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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2157
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2165-2170
Copyright © 2006 by The Endocrine Society

Mitotane Has an Estrogenic Effect on Sex Hormone-Binding Globulin and Corticosteroid-Binding Globulin in Humans

Nancy Nader, Gérald Raverot, Agnès Emptoz-Bonneton, Henri Déchaud, Marc Bonnay, Eric Baudin and Michel Pugeat

Institut National de la Santé et de la Recherche Médicale ERM 329 (N.N., A.E.-B., H.D., M.P.), Hôpital Debrousse, 69005 Lyon, France; Fédération d’Endocrinologie (G.R., A.E.-B., M.P.) and Laboratoire de Radiopharmacie et de Radioanalyse (H.D.), Centre de Medecine Nucleaire, Hopital Neurologique et Cardiologique, 69677 Bron Cedex 03, France; and Institut Gustave Roussy (M.B., E.B.), 94805 Villejuif Cedex, France

Address all correspondence and requests for reprints to: Michel Pugeat, M.D., Fédération d’Endocrinologie du Pôle Est, 59 bd Pinel, 69677 Bron Cedex, France. E-mail: michel.pugeat{at}chu-lyon.fr.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Side effects of mitotane (o,p'-DDD) have suggested estrogenic effects.

Objective: The objective of the study was to explore o,p'-DDD potential estrogenic effect on SHBG and corticosteroid-binding globulin (CBG).

Design: Human hepatoma cell lines (HepG2), lacking estrogen receptor (ER)-{alpha}, and Hep89, stably transfected by ER{alpha}, were used.

Setting: The study was conducted at an academic research laboratory and medical center.

Patients and Other Participants: The study included 10 male patients with recurrent adrenal carcinoma, receiving mitotane (4–6.5 g daily) for more than 6 months.

Main Outcome Measures: The main outcome measures were SHBG/CBG mRNA levels measured by real-time PCR, culture medium SHBG/CBG concentrations measured by specific immunoassays, and transient transfection experiments with human SHBG proximal promoter reporter constructs.

Results: Increased serum SHBG and CBG concentrations, which exceeded normal male limits, were observed in most mitotane-treated patients. In the HepG2 cell line, 17ß-estradiol (E2) or o,p'-DDD treatment had no effect on mRNA or SHBG/CBG concentrations. In contrast, in the Hep89 cell line, E2 increased concentrations of SHBG (r = 0.44, P < 0.0001) and CBG (r = 0.585, P < 0.0001) secreted into culture media in a dose-dependent manner. o,p'-DDD significantly increased SHBG (150% vs. control, P < 0.05) and CBG (184% vs. control, P < 0.05) production by Hep89 cells, at a concentration of 2 x 10–5 M. Transient transfection experiments in Hep89 cells showed that E2 or o,p'-DDD treatment did not increase the transcriptional activity of the minimal proximal promoter of human SHBG gene.

Conclusions: Mitotane increased SHBG/CBG gene expression and liver production by mechanisms requiring the presence of ER{alpha}.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
MITOTANE (1,1'-dichlorodiphenyldichloroethane) (o,p'-DDD) is the only adrenal-specific agent available for treatment of adrenal carcinoma in patients who cannot be cured by surgery (1). Mitotane exerts a specific, direct, cytotoxic effect on adrenocortical mitochondria (2) and impairs adrenal steroidogenesis by mechanisms that are not yet fully elucidated. The adrenolytic effect of mitotane has been attributed to its main metabolites, o,p'-DDA and o,p'-DDE (3, 4). Numerous side effects of mitotane administration have limited long-term clinical tolerance of mitotane administration. The efficacy of an attempted low-dose mitotane protocol (5, 6, 7) was not conclusive, and it is generally accepted that antitumor efficacy is observed only with doses reaching plasma o,p'-DDD concentrations higher than 14 mg/liter (8).

