Metabolic Activation and Binding of Mitotane in Adrenal Cortex Homogenates
W. CAIt, R. E. COUNSELL+, T. DJANEGARAt, D. E. SCHTEINGARTS, J. E. SINSHEIMER1X, AND L. L. WOTRING+
Received September 9, 1994, from the +College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, and Departments of #Pharmacology and $Internal Medicine, Medical School, University of Michigan, Ann Arbor MI 48109-0624. Accepted for publication November 30, 1994®
Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloro- ethane, o,p’-DDD] is an adrenocorticolytic agent of value in the treatment of adrenocortical carcinoma and Cushing’s syndrome. In support of a program to develop agents superior to mitotane, it is the purpose of this study to explore the relationship of the metabolism of mitotane to its binding to adrenal cortex tissue from several sources. The objective was to detect the mitotane moiety responsible for its covalent binding in various test systems. Studies were conducted with an 1251-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, prior to a comparison to results with lower specific activity [14C]mitotane. With dog adrenal cortical whole homogenates, the majority of covalent binding was to proteins with an additional one-sixth of the total bound radioactivity associated with a phospholipid fraction. No radioactivity was associated with DNA. The rank order of species in regard to metabolism and protein binding was bovine > dog > rat adrenal homogenates > human normal adrenal or tumor homogenates. The percentage of radioactivity recovered from the hydrolysates of those fractions was uniformly high. In addition, the only metabolite present in the hydrolysates corresponded to 1-(2- chlorophenyl)-1-(4-iodophenyl)acetic acid from the iodo analog of o,p- DDD and the corresponding o,p’-dichlorodiphenylacetic acid (o,p’-DDA) from o,p’-DDD. Our results are consistent with an acyl chloride being the reactive intermediate formed from the dichloromethyl moiety of mitotane, which leads to both DDA metabolite formation and binding to adrenal cortical bionucleophiles.
Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichlo- roethane, o,p’-DDD] is the only FDA-approved adrenocorti- colytic agent currently available that has extended survival in patients with adrenocortical carcinoma.1 It is also used in the treatment of benign Cushing’s syndrome.2 The metabo- lism of mitotane both in vivo by humans3,4 and rats5 and in vitro in adrenal preparations6-8 has been studied in our laboratories in order to understand the adrenocorticolytic activity of mitotane. Martz and Straw9 have reported that the level of metabolism and covalent binding of mitotane in dog, guinea pig, rabbit, rat, and human adrenal mitochondrial incubations correlated with the known sensitivity of these species to the adrenolytic effect of the drug. However, the nature of this binding and its relationship to the metabolism of mitotane was not established.
In support of a program to design a drug with improved therapeutic efficacy over that of mitotane, it was important to understand the relationship between metabolism and binding as well as to determine whether such metabolic activation was the same in the several test systems we could employ in the evaluation of candidate drugs. The major metabolites of mitotane in vitro and in vivo are 2,4-dichlo- rodiphenylacetic acid (o,p’-DDA) and its hydroxylated deriva- tives in their free and conjugated forms.4-8 On the basis of what has been established for mitotane and its isomer, p,p’- DDD, there are at least three pathways for the generation of active metabolic intermediates that could lead to their cova- lent binding of adrenal bionucleophiles and to the formation
of metabolites. These are (1) arene oxide formation prior to para hydroxylation,4,5,8 (2) aliphatic epoxide formation with the unsaturated side chain metabolites of DDD10-12 leading to aliphatic hydroxylation6,8 as well as o,p’-DDA formation,3-9 and (3) formation of an acyl chloride intermediate of DDA.10-12
Gold and Brunk10-12 have conducted in vivo metabolism studies on p,p’-DDD as it might relate to tissue binding and they found that epoxidation of the ethylenic metabolic inter- mediates of p,p’-DDD did lead to DDA. However, the more important pathway was through an acyl chloride intermediate of DDA. That is, as has been established for other compounds containing a dihalogenated methyl moiety,13 enzymatic hy- droxylation of the methine hydrogen could be followed by spontaneous dehydrohalogenation, leading to acyl halide formation. This active intermediate in turn could lead to acylation of bionucleophiles or in the presence of water to an acid metabolite, as represented below for mitotane.
