBP and TBDD suppressed CYP17 gene expression, whereas 2-BP, 2,4-DBP, PBP, and PBB resulted in up-regulation. Nakajin et al. [30] showed that some alkyl- phenols, such as 4-t-octylphenol and 4-nonylphenol inhibited CYP17 enzyme activity in H295R cells. Although fewer stud- ies have addressed the effects of brominated compounds on CYP17, Moran et al. [38] showed that 2,3,7,8-TCDD signif- icantly inhibited CYP17 activity, which in turn led to a pro- portional decrease in estradiol secretion in human luteinizing granulosa cells. A more recent study [20] showed that CYP17 activity was significantly inhibited by almost all tested hy- droxylated PBDEs (e.g., 4-OH-BDE 49, 6-OH-BDE 47, and 6-OH-BDE 99) in H295R cells, and those authors suggested
that the inhibition of CYP17 activity would result in a decrease of the synthesis of weak androgens and, consequently, affect testosterone and estradiol production in the testis and ovary, respectively.
The CYP21 gene product is required for the synthesis of both aldosterone and corticosteroids. The suppression of this gene would likely result in deficiencies of both cortisol and aldosterone and be accompanied by overproduction of andro- gens. In the present study, 2,4,6-TBP moderately suppressed, and PBP induced, CYP21 gene expression, whereas other bromophenols showed no significant responses, suggesting that CYP21 generally was not sensitive to bromophenols. However, TBDF and TBDD suppressed CYP21 gene expres- sion, but PBB 77, PBB 169, and PBP resulted in significantly increased expression. An increase in CYP21 gene expression may lead to an increase in the synthesis of cortisol and al- dosterone, which could result in an increase in the substrate available for androgen and estrogen production. Nakajin et al. [30] showed that 4-t-octylphenol and 4-nonylphenol inhibited CYP21 enzyme activity in H295R cells, indicated that some alkylphenols behaved as inhibitors of cortisol synthesis, and showed good correlation between cortisol secretion and in- hibition of the enzyme activity. Another study showed that CYP21 gene expression also was significantly suppressed on exposure to sediment extracts in H295R cells [33]. In contrast, CYP21 gene expression was induced by PCBs congeners (e.g., PCBs 101, 110, and 149) and by some of the metabolites (methylsulfonyl PCBs) in H295R cells [32].
In addition to expression of the 3BHSD2 gene, expression of the 17BHSD gene also was significantly affected by bromo- phenols. The 17HSD enzyme plays a key role in the final step of sex steroid biosynthesis, which controls estrogen and androgen concentrations. Up-regulation of this gene may lead to elevated estrogen production. Likewise, depending on the number and position of bromines, the levels of up-regulation significantly varied for 17BHSD1 and 17HSD4. In the present study, we observed that 2,4-DBP and PBP induced greater gene expression, whereas 2,4,6-TBP showed no induction for both 17BHSD1 and 17BHSD4 and PBP had no effect on 17BHSD4. In addition, PBB 77 significantly induced expres- sion of the two genes. Hence, increased gene expression would likely affect estrogen biosynthesis and could lead to elevated estrogen production. In addition, 2,3,7,8-TBDD resulted in a slight but significant suppression of 17ßHSD4 gene expres- sion.
Among the examined genes, CYP19 (aromatase) has re- ceived great attention. The CYP19 enzyme catalyzes the con- version of androgens to estrogens, is expressed in various tis- sues, and is directly involved in reproduction, behavior, de- velopment, and estrogen-dependent carcinogenesis [19]. Hence, it has been hypothesized that the ability of chemicals to alter CYP19 enzyme activity represents a potential mech- anism of endocrine disruption [31]. Indeed, the effects of a wide range of chemicals on this enzyme have been studied extensively in H295R cells [19,24,28]. One recent study showed that 0.5 to 7.5 µM 2,4,6-TBP caused a concentration- dependent induction of aromatase activity, whereas 2.5 to 7.5 LM 4-BP did not show any induction in H295R cells [19]. In the present study, most bromophenols significantly induced CYP19 gene expression. The present results therefore showed that bromophenols potentially interfere in steroidogenesis in vitro. In the present study, no gene induction was observed after 2,4-DBP and 2,6-DBP treatment, suggesting different
responses caused by different bromine substitution. Addition- ally, Cantón et al. [19] measured CYP19 activity in H295R cells exposed to 30 PBDE congeners (7.5 M) and found that only BDEs 19 and 28 slightly induced, and BDEs 206 and 209 slightly inhibited, the activity of this enzyme. In the pres- ent study, PBB 77, PBB 169, and TBDF all significantly in- duced CYP19 gene expression, whereas gene expression in the TBDD treatment remained unchanged, suggesting that TBDF is a more potent inducer than TBDD. Although measurement of gene expression patterns offers information at the mRNA level, measurement of gene expression together with enzyme activities and steroid hormone concentrations, such as testos- terone and estradiol, would provide more detailed information regarding the effects of chemicals on steroidogenesis. There- fore, based on the present results, further research may be needed to examine whether these compounds will affect the same pathway in vivo.
