RESEARCH ARTICLE
Chiral effects in adrenocorticolytic action of o,p’-DDD (mitotane) in human adrenal cells
V. Asp1*, T. Cantillana2*, Å. Bergman2, and I. Brandt1
1Environmental Toxicology, Uppsala University, Uppsala, Sweden, and 2Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden
Abstract
1. Adrenocortical carcinoma (ACC) is a rare malignant disease with poor prognosis. The main pharmaco- logical choice, o,p’-DDD (mitotane), produces severe adverse effects.
2. Since o,p’-DDD is a chiral molecule and stereoisomers frequently possess different pharmacokinetic and/or pharmacodynamic properties, we isolated the two o,p’-DDD enantiomers, (R)-(+)-o,p’-DDD and (S)-(-)-o,p’-DDD, and determined their absolute structures.
3. The effects of each enantiomer on cell viability and on cortisol and dehydroepiandrosterone (DHEA) secretion in the human adrenocortical cell line H295R were assessed. We also assayed the o,p’-DDD racemate and the m,p’- and p,p’-isomers.
4. The results show small but statistically significant differences in activity of the o,p’-DDD enantiomers for all parameters tested. The three DDD isomers were equally potent in decreasing cell viability, but p.p’-DDD affected hormone secretion slightly less than the o,p’- and m,p’-isomers.
5. The small chiral differences in direct effects on target cells alone do not warrant single enantiomer administration, but might reach importance in conjunction with possible stereochemical effects on pharmacokinetic processes in vivo.
Keywords: Adrenocortical cancer (ACC); mitotane; chirality; steroidogenesis
Introduction
Adrenocortical carcinoma (ACC) is a rare and highly malignant disease with poor prognosis. The incidence is approximately 0.5-2.0 per million population per year, and the disease is slightly more common in women than in men (Ahlman et al. 2001). Over 60% of the tumours overproduce adrenocortical hormones, most commonly corticosteroids, alone or in combination with androgens (Icard et al. 2001; Tauchmanova et al. 2004; Terzolo et al. 2007). Complete surgical removal of the tumour offers the best chance for long-term survival (Ahlman et al. 2001). If complete surgical removal is not possible, the main pharmacological choice is o,p’-DDD (mitotane), which has been used since 1960 to decrease cortisol hypersecretion and inhibit tumour growth. o,p’-DDD is
also used in the treatment of Cushing’s syndrome, that is, overexposure to cortisol (Newell-Price et al. 2006). However, the efficacy of o,p’-DDD ACC therapy in ulti- mately prolonging patient survival is unclear and still under debate (Ahlman et al. 2001; Bergenstal et al. 1960; Huang & Fojo 2008; Terzolo et al. 2007; Terzolo & Berruti 2008). A compilation in 2005 of results from different studies concluded that o,p’-DDD treatment helped control excess hormone secretion in most cases, but led to tumour regression in only 25% of patients (Hahner & Fassnacht 2005). Daily mitotane doses of several grams per day, in combination with careful monitoring of serum levels, are needed to reach even the lowest therapeutic level of 14 µg ml-1 (44 µM) (Baudin et al. 2001; Haak et al. 1994; Terzolo & Berruti 2008; van Slooten et al. 1984). Moreover, mitotane has a narrow therapeutic window;
*V. Asp and T Cantillana contributed equally to this work.
at least 80% of patients experience adverse side effects, and serum levels greater than 20 µg ml-1 increase the risk for adverse effects (Allolio & Fassnacht 2006). The adverse effects are mainly gastrointestinal and neuro- toxic in nature, and are often dose-limiting (Allolio & Fassnacht 2006).
o,p’-DDD is a chiral molecule with two enantiomers: (R)-(+)-o,p’-DDD and (S)-(-)-o,p’-DDD (Figure 1). As a registered drug for ACC treatment, o,p’-DDD is admin- istered as a racemic mixture with an enantiomeric ratio (ER) of 1 (Cantillana et al. 2009). Enantiomers may pos- sess different pharmacokinetic and/or pharmacody- namic properties: examples include thalidomide, where (S)-thalidomide is the more active enantiomer while most of the adverse effects are related to (R)-thalidomide (reviewed in Eriksson et al. 2001), and o,p’-DDT, where (-)-o,p’-DDT interacts with the human oestro- gen receptor and hence mediates oestrogenic effects while (+)-o,p’-DDT possesses very little such activity (Hoekstra et al. 2001). We recently presented results from a pharmacokinetic study in Göttingen minipigs that indicate polymorphisms in the disposition of o,p’- DDD enantiomers (Cantillana et al. 2009). Moreover, a Finnish study on placentas from healthy women (not treated with mitotane but exposed to o,p’-DDD present in the environment) found an enantiomeric ratio of below 1, implying that the distribution and/or kinetics of the two enantiomers were different (Shen et al. 2006). To our knowledge, the contribution of each o,p’-DDD enantiomer to the pharmacological effects and/or the severe adverse effects of o,p’-DDD has never been studied. If, however, the two enantiomers would indeed possess markedly different pharmacological properties, administration of only one enantiomer might improve o,p’-DDD therapy.