CI
CH
CI
CH
- HCI
1
CH
1
C H
C
0=0
C1
CI
L
CI
OH
C1
CI
+H20
-CH-
0=0
acylation of bionucleophiles
OH
op’ - DDA
It follows that if this biotransformation to the acyl chloride occurs for mitotane in the adrenal cortex and results in covalent binding to the bionucleophiles present, the hydrolysis of the bound adrenal cortex tissue should yield o,p’-DDA. The purpose of this investigation was to test this hypothesis with transformations in adrenal cortex preparations from various species using the 125I-labeled o-Cl, p’-I analog of DDD, 1-(2- chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane,7,14 as well as 14C-labeled mitotane.
Experimental Section
Materials-Nicotinamide adenine dinucleotide phosphate (NADP+), glucose 6-phosphate, glucose-6-phosphate dehydrogenase (type IX), N-(2-hydroxyethyl), piperazine-N’-2-ethanesulfonic acid (HEPES), and HEPES sodium were purchased from Sigma Chemical Co. (St. Louis, MO).
The reference compound 2,4-dichlorodiphenylacetic acid (o,p’-DDA) was obtained from the environmental Protection Agency (Research Triangle Park, NC), and 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2- dichloroethane (o,p’-DDD, mitotane) was purchased from Aldrich Chemical Co. (Milwaukee, WI). The synthesis of [14C]-o,p’-DDD, uniformly labeled in the 4-chlorophenyl ring, was previously reported by Counsell and Willette.15 The specific activity and GC analysis of purity for o,p’-DDD were 83.2 uCi/mmol and 94.0%.
1-(2-Chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane radiolabeled with iodine-125 was prepared as previously described14 except for the use of an improved method for radioiodination.16 A single radioactive
® Abstract published in Advance ACS Abstracts, January 1, 1995.
C 1995, American Chemical Society and American Pharmaceutical Association
peak corresponding to unlabeled standard was obtained as >99.6% pure on HPLC. The specific activity ranged between 1.1 and 3.4 Ci/ mmol.
1-(2-Chlorophenyl)-1-(4-iodophenyl)acetic Acid-Concentrated sul- furic acid (20 mL) was added dropwise to a well-stirred mixture of 8.0 g (0.043 mol) of o-chloromandelic acid and 10.6 g (0.052 mol) of iodobenzene cooled in an ice bath. The required o-chloromandelic acid was prepared from o-chlorobenzaldehyde and potassium cyanide in concentrated hydrochloric acid using the method of Jenkins.17 Stir- ring was continued for 1 h at 0 ℃ and then 3 h at room temperature. The mixture was poured on ice and extracted with 3 x 50 mL of ether, and the combined extracts were washed with 50 ml of water. The ethereal solution was extracted with 10% sodium hydroxide (2 x 50 mL) and the basic extract was acidified with 3 N hydrochloric acid and extracted with 3 x 100 mL of ether. The dried ethereal extract, redissolved in a small amount of ether, was separated on a silica gel (70 to 230 mesh, 60 Å) column with a hexane-ethyl acetate-acetic acid (90:9:1) solvent system. The purified compound was recrystal- lized from 70% ethanol to produce a 9.0 g (56.2%) yield. The white sheetlike crystals obtained showed the following properties: mp 119 — 120.5 °C; IR (KBr) 2500-3300 cm-1 (0-H), 1708 cm-1 (C=O), 1486, 1470 cm-1 (C=C, Ar), 1218 cm-1 (C-O), 805 cm-1 (1,4 disubstituted Ar-H), 735 cm-1 (1,2 disubstituted Ar-H); 1H NMR (CDC13) 8 5.45 (s, 1H, C-H), 8 7.03-7.10 (d, 2H, Ar-H), 8 7.23-7.29 (m, 3H, Ar-H), 8 7.38-7.44 (m, 1H, Ar-H), 8 7.66-7.72 (d, 2H, Ar-H); 13C NMR (CDC13) ở 177.2 (COOH), 8 93.6 (C-I, Ar), 8 53.4 (C-H); MS, m/z (relative intensity): 375 (5) [M + 3], 374 (26) [M + 2], 373 (12) [M + 1], 372 (61) [M], 330 (8) [M + 2 - CO2], 329 (47) [M + 2 - COOH], 328 (24) [M - CO2], 327 (100) [M - COOH], 293 (7) [M - CO2 - CI], 201 (9) [M - CO2 - I], 200 (7) [M - COOH - I] 199 (9) [M - COOH - HI] 166 (24) [M - CO2 - CI - I], 165 (79) [M - COOH - CI - I]. Anal. Calcd for C14H1002CII: C, 45.13; H, 2.70. Found: C, 45.03; H, 2.67.
