ELSEVIER
Contents lists available at ScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
T
Toxicology and Applied Pharmacology
Biphasic hormonal responses to the adrenocorticolytic DDT metabolite 3-methylsulfonyl-DDE in human cells
Vendela Asp ª, Erik Ullerås b, Veronica Lindström ª, Ulrika Bergström ª, Agneta Oskarsson b, Ingvar Brandt a,*
a Department of Environmental Toxicology, Uppsala University, Norbyvägen 18 A, SE-752 36 Uppsala, Sweden
b Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden
ARTICLE INFO
Article history: Received 13 August 2009 Revised 19 October 2009
Accepted 23 October 2009 Available online 6 November 2009
Keywords:
Adrenocorticolytic DDT metabolites Endocrine disruption Adrenocortical cancer (ACC) Biphasic responses Steroidogenesis H295R
ABSTRACT
The DDT metabolite 3-methylsulfonyl-DDE (3-MeSO2-DDE) has been proposed as a lead compound for an improved adrenocortical carcinoma (ACC) treatment. ACC is a rare malignant disorder with poor prognosis, and the current pharmacological therapy o,p’-DDD (mitotane) has limited efficacy and causes severe adverse effects. 3-MeSO2-DDE is bioactivated by cytochrome P450 (CYP) 11B1 in mice and causes formation of irreversibly bound protein adducts, reduced glucocorticoid secretion, and cell death in the adrenal cortex of several animal species. The present study was carried out to assess similarities and differences between mice and humans concerning the adrenocorticolytic effects of 3-MeSO2-DDE. The results support previous indications that humans are sensitive to the adrenocorticolytic actions of 3-MeSO2-DDE by demonstrating protein adduct formation and cytotoxicity in the human adrenocortical cell line H295R. However, neither the irreversible binding nor the cytotoxicity of 3-MeSO2-DDE in H295R cells was inhibited by the CYP11B1 inhibitor etomidate. We also report biphasic responses to 3-MeSO2-DDE in cortisol and aldosterone secretion as well as in mRNA levels of the steroidogenic genes StAR, CYP11B1 and CYP11B2. Hormone levels and mRNA levels were increased at lower concentrations of 3-MeSO2-DDE, while higher concentrations decreased hormone levels. These biphasic responses were not observed with o,p’-DDD or with the precursor DDT metabolite p,p’-DDE. Based on these results, 3-MeSO2-DDE remains a viable lead compound for drug design, although the adrenocorticolytic effects of 3-MeSO2-DDE in human cells seem more complex than in murine cells.
@ 2009 Elsevier Inc. All rights reserved.
Introduction
The DDT metabolite 3-methylsulfonyl-DDE (3-MeSO2-DDE; Fig. 1) has been proposed as a lead compound for developing an improved chemotherapy for adrenocortical cancer (ACC; Lindhe et al., 2002). ACC is a rare but highly malignant disorder, affecting approximately 0.5-2 per million population per year (Ahlman et al., 2001). The majority of patients develop hypersecretion of adrenocortical hor- mones (Tauchmanova et al., 2004). Radical tumor resection offers the best prognosis for long-term survival, but tumors frequently recur and only 16% to 38% of patients survive 5 years or more after diagnosis (Allolio and Fassnacht, 2006). The main pharmacological treatment, the DDT metabolite o,p’-DDD (mitotane, Lysodren; Fig. 1), suffers from low efficacy and is frequently associated with severe adverse effects (Allolio and Fassnacht, 2006).
The human adrenal cortex synthesizes three classes of steroid hormones: glucocorticoids (mainly cortisol) and androgens in the zona fasciculata/reticularis, and mineralocorticoids (aldosterone) in the zona glomerulosa. The first step in cortisol and aldosterone biosynthesis is the transportation of cholesterol across the mitochon-
drial membranes with the aid of steroidogenic acute regulatory (StAR) protein. At the inner mitochondrial membrane, the cholesterol side chain is cleaved off by cytochrome P450 (CYP) 11A1 to form pregnenolone. The microsomal CYP17, CYP21 and 30-hydroxysteroid dehydrogenase (3BHSD) convert pregnenolone into 11-deoxycortisol and 11-deoxycorticosterone (DOC). Finally, CYP11B1 converts 11- deoxycortisol into cortisol and DOC into corticosterone in the zona fasciculata/reticularis, while CYP11B2 converts DOC into aldosterone in the zona glomerulosa. The region-specific hormone secretion is a result of zone-specific expression of CYP17, CYP11B1, and CYP11B2. Cortisol synthesis in vivo is stimulated by adrenocorticotrophic hormone (ACTH) acting via cyclic AMP (cAMP), while aldosterone synthesis is stimulated by angiotensin II and potassium ions through increased intracellular calcium levels. The human adrenocortical cell line H295R expresses the enzymes needed to synthesize all three classes of adrenocortical steroid hormones and so does not represent any single zona (Gazdar et al., 1990; Rainey et al., 1993). The cells respond well to angiotensin II, but due to a poor response to ACTH, glucocorticoid synthesis may be stimulated with the cAMP-inducing agent forskolin (Bird et al., 1993; Rainey et al., 1993).
3-MeSO2-DDE selectively accumulates and causes necrosis in the adrenocortical zona fasciculata of mice after a single dose in vivo (Lund et al., 1988). The compound is bioactivated by CYP11B1 to a reactive intermediate, which forms irreversibly bound protein
* Corresponding author. Fax: +46 184716425.