The StAR protein transports cholesterol across the mito- chondrial membrane for conversion to pregnenolone by CYP11A and, thus, controls the rate-limiting step in steroido- genesis. In the present study, most bromophenols induced up- regulation of StAR gene expression. However, PBB 169 in- duced the StAR gene at 0.001 µM, whereas a down-regulation of this gene was clearly observed with exposure to greater concentrations (0.06 an d 4.0 µM). These responses may be a biphasic response of the gene: The lesser concentration of the test chemical induced increased gene expression, whereas at greater concentrations, gene expression was suppressed. Su- gawara et al. [39] reported that StAR promoter activity in- creased with ß-naphthoflavone concentration, reached a peak, and then subsequently decreased at increased concentrations. A recent study showed that StAR gene expression was sup- pressed by several PCBs [31]. Although StAR gene expression was up- or down-regulated after exposure to toxicants, Su- gawara and Fujimoto [40] demonstrated that the increase in StAR protein level in monkey kidney cell line (COS-1) is not a result of an increase in StAR gene expression but, rather, is a result of both an increase in translation and a longer half- life of the StAR protein.
The HMGR enzyme catalyzes the rate-limiting reaction of cholesterol biosynthesis, and its expression is regulated by various hormonal factors, such as concentrations of choles- terol, thyroid hormone, and estradiol [41]. Therefore, HMGR functions as a cholesterol regulator. Likewise, several of the tested compounds affected its gene expression depending on the nature of toxicants, dose concentrations, and substituted bromines. Among the tested compounds, TBDF induced the greatest responses, whereas no significant effect of TBDD was observed. A recent study showed that HMGR gene expression remained relatively unchanged after exposure to PCBs [32]. In general, transcriptional levels of HMGR and StAR mRNA were not markedly altered. This is consistent with the results of a previous study [25], in which the authors hypothesized that the basal abundance of the mRNA was relatively small. Because small changes in gene expression may lead to large changes in the steroidogenic pathway and complex control points and because multiple enzymes are involved, examina- tion of gene expression together with the measurement of en- zyme activity and hormone levels would provide more infor- mation regarding the effects of chemicals on steroidogenesis.
In conclusion, the present study demonstrated that some BFRs are able to alter steroidogenic gene expression in the H295R human adrenocortical carcinoma cells. Most of the
genes were either up- or down-regulated, to some extent, by exposure to the compounds, and 3HSD2 induction was the most pronounced. The present study further supports the mech- anism of chemical-induced endocrine disruption via modula- tion of steroidogenic genes, which could interfere with steroid hormone production and, in turn, affect hormone balance and immune function. The H295R cell line provided a good in vitro model for screening endocrine-disruptive chemicals. Al- though gene expression was significantly altered by many of the tested compounds, measurement of the activities of the relevant enzymes involved in steroidogenesis along with mea- surement of specific hormone concentrations would provide more information regarding chemical-induced endocrine dis- ruption. The ubiquity of BFRs, including PBDEs and their metabolites, new emerging bromodioxins/furans, and mixtures of chlorodioxins/furans, warrants further study for testing their endocrine activities. In addition, examining the potential en- docrine activities of these compounds in vivo will improve our understanding of their effects.
Acknowledgement-We would like to thank the Knowledge Innova- tion Program from the Chinese Academy of Sciences (project KSCX2- SW-128), and the City University of Hong Kong matching fund to Area of Excellence Scheme under the University Grants Committee of the Hong Kong Special Administrative Region, China (project 9400001), to R. Wu for financial support.
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