The high incidence of gynecomastia in male patients receiving o,p'-DDD has been associated with an increased binding capacity of SHBG (9), the specific transport protein that retains 17ß-estradiol and testosterone in the plasma compartment and modulates their disposal for target cells (10). On the other hand, cortisol metabolism has been reported to be altered in patients receiving o,p'-DDD treatment, with induction of hepatic microsomal enzymes (11, 12). Finally, increased serum corticosteroid-binding globulin (CBG) concentrations have also been reported in mitotane-treated patients (13). Because both SHBG and CBG are estrogen-sensitive liver proteins (10, 14), we hypothesized that o,p'-DDD may have an estrogenic action on SHBG and CBG gene expression. We aimed in this study to further understand the molecular mechanism(s) by which o,p'-DDD increases steroid-binding protein concentrations, using a human hepatoma cell line model.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Materials

o,p'-DDD was kindly provided by Roussel-Uclaf Laboratories (Paris, France). The restriction enzyme XhoI and cell culture reagents were purchased from Life Technologies (Grand Island, NY). PCR markers, reverse transcriptase kit, and the kit for luciferase and ß-galactosidase activity measurement were obtained from Promega (Madison, WI). PCR primers were purchased from Operon (Seraing, Belgium) and RNA extraction solution (RNA plus) from Q-Biogen (Cologne, Germany). Restriction enzymes KpnI and HindIII were from New England Biolabs (Beverly, MA). Culture flasks and dishes were provided by Falcon Plastics (Oxnard, CA).

Patients

Serum samples from 10 male patients receiving o,p'-DDD (mitotane), for more than 6 months at doses ranging from 4 to 6.5 g daily for recurrent adrenal carcinoma were provided by Gustave Roussy Institute, Villejuif, France. Total testosterone and estradiol were measured by an in-house RIA after organic extraction and celite chromatography (15) and SHBG by using an immunoradiometric assay (SHBG-RIACT) from Cis Bio International (Gif-sur-Yvette, France), with a detection limit of 3 nmol/liter. The method was adapted to increase the assay medium culture detection limit to 25 pmol/ml. Using the respective values for measured total testosterone and SHBG concentrations, the non-SHBG bound testosterone was calculated according to Vermeulen et al. (16). CBG was measured by RIA, as previously described (17). The method was modified to raise the sensitivity to 0.01 mg/ml.

Serum concentrations of o,p'-DDD and o,p'-DDE were measured using HPLC as described previously (8).

17ß-Estradiol and o,p'-DDD preparation

o,p'-DDD was diluted in ethanol at 2 x 10–4, 2 x 10–5, and 2 x 10–6 M; then 1 ml from each solution was evaporated (using an evaporating nitrogen system), and then adequate quantities of phenol red-free DMEM were supplemented with 10% stripped fetal calf serum (FCS) (for dose-response experiments) or OPTI-MEM supplemented with 10% stripped FCS (for transfection experiments) were added. 17ß-Estradiol solutions were prepared similarly and used at final concentrations of 10–10 to 10–5 M. Controls, using support medium only, were also performed.

Cell culture

The HepG2 cell line was a gift from Dr. C. Trepo (Institut National de la Santé et de la Recherche Médicale U 271, Lyon, France), and the Hep89 cell line [HepG2 stably transfected with human (h) estrogen receptor (ER)-{alpha}] was kindly provided by Dr. K. Karathanasis (Department of Nuclear Receptors, Wyeth-Ayerst Research, Radnor, PA) (18). Cells were grown at 37 C in a 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% FCS, L-glutamine (300 mg/liter), penicillin (100 U/mol), streptomycin (100 µg/ml), and amphotericin B (25 µg/ml).

Cells (~106) were plated in T25 flasks with 5 ml medium supplemented as above. Near confluence, cells were washed twice with PBS and incubated in phenol red-free DMEM, supplemented as above for 48 h. Then cells were incubated for a further 48 h in the same medium without, or in the presence of various concentrations of 17ß-estradiol (10–10 to 10–5 M) or o,p'-DDD (2 x 10–6 to 2 x 10–4 M). For each concentration of 17ß-estradiol or o,p'-DDD and controls, three culture flasks were prepared. In addition, three series of experiments were performed with HepG2 and Hep89 cells.