E-mail address: ingvar.brandt@ebc.uu.se (I. Brandt).
CI
CI
CI
CI
SO2CH3
CI
CI
CI
CI
3-MeSO2-DDE
o,p’-DDD
CI
CI
CI
CI
p,p’-DDE
adducts (Lund et al., 1988; Lund and Lund, 1995). 3-MeSO2-DDE, or its bioactivated counterpart, also reduces glucocorticoid secretion and causes mitochondrial degeneration in mice in vivo (Jönsson et al., 1991; Brandt et al., 1992). Observations that 3-MeSO2-DDE was more efficient than o,p’-DDD in producing irreversibly bound protein adducts in human adrenal homogenates and tissue slices lead to the suggestion that the 3-MeSO2-DDE molecular structure may constitute the backbone of more potent pharmaceuticals against ACC (Jönsson and Lund, 1994; Lindhe et al., 2002).
The adrenocorticolytic activity of o,p’-DDD has been studied experimentally mainly in dogs and comprises decreased glucocorti- coid biosynthesis, irreversibly bound protein adducts in mitochondria, and adrenocortical cell death (Hart et al., 1973; Martz and Straw, 1977). The compound is bioactivated to a reactive acyl chloride through a CYP-catalyzed reaction in the adrenal cortex of dogs and humans (Martz and Straw, 1980; Cai et al., 1995).
Although 3-MeSO2-DDE is a potent adrenocorticolytic compound in mice, the toxicity of the compound exhibits considerable species variation (Lindström et al., 2008). We recently reported cytotoxicity, decreased corticosterone production, and formation of irreversibly bound protein adducts by 3-MeSO2-DDE in murine adrenocortical Y-1 cells, thereby reproducing previous murine in vivo data in an in vitro model (Hermansson et al., 2007; Asp et al., 2009). The objective of the present study was to provide a mice/human in vitro interspecies link concerning the adrenocorticolytic effects of 3-MeSO2-DDE, thereby laying a foundation for further human sensitivity assessment and future ACC drug design.
In this study, we compare the effects of 3-MeSO2-DDE and o,p’- DDD on cell viability, irreversible protein binding, hormone secretion and expression of steroidogenic genes in the H295R cell line. Moreover, since previous studies have shown that the methylsulfonyl moiety of 3-MeSO2-DDE is essential for its effects in mice in vivo and in murine cells in vitro (Lund et al., 1988; Lund and Lund, 1995; Hermansson et al., 2007; Asp et al., 2009), we also examine the effects on cell viability and hormone secretion of the parent structure, the persistent DDT metabolite p,p’-DDE (Fig. 1).
Methods
Chemicals and reagents. The chemical structures of the test compounds are presented in Fig. 1. 2-(3-Methylsulphonyl-4- chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene (3-MeSO2- DDE, purity >99%) was synthesized by Synthelec (Lund, Sweden). 2,2-Bis(4-chlorophenyl)-1,1-dichloroethene (p.p’-DDE, purity >99%)
was from EGA-Chemie (Steinheim/Albuch, Germany). 3-MeSO2-[14C] DDE (specific activity 481 kBq/umol, purity >98%) and o,p’-[14C]DDD (specific activity 414 kBq/umol, purity >98%), were prepared as previously described (Bergman and Wachtmeister, 1977; Lindholm et al., 1987). 2-(2-Chloro-phenyl)-2-(4-chlorophenyl)-1,1-dichloroeth- ane (o,p’-DDD, purity >99%), forskolin, etomidate, angiotensin II, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Cell culture. The human adrenocortical cell line H295R (ATCC CRL- 2128) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in DMEM-F12 (1:1; Sigma-Aldrich), containing 15 mM HEPES and supplemented with 2.5 mM L-glutamine (Sigma-Aldrich), 1% ITS+ (BD Biosciences) and 2.5% NuSerum I (BD Biosciences; complete culture medium). The cells were kept in a humidified atmosphere at 37 ℃, 5% CO2. Cell stimulation with forskolin or angiotensin II was performed in complete culture medium, while exposure to test compounds and/ or etomidate was performed in DMEM-F12 supplemented only with 2.5 mM L-glutamine (assay medium). DMSO concentrations during incubations never exceeded 0.2%. No toxic effects were observed in the cells in response to 0.2% DMSO.
Irreversible binding assay. The time-dependent irreversible binding of 3-MeSO2-DDE and o,p’-DDD to H295R cellular proteins was determined, using a modified version of the method of Wallin et al. (1981) (Hermansson et al., 2007). Cells at 50% confluency in 6-well plates were preincubated for 48 h in complete culture medium containing 10 µM forskolin or the corresponding volume of DMSO. After preincubation, the cells were exposed to 3-MeSO2-[14C]DDE or o,p’-[14C]DDD (1.85 kBq/sample diluted with unlabeled substances to a total concentration of 10 uM) in assay medium without forskolin for 20 min- 24 h (37 ℃, 5% CO2). The concentration of 10 uM was selected based on concentration-response studies in murine cells (Hermansson et al., 2007). When etomidate was used, the cells were preincubated for 1 h with assay medium containing 10 uM etomidate or the corresponding volume of DMSO directly prior to the addition of test substances. Etomidate and DMSO were also re-added together with test substances. All substances used were dissolved in DMSO and added from 1000-fold stock solutions. Each compound and time point was assayed in triplicate and the experiments were repeated two (3- MeSO2-DDE) or three (o,p’-DDD) times. Sample treatments subse- quent to incubations were as in Hermansson et al. (2007). Briefly, the cells were disrupted by sonication and protein extracts were transferred onto glass microfiber filters. After extensive washing in organic solvents, the radioactivity on the filters was measured and the amount of irreversibly bound compound was calculated and expressed as pmol substance/mg protein.