Preparation of Adrenal Cortex Subcellular Fractions-Adrenal glands of mongrel dogs were removed and kept on ice enroute to the laboratory. The adrenal glands were trimmed of fat and connective tissue and sliced open, and the cortex was separated from the medulla. The cortices were weighed, minced, and homogenized in 5 volumes of 0.25 M sucrose-0.05 M HEPES buffer (pH 7.4) with a Teflon- glass Potter-Elvehjem homogenizer. The mixture was centrifuged (1000g, 4 ℃, 15 min). The supernatant was filtered through four layers of cheese cloth to remove fat, and to obtain whole homogenates, a portion of the supernatant was recentrifuged (109000g, 4 ℃, 20 min) so that the additonal fat floating on top of the supernatant could be discarded. The resulting pellet was resuspended in the defatted supernatant using the homogenizer to yield whole homogenates. To obtain subcellular fractions, the homogenate after its centrifugation at 1000g and filtration through cheese cloth was further centrifuged (10000g; 4 ℃, 20 min) to precipitate mitochondria which were washed in sucrose-HEPES buffer and recentrifuged. The 10000g supernatant was then centrifuged (109000g, 4 ℃, 60 min) to obtain microsomes and, after removal of a top fatty layer, to yield cytosolic fractions. The microsomal pellet was washed and recentrifuged. Homogenate fractions were adjusted with buffer to the same concentration as in the original homogenate and stored at -80 ℃ until use. To get a mixture of mitochondria and cytosol, the mitochondrial precipitate was resuspended in cytosol with a concentration equal to the original homogenate.
Whole homogenates of canine adrenal cortices were prepared from frozen glands by the same procedure as described above. Whole homogenates from bovine, rat, and human cortices were also prepared from frozen glands in a similar manner except that their lower fat content did not require filtration through cheese cloth nor centrifuga- tion at 109000g. Frozen canine and bovine adrenal glands were obtained from Pel-Freez Biologicals (Rogers, AR). Adrenal glands from 20 rats (Sprague-Dawley) were collected and stored at -20 ℃. Three human adrenal glands from liver transplant preparations and adrenal tumors of patients were provided by the University of Michigan Hospital and stored at -80 ℃. Protein concentrations of enzyme preparations were measured by the Lowry procedure as modified by Miller18 with BSA as the standard.
Incubations-Incubations in 15-mL centrifuge tubes consisted of the following: (A) 0.5 mL of HEPES buffer (pH 7.4) containing 0.82 µg (1.0 µM, 2.0 uCi) or 5.98 µg (7.0 uM, 2.0 uCi) of p’_125I analog of o,p’-DDD; (B) 0.25 mL of 44 umol/mL MgCl2; (C) 0.25 mL of NADPH- generating mixture (1.88 umol of NADP+, 22 umol of glucose
6-phosphate and 1.2 units of glucose-6-phosphate dehydrogenase in HEPES buffer); and (D) 1 mL of whole homogenate or homogenate fraction as the enzyme source. Incubations of [14C]-o,p’-DDD (0.14 mM, 0.045 „Ci) were as described above but at an increased volume (4 mL). The mixtures were incubated at 37 ℃ for 2 h and then stored at -20 ℃ prior to freeze drying or extraction.