RNA extraction and real-time RT-PCR: Light Cycler technique

Total RNA was extracted according to the published modification (19) of the method of Chomczinsky and Sacchi (20). For each T25 flask and after removing mediums, the cells were lysed with 1 ml RNAplus (Q-Biogen) solution. After extraction, the final RNA pellet was dissolved in 100 µl H2O and stored at –80 C until used. The RNA concentration was determined by absorbance at 260 nm.

RNA extracted from HepG2 and Hep89 cells was reverse transcribed with Moloney murine leukemia virus reverse transcriptase using a reverse transcriptase kit. The primer sequences for SHBG (5'-aac-tta-aga-cgg-act-cag-gg and 3'-gag-tat-gtc-cag-ggt-ggt-ctt) and CBG (5'-cac-caa-cca-ggc-aaa-ttt-ct and 3'-gga-cgt-cag-gtt-tag-ggt-ga) produced a PCR fragment of 263 bp for SHBG and 252 bp for CBG. The quality of RNA and cDNA synthesis was ascertained by amplification of the homosapiens ribosomal protein (P36B4) gene as the internal control. The primer sequences for the P36B4 primers were: 5'-tac-cac-aag-aac-ggg-tag-tc and 3'-cac-taa-aat-ctc-cag-ggg-ca and produced a PCR fragment of 292 bp. Real-time RT-PCR was performed with a Light Cycler (Roche Molecular Biochemicals, Mannheim, Germany) in Light Cycler capillaries using a commercially available master mix containing Taq DNA polymerase and SYBR-Green I deoxyribonucleoside triphosphates (Light Cycler DNA master SYBR-Green I, Roche Molecular Biochemicals). After addition of primers (final concentration: 0.4 µM), MgCl2 (3 mM), and template cDNA to the master mix, 30 cycles of denaturation (94 C for 15 sec), annealing (60 C for 5 sec), and extension (72 C for 12 sec) were performed. All samples were assayed in duplicate. For each PCR run, a standard curve was constructed from serial dilutions of cDNA from HepG2 and Hep89 cells lines. The level of expression of SHBG and CBG mRNA was given as relative copy numbers normalized against P36B4 mRNA and shown as mean ± SD. Relative SHBG and CBG mRNA expression were calculated using the formula (A/G) x 1000, where A is the relative copy number of SHBG or CBG mRNA and G is the relative copy number of P36B4 mRNA.

SHBG/CBG and intracellular protein assays

Cell culture media were collected to measure SHBG/CBG concentrations. The results were correlated with the intracellular protein concentration and expressed as a percentage of control values. After removing medium from each flask, cells were collected using 0.25% trypsin-EDTA solution and resuspended in RPMI 1640 supplemented with 10% FCS. Cells were centrifuged (800 rpm, 5 min), and the cell pellets were resuspended, again in 5 ml RPMI 1640 (10% FCS). Using a sonifier cell disrupter, 1 ml of this solution was sonicated and used for Bradford dye binding assay (protein assay, Bio-Rad Laboratories, Marnes-la-Coquette, France) to determine intracellular protein concentrations. BSA was used as standard.