Cell viability/toxicity assay. The MTT assay (Mosmann, 1983) was performed with the modifications proposed by Tada et al. (1986). Cells at 50% confluency in 96-well plates were preincubated for 1 h with assay medium containing 10 uM etomidate or the corres- ponding volume of DMSO. The preincubation solutions were then removed and replaced with 100 ul test solutions. Test solutions of 3-MeSO2-DDE, p,p’-DDE and o,p’-DDD from 1000-fold stock solu- tions were prepared in assay medium containing 10 uM etomidate in DMSO or the corresponding volume of DMSO. The total DMSO concentration was 0.2%. Final concentrations of test compounds were 1.25, 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 uM. These concen- trations were well below the therapeutic mitotane threshold serum concentration of 14 µg/ml ( 44 µM) (van Slooten et al., 1984). Each compound and concentration was assayed in triplicate and the experiment was repeated three times. The cells were incubated at 37 ℃, 5% CO2 for a total of 72 h. The cell viability was determined
| Gene | Primer (sense) | Primer (antisense) | Probe |
|---|---|---|---|
| StAR CYP11A1 CYP17 HSD3B2 CYP21 CYP11B1 CYP11B2 | TTGCTTTATGGGCTCAAGAATG CTTCTTCGACCCGGAAAATTT GCTGACTCTGGCGCACACT GCGGCTAATGGGTGGAATCTA TCCCAGCACTCAACCAACCT TCCCGAGGGCCTCTAGGA TTGTTCAAGCAGCGAGTGTTG | GGAGACCCTCTGAGATTCTGCTT CCGGAAGTAGGTGATGTTCTTGT CCATCCTTGAACAGGGCAAA CCTCATTTATACTGGCAGAAAGGAAT CAGCTCAGAATTAAGCCTCAATCC GGGACAAGGTCAGCAAGATCTT GCATCCTCGGGACCTTCTC | CATGCGCTGGCAGTACATGTGCAC CCCAACCCGATGGCTGAGCAA TCGCCAGCCTTCGATGCAGCT TGATACCTTGTACACTTGTGCGTTAAGACCCA CTCCCTTCCTGACCCTCCGCTGC TGCTGCTTAGCCTGGCAAACCCTG TCCTCTGCTTCCTGAGCTGTCCCCT |
+ Originally published in Oskarsson et al. (2006).
during the last 3 h of incubation by the addition of 20 ul MTT (5 mg/ml in PBS) to each well. Formed formazan crystals were dissolved by the addition of 100 ul 10% SDS in 0.01 M hydrochloric acid and overnight incubation at 37 ℃, 5% CO2. Absorbance was measured at 560 nm with a reference measurement at 750 nm. The absorbance at 750 nm was subtracted from the 560-nm reading. Cell viability was expressed as percent viability relative to the respective DMSO control cells.
Hormone secretion assay. Hormone secretion experiments were performed under subtoxic conditions, defined as cell viability ≥75% of control at the end of the experiment. Cells at 60% confluency in 24- well plates were preincubated for 48 h in complete culture medium containing either 10 uM forskolin or the corresponding volume of DMSO, or 10 nM angiotensin II or the corresponding volume of water. Test solutions ( 500 ul) of 3-MeSO2-DDE, p,p’-DDE and o,p’-DDD were prepared from 1000-fold stock solutions in DMSO in assay medium. Angiotensin II, but not forskolin, was re-added together with test compounds. Final concentrations of test compounds were 0.625, 1.25, 2.5, 5, 7.5 and 10 uM. These concentrations were well below the therapeutic mitotane threshold serum concentration of 14 µg/ml ( 44 „M; van Slooten et al., 1984). Each compound and concentration was assayed in triplicate and the experiment was repeated three times. The cells were incubated with test compounds at 37 ℃, 5% CO2 for 24 h, after which the test solutions were replaced with fresh assay medium ( 500 ul) without test compounds, forskolin or angiotensin II. After 3 h the cell medium was collected and stored at -20 ℃ until hormone measurements. The cells in one replicate well were then incubated for 3 h with 500 ul assay medium and 100 ul MTT ( 5 mg/ ml in PBS) and viability was assessed using the protocol described above. The cells from the other two replicate wells were pooled and used for RNA preparation.
Hormone level measurements. Cortisol and aldosterone levels were measured using ELISA kits from Neogen (Lansing, MI, USA) and Demeditec (Kiel-Wellsee, Germany), respectively, according to the manufacturer’s instructions. Hormone levels were expressed as percent of DMSO control levels.
RNA preparation and real-time RT-PCR. Total RNA was prepared using the Absolutely RNA Microprep kit (Stratagene) and reverse transcribed into cDNA by the AffinityScript Multiple Temperature enzyme (Stratagene, La Jolla, CA, USA) at 42 ℃ using oligo(dT) primers. The purity and quantity of the RNA were determined with a spectrophotometer and the integrity of selected RNA samples was assessed using the Agilent RNA 6000 Nano LabChip kit in a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Quantitative RT-PCR reactions were set up using cDNA corresponding to 250 ng total RNA with dual-labeled probe chemistry and performed using Rotorgene3000. Gene-specific primers and probes for StAR, CYP11A1, CYP17, HSD3B2, CYP21, CYP11B1 and CYP11B2 have been described previously (Oskarsson et al., 2006) and are given in Table 1. Gene expression levels were calculated from a standard curve. Changes in
gene expression levels were then calculated relative to the respective DMSO controls.