o,p’-DDD is bioactivated in the adrenal cortex of dogs to a reactive acyl chloride that gives rise to irre- versibly bound mitochondrial protein adducts, induces adrenocortical cell death, and decreases glucocorticoid production (Hart et al. 1973; Martz & Straw 1977). The acyl chloride is formed through cytochrome P450 (CYP)- catalysed hydroxylation and subsequent spontaneous dehalogenation at the ß-carbon in the dichloroethane side chain (Cai et al. 1995b). Bioactivation of o,p’-DDD
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
Cl
Cl
(R)-(+)-o,p’-DDD)
(S)-(-)-o,p’-DDD)
also occurs in adrenal homogenates from humans, cows, rabbits and rats (Cai et al. 1995b; Martz & Straw 1980). Besides its adrenocorticolytic activity, o,p’-DDD directly affects glucocorticoid secretion by inhibiting the ster- oidogenic enzymes CYP11A1 and CYP11B1 (Asp et al. 2009; Hart & Straw 1971; Hart et al. 1971).
In order to assess the direct effects of each o,p’-DDD enantiomer in target cells, we isolated the enantiomers by high-performance liquid chromatography (HPLC) and determined their effects on hormone secretion and cell viability in the human adrenocortical cell line H295R. We compared the enantiomer effects with those of the racemic mixture and to those of the m,p’- and p,p’-DDD isomers.
Materials and methods
Chemicals
o,p’-DDD (purity 99%) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). p,p’- DDD (99%) was from EGA-Chemie (Steinheim/Albuch, Germany). m,p’-DDD (99%) was synthesized as described elsewhere (Lindholm et al. 1987). The cell culture tested 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St Louis, MO, USA).
Enantiomer separation and identification
The procedures for semi-preparative enantioselec- tive separation of the two o,p’-DDD enantiomers by HPLC and subsequent crystallization and identification by X-ray diffraction have been described previously (Cantillana & Eriksson 2009; Cantillana et al. 2009). Briefly, the enantiomers were isolated by repeated injec- tion and fractionation cycles using a semi-preparative permethylated y-cyclodextrin column (Nucleodex, Macherey-Nagel, Germany) with methanol:water (8:2) as the mobile phase. The isolated enantiomers were crys- tallized in ethanol and the absolute configuration was determined by X-ray crystallography. The specific rota- tion, [a], was assessed with a polarimeter with a sodium lamp at room temperature. The first eluting enantiomer was identified as (S)-(-)-o,p’-DDD and hence the second eluting enantiomer is (R)-(+)-o,p’-DDD (Cantillana & Eriksson 2009).
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 Dulbecco’s modified Eagle’s medium (DMEM)-F12
(1:1) (Sigma-Aldrich), containing 15 mM HEPES and sup- plemented with 2.5 mM L-glutamine (Sigma-Aldrich), 1% ITS+ (BD Biosciences, Franklin Lakes, NJ, USA) and 2.5% NuSerum I (BD Biosciences). The cells were kept in a humidified atmosphere at 37℃, 5% CO2.
MTT cell viability/cytotoxicity assay
The MTT assay was performed essentially as described in Tada et al (1986). For cytotoxicity testing, cells were seeded in 96-well plates 24h before assay. DDD isomers and o,p’-DDD enantiomers were dissolved in DMSO and added to assay medium (DMEM-F12 medium supple- mented with glutamine but without ITS+ and NuSerum) from 1000-fold stock solutions, yielding final concentra- tions of 1.25, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, and 20 uM. 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 72h. Cell viability was determined during the last 2 h of incubation by the addition of one-fifth volume MTT (5 mg ml-1 in phosphate-buffered saline (PBS)) to each well. Formed formazan crystals were dissolved by the addition of 10% sodium dodecylsulphate (SDS), 0.01 M hydrochloric acid and overnight incubation at 37℃, 5% CO2. Absorbance values at 560 nm were normalized against DMSO con- trols after subtraction of background absorbance at 750 nm. Cell viability was expressed as per cent viability of DMSO control cells. For viability assessment during the hormone assay, fresh assay medium containing one- fifth volume MTT was added for 2h after the collection of cell medium for cortisol measurement.