Transient transfection experiments

Human SHBG proximal promoters (–801/+7, – 299/+7, –137/+7) fused to luciferase reporter gene in PGL2 were kindly provided by Dr. G. Hammond (Research Institute for Children’s and Women’s Health, Vancouver, Canada) (21). These reporter plasmids were digested with XhoI/HindIII (–801/+7) and KpnI/HindIII (–299/+7, –137/+7), and released inserts were subcloned into the promoterless PGL3 basic luciferase reporter plasmid to increase the sensitivity of luciferase activity measurement. Hep89 cells were grown in 6-well or 35-mm tissue culture plates in the order of 3 x 105 cells/well in 2 ml defined DMEM medium. Near confluence, transient transfection of each human SHBG promoter reporter plasmid together with a pSVLacZ control plasmid was carried out using LipofectAMINE reagent according to Life Technologies. In transactivation experiments, reporter plasmids (2 µg) were mixed with 1 µg of pSVLacZ control plasmid using OPTI-MEM medium supplemented with 10% stripped FCS. Approximately 48 h after the start of transfection, the cells were treated for 2 d with 17ß-estradiol (10–5 M) or o,p'-DDD (2 x 10–4 M) in OPTI-MEM medium (10% FCS). After 48 h of treatment, the cells were washed twice with PBS and lysed by a reporter lysis buffer (Promega) in three cycles of freeze-thawing. The cells were harvested (1200 g, 1 min), and cell pellets were used to measure luciferase and ß-galactosidase activity, as recommended by the manufacturer. To correct for transfection efficiency, light units from the luciferase assay were divided by the OD reading from the ß-galactosidase assay.

Statistical analysis

Data were expressed as mean and SD (± SD) for three independent experiments performed with HepG2 or Hep89 cells and were analyzed by ANOVA. Statistical difference between treated and untreated culture flasks or dishes was assessed by the Student t test, with a significance threshold set at P < 0.05. Dose-dependent effects of 17ß-estradiol and o,p'-DDD were studied by the least square regression method. For multiple transfection experiments, data were analyzed by ANOVA. Relationships between SHBG/CBG/o,p'-DDA/o,p'-DDE in sera patients were sought by Spearman tests and simple linear regression analysis.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Sex steroid and binding protein concentrations in mitotane-treated male patients

The concentrations of testosterone, non-SHBG bound testosterone, 17ß-estradiol, o,p'-DDD, o,p'-DDE, SHBG, and CBG in 10 male patients are given in Table 1Go. Estradiol concentration was within the normal range except in patient 2. In contrast, total testosterone concentration was lower than 300 ng/dl in four of 10 patients and non-SHBG bound testosterone concentration was lower than 130 ng/dl in seven of 10 patients. Steroid-binding protein levels exceeded normal male limits in eight of 10 patients for SHBG (>55 nmol/liter) and nine of 10 for CBG (>54 mg/liter), and SHBG and CBG levels were significantly correlated (R2 = 0.504, P < 0.02). o,p'-DDD concentration was not associated with SHBG (Fig. 1AGo) but correlated significantly with CBG levels (R2 = 0.513, P < 0.02) (Fig. 1BGo). o,p'-DDE levels were detectable in only three patients.


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  		TABLE 1. Steroids and steroid-binding proteins, o,p'-DDD and o,p'-DDE concentrations in serum samples from 10 male patients under o,p'-DDD treatment (4.5–6 g daily)

 

Figure 1
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  		FIG. 1. Relationship plot between SHBG and o,p'-DDD levels (A) and CBG and o,p'-DDD levels (B) in 10 male patients receiving mitotane.

 
Effect of o,p'-DDD on SHBG/CBG mRNA expression

The real-time PCR technique showed a significantly increased expression of SHBG/CBG mRNA levels in Hep89 cells from an o,p'-DDD concentration of 2 x 10–5 M (6.4 mg/liter) up to 2 x 10–4 M (64 mg/liter) (153% vs. control, P < 0.05 for SHBG; 150% vs. control, P < 0.05 for CBG). In contrast, no significant effect was observed in HepG2 cells (Fig. 2Go).


Figure 2
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  		FIG. 2. Effect of o,p'-DDD on SHBG (A) and CBG (B) mRNA expression by Hep89 and HepG2 cells using the real-time PCR technique. Cells were treated for 48 h with support medium as control, o,p'-DDD at 2 x 10–6 M, 2 x 10–5 M, and 2 x 10–4 M. The level of expression of SHBG/CBG mRNA is given as relative copy numbers normalized against P36B4 mRNA and shown as the mean ± SEM of three independent experiments, each performed in triplicate flasks. *, P < 0.05 vs. control.