Statistical methods. Mean values of the triplicates from each experiment were used for statistical analysis. Statistical analyses were performed using GraphPad Prism version 5.01 for Windows. The effect of etomidate on irreversible binding by o,p’-DDD was analyzed with a paired ratio t-test. For cell viability data, variable slope concentration-response curves were fitted and the influence of etomidate was assessed by an F-test. Hormone levels and real-time RT-PCR results were subjected to one-way ANOVA followed by Dunnett’s multiple comparison test. The level of significance in all tests was set to 0.05.
Results
Formation of irreversibly bound protein adducts
We compared the ability of 3-MeSO2-[14C]DDE and o,p’-[14C]DDD to form irreversibly bound protein adducts in the H295R cell line. Both compounds, or their enzymatically transformed counterparts, pro- duced a pronounced non-extractable binding to cellular protein (Table 2). The binding of both compounds increased with time, with approximately half of the binding occurring already within 20-min incubation (Fig. 2). The binding curves of the two compounds had similar shapes (Fig. 2), although o,p’-[14C]DDD reached higher binding levels than 3-MeSO2-[14C]DDE (Table 2). Binding levels are shown for cells preincubated with 10 uM forskolin, however the omittance of the preincubation step had no significant impact on binding levels (data not shown). The addition of the CYP11B1-inhibitor etomidate ( 10 µM) reduced the binding of o,p’-[14C]DDD after 24 h to 29% while not significantly affecting the binding levels of 3-MeSO2-[14C]DDE (Table 2).
Cytotoxicity of test compounds and the modulating effect of etomidate
3-MeSO2-DDE, o,p’-DDD and p,p’-DDE all decreased the viability of H295R cells in a concentration-dependent manner as measured by MTT reduction after 72 h (Fig. 3). The three compounds generated
| Test compound | Binding1 | |
|---|---|---|
| pmol/mg protein | % of binding at 24 h | |
| 10 µM 3-MeSO2-[14C]DDE | 132±17 | 100 |
| + 10 uM etomidate | 159±63 | 116±32 |
| 10 um o,p'-[14C]DDD | 175±20 | 100 |
| + 10 µM etomidate | 51 ±82 | 29±42 |
+ Data presented as mean ± standard error of two (3-MeSO2-[14C]DDE) or three (o,p’-[14C]DDD) independent experiments.
a p<0.05 vs. the corresponding controls.
3-MeSO2-DDE
o,p’-DDD
125
100
% of binding at 24h
75
50
25
0
0
3
6
9
12
15
18
21
24
Time (h)
significantly different concentration-response curves, with EC50 values (and 95% confidence intervals) of 6.8 µM (5.6-8.1 µM) for 3-MeSO2-DDE, 10.6 µM (9.0-12.4 µM) for o,p’-DDD and 13.0 µM (11.6-14.5 µM) for p,p’-DDE. 3-MeSO2-DDE was thus the most potent compound in decreasing cell viability. Etomidate significantly reduced the cytotoxicity of o,p’-DDD, shifting the concentration- response curve to the right (Fig. 3B). In contrast, 10 uM etomidate had no significant effect on 3-MeSO2-DDE or p,p’-DDE cytotoxicity (Fig. 3A and C).
Effects on cortisol and aldosterone secretion
All hormone secretion experiments were performed under subtoxic conditions as defined in the Methods section. Unstimulated H295R cells secreted cortisol at 7.5±0.7 ng/ml and aldosterone at 250±24 pg/ml (collected during 3 h subsequent to 24-h incubation with 0.1% DMSO, mean ± standard error). In unstimulated cells, 3- MeSO2-DDE generated a biphasic concentration-dependent response in both cortisol and aldosterone secretion (Figs. 4A and B): lower concentrations of the compound increased hormone levels while higher concentrations decreased hormone levels. Unstimulated cortisol and aldosterone levels were increased to 189% and 163% of control, respectively, by 1.25 uM 3-MeSO2-DDE while 10 µM, the highest concentration tested, decreased the cortisol and aldosterone levels to 26% and 40%, respectively (Fig. 4A and B). o,p’-DDD inhibited both cortisol and aldosterone secretion in a linear concentration- dependent manner, while p,p’-DDE had no significant effects on hormone secretion in unstimulated H295R cells.