Hormone assay and cortisol measurements
Cells were seeded in 24-well plates 48 h before assay. DDD isomers and o,p’-DDD enantiomers were added to assay medium from 1000-fold stock solutions in DMSO, yielding final concentrations of 0.625, 1.25, 2.5, 5, 7.5, 10 and 12.5 uM. Each compound and concentration was assayed in triplicate and the experiment was repeated three times. The cells were incubated at 37℃, 5% CO, for 24 h. The medium was then removed and replaced with 500 ul fresh assay medium without test compounds and incubated for a further 3 h to allow hormone secretion. The cell medium was collected and stored at -20℃ until analysis. Cortisol and dehydroepiandrosterone (DHEA) levels were measured using EIA kits from Neogen (Lansing, MI, USA) and Demeditec (Kiel-Wellsee, Germany), respectively, according to the manufacturers’ instructions. Hormone levels were expressed as per cent of DMSO control levels. Cell viability during hormone assays was determined by MTT reduction and results were only included in the analysis if viability was greater
than or equal to 75% of control cells at the end of the experiment.
Statistical analyses
Cell viability and hormone data for each test compound were fitted with a non-linear concentration-response curve (variable slope) using GraphPad Prism version 5.01 for Windows. Significant differences between the concentration-response curves were assessed by the extra sum-of-squares F-test. The level of significance was set to 0.05.
Results
Effects of test compounds on cell viability
Cell viability was measured by MTT reduction after 72-h incubation with test compounds. The o,p’-DDD racemic mixture as well as the two o,p’-DDD enantiomers (R)- (+)-o,p’-DDD and (S)-(-)-o,p’-DDD all decreased H295R cell viability in a concentration-dependent manner. (R)-(+)-o,p’-DDD and (S)-(-)-o,p’-DDD generated significantly different concentration-response curves, as calculated using the extra sum-of-squares F-test (p = 0.042) (Figure 2A). EC50 values (and 95% confi- dence intervals (CI)) were 13.5 µM (5.8-31.4 uM) for (R)-(+)-o,p’-DDD and 10.5 µM (9.3-11.8 µM) for (S)- (-)-o,p’-DDD. Interestingly, the concentration-response curve for the o,p’-DDD racemic mixture lay below both enantiomer curves but was not parallel to either of them (Figure 2B). The m,p’- and p,p’-DDD isomers also caused concentration-dependent decreases in cell viability, but displayed identical concentration-response relation- ships and cytotoxic potency as the racemic o,p’-DDD (p=0.62) (Figure 2C).
Effects of test compounds on cortisol and DHEA secretion
Cortisol and DHEA secretion was measured in H295R cells after 24-h incubation with non-cytotoxic con- centrations of test compounds (non-cytotoxic condi- tions defined as at least 75% of viability of control cells remaining at the end of the experiment) followed by a 3-h secretion period.
Cortisol secretion was decreased in a concentra- tion-dependent way by all test compounds. The con- centration-response curve for (R)-(+)-o,p’-DDD had a steeper slope and lay consistently below the curve for (S)-(-)-o,p’-DDD (Figure 3A). The two curves were significantly different according to the extra sum-of- squares F-test (p=0.0003). IC50 values (and 95% CI) were 4.3 µM (3.7-5.1 µM) for (R)-(+)-o,p’-DDD and 6.2 µM
(4.2-9.3 µM) for (S)-(-)-o,p’-DDD. In contrast to the cell viability data, the concentration-response curve for the racemic o,p’-DDD lay between the two enantiomer curves (Figure 3B). The concentration-response curve
(A)
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for the racemic o,p’-DDD could not be separated from the m,p’-DDD curve by statistical analysis (p=0.30) but was significantly different from the p,p’-DDD curve (p=0.035) (Figure 3C).
All test compounds also decreased DHEA secretion by the H295R cells in a concentration-dependent man- ner. The concentration-response curves for (R)-(+)-o,p’-
(A)
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DDD and (S)-(-)-o,p’-DDD were significantly different according to the extra sum-of-squares F-test (p<0.0001) (Figure 4A). The IC50 value (and 95% CI) for (R)-(+)-o,p’- DDD was 5.0 µM (4.1-6.1 µM). No useful IC50 value could be calculated from the (S)-(-)-o,p’-DDD data. As for the
(A)
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DHEA (% of control)
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cortisol results, the concentration-response curve for the racemic o,p’-DDD lay between the two enantiomer curves (Figure 4B). The concentration-response curves for the o,p’- and m,p’-DDD isomers could not be sepa- rated by statistical analysis (p=0.45) (Figure 4C). Our software could not fit a concentration-response curve to the DHEA data for p,p’-DDD (Figure 4C).