 
Effect of 17ß-estradiol and o,p'-DDD on SHBG/CBG HepG2/Hep89 production

In HepG2 cells, SHBG/CBG production was not significantly affected by exposure to 17ß-estradiol concentrations up to 10–5 M or o,p'-DDD concentrations up to 2 x 10–4 M (64 mg/liter). In contrast, 17ß-estradiol increased SHBG (r = 0.44, P < 0.0001) and CBG (r = 0.585, P < 0.0001) concentrations dose dependently in Hep89 culture cell medium. The minimum effective dose of 17ß-estradiol was 10–7 M, leading to a significant increase of 181% for SHBG (vs. control; P < 0.05) and 230% (vs. control; P < 0.05) for CBG at 10–5 M (Fig. 3Go). Higher doses of o,p'-DDD (2 x 10–5 M; 6.4 mg/liter) than 17ß-estradiol were required to significantly increase SHBG (150% vs. control, P < 0.05) and CBG (184% vs. control, P < 0.05) in Hep89 cell medium. Above this concentration, the increase in SHBG/CBG production tended to plateau (Fig. 4Go). In both cell types and all conditions, the intracellular protein quantities remained unchanged (data not shown).


Figure 3
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  		FIG. 3. Effect of estradiol on SHBG (A) and CBG (B) secretion in HepG2 and Hep89 cells at 48 h of culture. Cells were treated for 48 h with support medium as control, estradiol at 10–10 M, 10–9 M, 10–8 M, 10–7 M, 10–6 M, and 10–5 M. Data are expressed as the mean ± SEM of three independent experiments, each performed in triplicate flasks and correlated to the intracellular protein content and expressed as a percentage of the control value. *, P < 0.05 vs. control.

 

Figure 4
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  		FIG. 4. Effect of o,p'-DDD on SHBG (A) and CBG (B) secretion in HepG2 and Hep89 cells at 48 h of culture. Cells were treated for 48 h with support medium as control, o,p'-DDD at 2 x 10–6 M, 2 x 10–5 M, and 2 x 10–4 M. Data are expressed as the mean ± SEM of three independent experiments, each performed in triplicate flasks and correlated to the intracellular protein content and expressed as a percentage of the control value. *, P < 0.05 vs. control.

 
Effect of 17ß-estradiol and o,p'-DDD on transcriptional activity of SHBG gene promoter

Independently of hormonal treatment, there was a significant difference (P < 0.05 vs. basic PGL3) in base-line SHBG promoter activity according to size: the baseline transcriptional activity of the shortest promoter, –137/+7, was 3 times as strong as the –298/+7 and –801/+7 promoters, as described earlier (15), and 12 times as strong as control plasmid PGL3basic (Fig. 5Go).


Figure 5
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  		FIG. 5. Analysis of human SHBG promoter activity in Hep89 cells treated with estradiol and o,p'-DDD. Hep89 cells were transiently transfected with three different SHBG promoter-luciferase reporter gene constructs (–801/+7, –299/+7, –137/+7) and treated for 48 h with estradiol 10–5 M (A) or o,p'-DDD 2 x 10–4 M (B). Transfection efficiency was corrected by cotransfection with pCMVLacZ control vector and measuring the resultant ß-galactosidase activity. Luciferase was assayed 48 h after transfection. Data are expressed as the mean of three independent experiments, each performed in triplicate dishes. *, P < 0.05 vs. empty PGL3. {dagger}, P < 0.05 vs. PGL3 + 800. {clubsuit}, P < 0.05 vs. PGL3 + 299.