Cell stimulation increased hormone secretion approximately three-fold. Cells treated for 48 h with 10 uM forskolin increased cortisol secretion 3.2±0.2 while 10 nM angiotensin II increased aldosterone secretion 2.8±0.1 (mean fold induction ±standard error). In stimulated cells, 3-MeSO2-DDE and o,p’-DDD significantly reduced both cortisol and aldosterone secretion in a concentration-
· DMSO
etomidate (10 µM)
(A)
120
3-MeSO2-DDE
100
Viability (% of control)
80
60
40
HỌH
HOOH
HOH
20
0
0
5
10
15
20
conc (µM)
(B)
120
o,p’-DDD
100
Viability (% of control)
80
60
40
20
0
0
5
10
15
20
conc (uM)
(C)
120
p,p’-DDE
100
Viability (% of control)
80
60
40
20
0
0
5
10
15
20
conc (uM)
-O- 3-MeSO2-DDE -.- o,p’-DDD -*- p,p’-DDE
(A)
250
a
a
200
Cortisol (% of control)
150
100
*
50
a
0
0.0
2.5
5.0
7.5
10.0
conc (µM)
(B)
250
Aldosterone (% of control)
200
C
O
a
150
100
T
b
50
b
b
C
0
0.0
2.5
5.0
7.5
10.0
conc (µM)
dependent manner (Figs. 5A and B). At 10 µM both compounds reduced cortisol secretion to 16% of control levels, however the concentration-response curve for 3-MeSO2-DDE was slightly steeper than the curve for o,p’-DDD at lower concentrations (Fig. 5A). For aldosterone secretion, the concentration-response curves for 3- MeSO2-DDE and o,p’-DDD were both nearly linear, 10 uM of each compound reducing the aldosterone levels to 37% and 25% of control, respectively (Fig. 5B). p,p’-DDE had no significant effect on cortisol secretion in stimulated cells, but caused a concentration-dependent reduction in aldosterone secretion (Fig. 5B), 10 uM reducing the aldosterone levels to 58% of control.
Effects on steroidogenic gene expression
In unstimulated cells, 1.25 uM 3-MeSO2-DDE strongly increased mRNA levels of the genes for CYP11B1, CYP11B2, and steroidogenic
acute regulatory protein (StAR) after 24 h, while 5 and 10 µM did not significantly affect mRNA levels (Figs. 6A-C). CYP11B1 levels increased more than 5-fold, CYP11B2 levels increased more than 14-fold, and StAR levels approximately doubled. In addition, 5 µM 3-MeSO2-DDE decreased CYP21 mRNA levels (Fig. 6F). 3-MeSO2-DDE had no significant effects on the mRNA levels of CYP11A, CYP17, or HSD3B2 in unstimulated cells (Figs. 6D, E, G). Ten micromolar o,p’-DDD increased CYP11B1 mRNA levels and decreased HSD3B2 mRNA levels in unstimulated cells (Figs. 6A, G). o,p’-DDD had no significant effects on the mRNA levels of StAR, CYP11B2, CYP11A, CYP17, or CYP21 in unstimulated cells (Figs. 6B-F).
Since the major differences in effects on steroidogenic gene expression between 3-MeSO2-DDE and o,p -DDD were found in CYP11B1 and CYP11B2 levels, we analyzed these genes further in stimulated cells. The biphasic pattern caused by 3-MeSO2-DDE in unstimulated cells was reproduced in stimulated cells, although the
-O- 3-MeSO2-DDE -.- o,p’-DDD x- p,p’-DDE
(A)
forskolin-treated cells
125
100
Cortisol (% of control)
T.
75
50
C
b
C
25
C
C
C
C
0
0.0
2.5
5.0
7.5
10.0
conc (µM)
(B)
angiotensin II-treated cells
125
100
H
Aldosterone (% of control)
a
Ho
b
1
75
C
C
C
C
50
C
C
C
25-
C
0
0.0
2.5
5.0
7.5
10.0
conc (μM)
(A)
CYP11B1
(B)
CYP11B2
8
Relative expression
☐ 3-MeSO2-DDE
20
☒ o,p’-DDD
Relative expression
C
C
6
15
4
10
C
2
5
0
0.00
1.25
5.00
10.00
0
0.00
1.25
5.00
10.00
conc (M)
conc (UM)
(C)
(D)
StAR
CYP11A
2.5
2.5
Relative expression
2.0
b
Relative expression
2.0
T
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.00
1.25
5.00
10.00
0.0
0.00
1.25
5.00
10.00
conc (UM)
conc (UM)
(E)
CYP17
(F)
CYP21
Relative expression
2.5
Relative expression
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
a
0.0
0.0
0.00
1.25
5.00
10.00
0.00
1.25
5.00
10.00
conc (UM)
conc (UM)
(G)
HSD3B2
Relative expression
2.5
2.0
1.5
1.0
a
0.5
0.0
0.00
1.25
5.00
10.00
conc (µM)
(A)
CYP11B1 F
(B)
CYP11B2 F
4
15
a
Relative expression
3-MeSO2-DDE
3
o,p’-DDD
Relative expression
10
2
1
5
0
0.00
1.25
5.00
10.00
0
0.00
1.25
5.00
10.00
conc (UM)
conc (UM)
(C)
CYP11B1 AT
(D)
CYP11B2 AT
2.0
2.0
Relative expression
Relative expression
b
1.5
1.5
1.0
1.0
0.5
a
0.5
b
0.0
0.00
1.25
5.00
10.00
0.0
0.00
1.25
5.00
10.00
conc (µM)
conc (UM)
changes were of lower magnitude (Fig. 7). In both forskolin- and angiotensin II-stimulated cells, 1.25 µM 3-MeSO2-DDE significantly increased CYP11B2 levels (Figs. 7B and D). Moreover, 10 uM 3- MeSO2-DDE significantly decreased both CYP11B1 and CYP11B2 levels in angiotensin II-stimulated cells (Figs. 7C, D). o,p-DDD had no significant effects on the mRNA levels of CYP11B1 or CYP11B2 levels in stimulated cells (Fig. 7).
The impact of p.p-DDE on steroidogenic gene expression was not assessed.