Discussion
Chiral pharmaceuticals such as o,p’-DDD (mitotane) might comprise stereoisomers with different pharmacoki- netic and/or pharmacodynamic properties. We therefore isolated the two o,p’-DDD enantiomers, (R)-(+)-o,p’-DDD and (S)-(-)-o,p’-DDD, and assessed their direct effects on viability and hormone secretion in human target adreno- cortical H295R cells. The results revealed small but statis- tically significant differences between (R)-(+)-o,p’-DDD and (S)-(-)-o,p’-DDD for cell viability/cytotoxicity as well as for cortisol and DHEA secretion. The concentration- response curves generated by the two compounds, as well as by the o,p’-DDD racemic mixture, were analysed using the extra sum-of-squares F-test. This test compares sev- eral parameters of the concentration-response curves and thus yields more information than single-parameter anal- ysis. The EC50/IC50 values are given for reference purposes, but are only one of the measures included in the analysis. It should be noted that all concentrations tested were well below the lowest therapeutic serum concentration, 14 μg ml-1 (44 μΜ).
The respective contribution of each o,p’-DDD enan- tiomer to the biological effects of the racemic mixture seemed to differ for the studied parameters. For cortisol and DHEA secretion, the impact of the racemate was largely the sum of the enantiomer effects at correspond- ing total concentrations (Figures 3B and 4B, respectively). For cell viability, however, the racemic mixture produced higher cytotoxicity than the sum of the two pure enantiom- ers at corresponding total concentrations (Figure 2B).
We also compared the effects of o,p’-, m,p’-, and p,p’-DDD. The three isomers displayed equal cytotoxic potency in our system (Figure 2C). This is in agreement with our previous findings that the four DDD isomers o,p’-, m,p’-, o,m’-, and p,p’-DDD all bind irreversibly in the murine lung following CYP-catalysed activa- tion, regardless of the chlorine substitution pattern in the phenyl rings (Lund et al. 1986, 1989). The lack of structure specificity complies with the notion that the metabolic activation takes place at the dichloroethane side-chain. Moreover, the o,p’- and m,p’-DDD iso- mers were equally potent in decreasing cortisol and DHEA secretion and their concentration-response curves could not be separated by statistical analysis (Figures 3C and 4C). However, p,p’-DDD had slightly
less impact on cortisol secretion than the o,p’- and m,p’-isomers and seemed also to have less effect on DHEA secretion, although the DHEA data for p,p’-DDD could not be subjected to statistical analysis. Taken together, the results are in slight contrast to those of Cai et al (1995a), who reported an o,p’ - > m,p’- > p,p’- DDD rank order for effects on both cortisol production and cell proliferation in NCI H295 cells (the adreno- cortical suspension cell line from which the adherent H295R cell line was adapted). However, Cai et al. used compound concentrations as high as 50 M, where DDD solubility is an issue, and incubations of 7 days, more than double the length of the present experiments.
The small differences in potency found in vitro for (R)-(+)-o,p’-DDD and (S)-(-)-o,p’-DDD do not warrant single enantiomer therapy in themselves. However, pharmacokinetic processes such as distribution and elimination in vivo might differ between the enanti- omers, affecting target site concentrations. Indeed, we recently observed interindividual differences in the disposition of o,p’-DDD enantiomers in minipigs that manifested themselves as different enantiomeric ratios in blood plasma and adipose tissue (Cantillana et al. 2009).
In summary, the results revealed small but statisti- cally significant differences in the effects on cell viability and cortisol and DHEA secretion in human H295R cells of the two o,p’-DDD enantiomers (R)-(+)-o,p’-DDD and (S)-(-)-o,p’-DDD. Moreover, all three DDD isomers affected cell viability equally, but p,p’-DDD affected hor- mone secretion slightly less than o,p’- and m,p’-DDD. However, although the differences in direct effects on target cells between (R)-(+)-o,p’-DDD and (S)-(-)-o,p’- DDD were small, their importance might increase due to different pharmacokinetic effects not addressed in this study.
Declaration of interest
Financial support was obtained from the Swedish Research Council Formas and from the Cancer and Allergy Foundation. I. Brandt is cofounder of OncoTargeting AB, a company created to develop drugs against cancer, including ACC. The authors alone are responsible for the content and writing of the paper.
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