 
Whatever the size of the promoter studied, cell treatment with 10–5 M 17ß-estradiol (Fig. 5AGo) or 2 x 10–4 M o,p'-DDD (Fig. 5BGo) did not significantly affect transcription activity, compared with controls.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The results of our study confirmed that most male patients under mitotane treatment in a dose ranging from 4 to 6.5 g daily had dramatic increase in SHBG and CBG levels, which correlated highly significantly with each other and only for CBG with o,p'-DDD levels. These results are in agreement with a previous kinetic study of three patients showing that circulating o,p'-DDD levels correlated with SHBG and CBG during the first month of treatment, whereas the significance of these correlations was lost in the subsequent months of treatment (13). There is no evidence in the literature demonstrating that liver cells can metabolize o,p'-DDD to o,p'-DDE. We tested this hypothesis by measuring o,p'-DDD and o,p'-DDE after cell treatment with o,p'-DDD. Although the o,p'-DDD was as expected, we were unable to detect o,p'-DDE in medium HepG2 cells (data not shown).

To further understand the potential mechanism(s) by which o,p'-DDD could induce hepatic SHBG/CBG production, we used the HepG2/Hep89 cell lines, which provided a unique opportunity to identify the ER{alpha}-dependent molecular effect of 17ß-estradiol and o,p'-DDD (18). We found that both 17ß-estradiol and o,p'-DDD increased SHBG/CBG production in HepG89 cells but not in the HepG2 cell line. However, whereas the effects of 17ß-estradiol on SHBG/CBG were dose dependent, high doses of o,p'-DDD were required, and we could not generate a significant dose-dependent effect in Hep89 cells. The limited solubility of o,p'-DDD under our experimental conditions might contribute to explaining these findings. In all treatment conditions, the baseline secretion of SHBG and CBG by HepG2 cells as well as total intracellular protein content were preserved, suggesting that a toxic effect of the highest used doses of o,p'-DDD on HepG2 cells was unlikely.

At doses equivalent to recommended therapeutic concentrations (8), o,p'-DDD significantly stimulated SHBG and CBG secretion and mRNA concentrations. These effects were obtained at a 2000-fold higher concentration than 17ß-estradiol. This difference in dose effect might be explained by a l000-fold weaker binding affinity of o,p'-DDD than 17ß-estradiol for recombinant hER{alpha}, as reported by Chen et al. (22).

Transient transfection experiments in Hep89 cells showed that 17ß-estradiol or o,p'-DDD treatment did not increase the transcriptional activity of the proximal human SHBG gene promoter. This region of the SHBG promoter contains binding sites for hepatocyte nuclear factor-4, which have been shown to markedly increase transcriptional activity. This region also contains a binding site for the nuclear transcription factor Sp1, which may be an alternative pathway for hER{alpha}-mediated activity, and a (TAAAA) repeat motif within an alu sequence (23). These observations suggest that responsive elements used by hER{alpha} are located in other zones of the SHBG gene and may pass through human ER{alpha}/protein interactions as demonstrated for some hER{alpha}-activated genes (24).

From a clinical point of view, the increase in SHBG levels in male patients receiving mitotane was in the order of magnitude that we reported in male hyperthyroid patients who also have decreased non-SHBG bound testosterone (25), suggesting mild hypogonadism. A prospective study will be necessary to find out the SHBG/CBG kinetic increase and the SHBG/CBG side effects under mitotane treatment.

In conclusion, our results show that o,p'-DDD increases SHBG and CBG expression and secretion by an ER{alpha}-dependent mechanism. Its intimate molecular effect remains to be elucidated.


   Acknowledgments
 
We are particularly indebted to Dr. Goeff Hammond for critical advice and Iain McGill and Kevin Hogeveen for help in revising the manuscript.


   Footnotes
 
This work was supported by Université Claude Bernard, Unité d’Enseignement Professionnelle Lyon-Nord, and Fondation de France. N.N. was supported by a grant from Association de Langue Française pour l’Etude du Diabète et des Maladies Métaboliques.

First Published Online March 21, 2006

Abbreviations: CBG, Corticosteroid-binding globulin; ER, estrogen receptor; FCS, fetal calf serum; h, human; o,p'-DDD, 1,1'-dichlorodiphenyldichloroethane.

Received September 29, 2005.

Accepted March 9, 2006.


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 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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