Discussion
In this report we demonstrate that 3-MeSO2-DDE, proposed as a lead compound in ACC drug design, generates irreversibly bound protein adducts and reduces cell viability in human adrenocortical cells. In addition, we report biphasic responses to 3-MeSO2-DDE in the secretion of adrenocortical hormones and in the expression of steroidogenic genes. The effects of 3-MeSO2-DDE were compared to the effects of the current ACC drug, o,p’-DDD (mitotane), and to the parent structure of 3-MeSO2-DDE, p,p’-DDE.
3-MeSO2-DDE, o,p’-DDD and p,p’-DDE all caused concentration- dependent cytotoxicity in the human H295R cell line. A previous study on H295R cells did not report cytotoxicity in response to 3- MeSO2-DDE (Johansson et al., 2002) but employed a different experimental protocol with shorter exposure times. The rank order in potency, based on EC50 values from the concentration-response curves, was 3-MeSO2-DDE>o,p’-DDD>p.p’-DDE. In contrast, p,p’- DDE had no significant effect on cell viability in the murine adrenocortical cell line Y-1 in concentrations up to 20 uM (Asp et
al., 2009). The MTT assay measures the reducing capacity of the cells, interpreted as viability, and does not take the underlying mechanisms of cell damage into account (Berridge et al., 2005). It is possible that p.p’-DDE was bioactivated to some extent by the H295R cells. It is also possible that the H295R cells are more sensitive to non-specific toxicity due to membrane partitioning effects (narcosis), than the Y-1 cells.
The time-dependent irreversible binding profiles for 3-MeSO2- DDE and o,p’-DDD both displayed an initial steep increase and flattened with time, suggesting that the protein adducts were generated through a saturable process (Hermansson et al., 2007). However, although both cytotoxicity and irreversible binding by o, p’-DDD could be counteracted with the CYP11B1 inhibitor etomidate, 10 uM etomidate did not affect either the cytotoxicity or the protein binding of 3-MeSO2-DDE. This is in contrast to previous in vitro and ex vivo studies, where the CYP inhibitor metyrapone inhibited irreversible binding by 3-MeSO2-DDE to human adrenal homogenates and human adrenal tissue slices (Jönsson and Lund, 1994; Lindhe et al., 2002). The murine CYP11B1 has been shown to metabolically activate 3-MeSO2-DDE (Lund and Lund, 1995), and etomidate did indeed decrease both irreversible binding and cytotoxicity of 3-MeSO2-DDE in murine Y-1 cells (Hermansson et al., 2007; Asp et al., 2009). CYP11B1 is presumably involved also in 3-MeSO2-DDE activation in human cells, since the irreversible binding observed by Lindhe et al. (2002) was localized to the adrenocortical regions where human CYP11B1 is expressed (zona fasciculata/reticularis). Our results therefore raise the question whether it is the parent 3-MeSO2-DDE molecule that is cytotoxic and binds to proteins in human cells or if bioactivation in
human adrenal cortex tissue is performed by other enzyme(s) in addition to CYP11B1. It should be noted that etomidate at the concentration used ( 10 uM) inhibits the enzymatic activities of both CYP11B1 and CYP11A1 (Dörr et al., 1984; Vanden Bossche et al., 1984; Varga et al., 1993).
In murine Y-1 cells, 3-MeSO2-DDE inhibited the CYP11B1- catalyzed conversion of 11-deoxycorticosterone to corticosterone, the main glucocorticoid in rodents, in a concentration-dependent manner (Asp et al., 2009). 3-MeSO2-DDE inhibited both unstimulated and forskolin-stimulated corticosterone secretion without affecting Cyp11b1 mRNA levels (Asp et al., 2009). This suggested that 3-MeSO2- DDE, or its bioactivated counterpart, interfered with CYP11B1 catalytic activity at the protein level. In the human H295R cells used in this study, however, the endocrine responses to 3-MeSO2-DDE appeared more complex, possibly since we measured the final output of cortisol/aldosterone and not the isolated 11ß-hydroxylation event. Forskolin- and angiotensin II-stimulated production of cortisol/ aldosterone were here nhibited in a concentration-dependent manner, in accordance with the inhibition of 11ß-hydroxylation by 3-MeSO2-DDE observed by Johansson et al. (2002). However, in the present study, unstimulated secretion of both cortisol and aldosterone exhibited a biphasic course where the lower concentrations of 3- MeSO2-DDE approximately doubled hormone secretion. This biphasic response was not observed for o,p’-DDD or p,p’-DDE.
The biphasic secretory responses to 3-MeSO2-DDE after 24 h were mirrored in the mRNA levels of StAR, CYP11B1 and CYP11B2, which were all increased at 1.25 uM in unstimulated cells but not at higher concentrations. CYP11B2 mRNA exhibited the greatest increase, however it was still present in very low copy number in the cells. StAR mRNA is present in much larger copy numbers, and so even a modest relative increase corresponds to a vast increase in copy number. Biphasic patterns in CYP11B1 and CYP11B2 mRNA levels were also seen in response to 3-MeSO2-DDE cells stimulated with forskolin and angiotensin II. Increased mRNA levels can be due to either increased transcription rates or increased mRNA stability. For example, PCB126 has been shown to increase CYP11B1 and CYP11B2 mRNA stability, thus raising unstimulated cortisol and aldosterone levels in H295R cells (Li et al., 2004; Li and Wang, 2005; Lin et al., 2006). Stimulation of the cells further increased aldosterone levels whereas stimulated cortisol levels were not affected by PCB126 (Li et al., 2004; Li and Wang, 2005). Moreover, PCB126 increased unstimu- lated but not stimulated CYP19 expression in mouse Leydig and human H295R cells (Li, 2007). Xu et al. (2006) also found upregula- tion of steroidogenic genes after exposing unstimulated H295R cells to PCBs and MeSO2-PCBs.
In contrast to the strong inducing effects of 1.25 uM 3-MeSO2- DDE on steroidogenic gene expression, o,p’-DDD affected mRNA levels only at 10 µM, when cortisol and aldosterone secretion was decreased.
Taken together, the cytotoxicity, hormone secretion and gene expression data suggest that 3-MeSO2-DDE acts as an inducer of StAR, CYP11B1 and CYP11B2 at low concentrations, leading to increased hormone secretion. However, at higher concentrations and/or longer exposure, the adrenocorticolytic activity of 3-MeSO2-DDE overpowers the cellular defenses, resulting in cytotoxicity.
In summary, although we found some differences in how 3- MeSO2-DDE affects human H295R cells in comparison to murine Y-1 cells, 3-MeSO2-DDE was cytotoxic and affected glucocorticoid production with similar potency in both species. The methylsulfonyl moiety seemed to be of less importance for cytotoxic and hormonal effects in H295R cells than in Y-1 cells, since p,p’-DDE decreased both cell viability and aldosterone secretion in this study. 3-MeSO2-DDE thus remains a viable lead compound in the search for safer and more efficient ACC drugs, although the biphasic nature of the hormonal responses presents a challenge in selecting test concentrations for high-throughput screening of drug candidates.
Conflict of interest statement
IBt is cofounder of OncoTargeting AB, a company created to develop drugs against cancer, including ACC.
Acknowledgments
Prof. Åke Bergman, Department of Environmental Chemistry, Stockholm University, is acknowledged for providing the radiolabeled compounds. Economic support was given by the Swedish Research Council Formas and the Swedish Animal Welfare Agency.
References
Ahlman, H., Khorram-Manesh, A., Jansson, S., Wangberg, B., Nilsson, O., Jacobsson, C.E., Lindstedt, S., 2001. Cytotoxic treatment of adrenocortical carcinoma. World J. Surg. 25, 927-933.
Allolio, B., Fassnacht, M., 2006. Clinical review: adrenocortical carcinoma: clinical update. J. Clin. Endocrinol. Metab. 91, 2027-2037.
Asp, V., Lindström, V., Olsson, J.A., Bergström, U., Brandt, I., 2009. Cytotoxicity and decreased corticosterone production in adrenocortical Y-1 cells by 3-methylsulfo- nyl-DDE and structurally related molecules. Arch. Toxicol. 83, 389-396.
Bergman, A., Wachtmeister, C.A., 1977. Synthesis of methanesulfonyl derivatives of 2,2- bis (4-chlorophenyl)-1,1-dichloroethylene (p.p’-DDE), present in seal from the Baltic. Acta Chem. Scand. 31, 90-91.
Berridge, M.V., Herst, P.M., Tan, A.S., 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11, 127-152.
Bird, I.M., Hanley, N.A., Word, R.A., Mathis, J.M., McCarthy, J.L., Mason, J.I., Rainey, W.E., 1993. Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin-II- responsive aldosterone secretion. Endocrinology 133, 1555-1561.
Brandt, I., Jönsson, C.J., Lund, B.O., 1992. Comparative studies on adrenocorticolytic DDT-metabolites. Ambio 21, 602-605.
Cai, W., Djanegara, T., Sinsheimer, J., Wotring, L., Counsell, E., Schteingart, D., 1995. Metabolic activation and binding of mitotane in adrenal cortex homogenates. Eur. J. Pharm. Sci. 84, 134-138.
Dörr, H.G., Kuhnle, U., Holthausen, H., Bidlingmaier, F., Knorr, D., 1984. Etomidate: a selective adrenocortical 11 beta-hydroxylase inhibitor. Klin. Wochenschr. 62, 1011-1013.
Gazdar, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., Triche, T.J., Myers, C.E., Chrousos, G.P., Brennan, M.F., Stein, C.A., La Rocca, R.V., 1990. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 50, 5488-5496.
Hart, M.M., Reagan, R.L., Adamson, R.H., 1973. The effect of isomers of DDD on the ACTH-induced steroid output, histology and ultrastructure of the dog adrenal cortex. Toxicol. Appl. Pharmacol. 24, 101-113.
Hermansson, V., Asp, V., Bergman, A., Bergström, U., Brandt, I., 2007. Comparative CYP- dependent binding of the adrenocortical toxicants 3-methylsulfonyl-DDE and o,p’- DDD in Y-1 adrenal cells. Arch. Toxicol. 81, 793-801.
Johansson, M.K., Sanderson, J.T., Lund, B.O., 2002. Effects of 3-MeSO2-DDE and some CYP inhibitors on glucocorticoid steroidogenesis in the H295R human adrenocor- tical carcinoma cell line. Toxicol. In Vitro 16, 113-121.
Jönsson, C.J., Lund, B.O., 1994. In vitro bioactivation of the environmental pollutant 3- methylsulphonyl-2, 2-bis(4-chlorophenyl)-1,1-dichloroethene in the human ad- renal gland. Toxicol. Lett. 71, 169-175.
Jönsson, C.J., Rodriguez-Martinez, H., Lund, B.O., Bergman, Å., Brandt, I., 1991. Adrenocortical toxicity of 3-methylsulfonyl-DDE in mice. II. Mitochondrial changes following ecologically relevant doses. Fundam. Appl. Toxicol. 16, 365-374.
Li, L.A., 2007. Polychlorinated biphenyl exposure and CYP19 gene regulation in testicular and adrenocortical cell lines. Toxicol. In Vitro 21, 1087-1094.
Li, L.A., Wang, P.W., 2005. PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells. Toxicol. Sci. 85, 530-540.
Li, L.A., Wang, P.W., Chang, L.W., 2004. Polychlorinated biphenyl 126 stimulates basal and inducible aldosterone biosynthesis of human adrenocortical H295R cells. Toxicol. Appl. Pharmacol. 195, 92-102.
Lin, T.C., Chien, S.C., Hsu, P.C., Li, L.A., 2006. Mechanistic study of polychlorinated biphenyl 126-induced CYP11B1 and CYP11B2 up-regulation. Endocrinology 147, 1536-1544.
Lindhe, Ö., Skogseid, B., Brandt, I., 2002. Cytochrome P450-catalyzed binding of 3- methylsulfonyl-DDE and o,p’-DDD in human adrenal zona fasciculata/reticularis. J. Clin. Endocrinol. Metab. 87, 1319-1326.
Lindholm, A., Klasson-Wehler, E., Wehler, T., Bergman, Å., 1987. Synthesis of 14C-labelled DDD isomers of high specific activity. J. Labelled Comp. Radiopharm. 24, 1011-1019. Lindström, V., Brandt, I., Lindhe, Ö., 2008. Species differences in 3-methylsulphonyl- DDE bioactivation by adrenocortical tissue. Arch. Toxicol. 82, 159-163.
Lund, B.O., Bergman, Å., Brandt, I., 1988. Metabolic activation and toxicity of a DDT- metabolite, 3-methylsulphonyl-DDE, in the adrenal zona fasciculata in mice. Chem .- Biol. Interact. 65, 25-40.
Lund, B.O., Lund, J., 1995. Novel involvement of a mitochondrial steroid hydroxylase (P450c11) in xenobiotic metabolism. J. Biol. Chem. 270, 20895-20897.
Martz, F., Straw, J.A., 1977. The in vitro metabolism of 1-(o-chlorophenyl)-1-(p- chlorophenyl)-2,2-dichloroethane (o,p’-DDD) by dog adrenal mitochondria and metabolite covalent binding to mitochondrial macromolecules: a possible mechanism for the adrenocorticolytic effect. Drug Metab. Dispos. 5, 482-486.
Martz, F., Straw, J.A., 1980. Metabolism and covalent binding of 1-(o-chlorophenyl)-1- (p-chlorophenyl)-2,2-dichloroethane (o,p’-DDD). Correlation between adrenocor- ticolytic activity and metabolic activation by adrenocortical mitochondria. Drug Metab. Dispos. 8, 127-130.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.
Oskarsson, A., Ullerås, E., Plant, K.E., Hinson, J.P., Goldfarb, P.S., 2006. Steroidogenic gene expression in H295R cells and the human adrenal gland: adrenotoxic effects of lindane in vitro. J. Appl. Toxicol. 26, 484-492.
Rainey, W.E., Bird, I.M., Sawetawan, C., Hanley, N.A., McCarthy, J.L., McGee, E.A., Wester, R., Mason, J.I., 1993. Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids. J. Clin. Endocrinol. Metab. 77, 731-737.
Tada, H., Shiho, O., Kuroshima, K., Koyama, M., Tsukamoto, K., 1986. An improved colorimetric assay for interleukin 2. J. Immunol. Methods 93, 157-165.
Tauchmanova, L., Colao, A., Marzano, L.A., Sparano, L., Camera, L., Rossi, A., Palmieri, G., Marzano, E., Salvatore, M., Pettinato, G., Lombardi, G., Rossi, R., 2004. Andreno-
cortical carcinomas: twelve-year prospective experience. World J. Surg. 28, 896-903.
Wallin, H., Schelin, C., Tunek, A., Jergil, B., 1981. A rapid and sensitive method for determination of covalent binding of benzo[a]pyrene to proteins. Chem .- Biol. Interact. 38, 109-118.
van Slooten, H., Moolenaar, A.J., van Seters, A.P., Smeenk, D., 1984. The treatment of adrenocortical carcinoma with o,p’-DDD: prognostic implications of serum level monitoring. Eur. J. Cancer Clin. Oncol. 20, 47-53.
Vanden Bossche, H., Willemsens, G., Cools, W., Bellens, D., 1984. Effects of etomidate on steroid biosynthesis in subcellular fractions of bovine adrenals. Biochem. Pharmacol. 33, 3861-3868.
Varga, I., Racz, K., Kiss, R., Futo, L., Toth, M., Sergev, O., Glaz, E., 1993. Direct inhibitory effect of etomidate on corticosteroid secretion in human pathologic adrenocortical cells. Steroids 58, 64-68.
Xu, Y., Yu, R.M., Zhang, X., Murphy, M.B., Giesy, J.P., Lam, M.H., Lam, P.K., Wu, R.S., Yu, H., 2006. Effects of PCBs and MeSO2-PCBs on adrenocortical steroidogenesis in H295R human adrenocortical carcinoma cells. Chemosphere 63, 772-784.