Cell Cultures-Cell cultures of a human adrenocortical carcinoma cell line [NCI H295, American Type Culture Collection (Rockville, MD)]19 were grown as previously described.20 Culture medium was removed when cells were estimated to number 1.5 x 107 cells per 75-cm2 flask. R5-HITES medium [RPMI 1640 supplemented with 5% fetal bovine serum and other components20] (15 mL) containing 50 AM of 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane and a trace amount of 125I-labeled compound (4.0-30.0 uCi) were added to each culture flask. Cell-free flasks were used as controls. All flasks were incubated (37 ℃, 95% air/5% CO2) with the continuous presence of compound and removed for analysis after 1, 4, or 7 days.
The condition of the cells was noted by microscopic examination and then the medium was removed from each flask to a 50-mL tube. The cells were trypsinized with 2 x 2.5 mL of trypsin-EDTA, resuspended in 10 mL of the R5-HITES medium, and combined with the R5-HITES medium in the 50-mL tube. The cells were sedimented by centrifugation at 200g for 10 min, and the medium was removed and kept for HPLC analysis of the metabolites. The cells were washed with 10 mL and then 30 mL of R-HITES medium (without serum). The cells, resuspended in 1.0 mL of water, were frozen and thawed five times to rupture cell membranes so as to provide cell lysates prior to their analysis.
Determination of Covalent Binding-The distribution of ra- dioactivity bound to bionucleophiles, in the incubation mixtures and in cell lysates, was found by the method of Lund et al.21 in which a phospholipid fraction was obtained by extraction with chloroform- methanol (2:1). This resulted in three-phase systems with any binding to phospholipids present in the chloroform-methanol layer formed below an aqueous-methanol layer, while precipitated proteins along with metabolites bound to’proteins were present at the interface of the two solvent layers. Phospholipids were recovered from silica gel added to the chloroform-methanol layer as described in the Lund et al.21 procedure. The phospholipids present before and after the procedure were measured by the method of Trudinger22 so that the radioactivity bound to phospholipids could be adjusted for loss during the isolation process.
The precipitated proteins were solubilized in 1 ml of 1% SDS, reprecipitated with 5 mL of acetone, and centrifuged (1500g, 10 min). The solubilization/precipitation procedure was repeated nine ad- ditional times to remove unbound radioactive substrate and metabo- lites. Aliquots of the protein solutions were taken for counting and adjusted for loss by a Lowry assay of proteins18 before and after the purification process. Binding to DNA was examined in a second aliquot (0.5 mL) of the incubation mixture, as noted by Lund et al.,21 in which any DNA bound activity was precipitated and separated from that bound to proteins with the perchloric acid procedure of Hellman and Ullberg.23 Quantitation of DNA before and after this isolation procedure was by a modified diphenylamine method.24
Determination of Unbound Metabolites-For biotransforma- tion studies of the incubation mixtures and cell lysates, aliquots of the combined solvent phases from the Lund et al.21 procedure, after removal of the precipitated proteins, were dried under nitrogen. These residues were dissolved in ethanol solutions containing sub- strate and metabolite standards prior to HPLC analysis. However, for cell-culture media, where binding studies were not applicable, the media was freeze-dried directly. The residues were moistened with 0.1 mL of water, acidified with 10% trichloroacetic acid (0.5 mL), and extracted with 2 x 5 mL of methanol. The combined extracts were evaporated to dryness under nitrogen and dissolved in the ethanol standard-compound solution prior to HPLC analysis.
Chemical Hydrolysis of Radiolabeled Macromolecules-Acid Hydrolysis-Lipid (0.13 umol) or protein samples (0.2-1.0 mg) were hydrolyzed with 1.5 mL of 6 N HCI at 110 ℃ under an atmosphere of nitrogen for 24 h. The reaction mixture was extracted with 5 x 5 mL of ether. The extracts were combined and evaporated to dryness under nitrogen. The dried residue from the protein sample was dissolved in 100-120 /L of an ethanol solution which also contained the iodo analog of o,p’-DDA as a reference metabolite. The ethanol solution (90-100 uL) was analyzed by reverse-phase HPLC. The
| Subcellular Fractionª | % Transformation | % Apparent Protein Bindingd | Hydrolysis of Protein Fractions | |||
|---|---|---|---|---|---|---|
| Acidb | Totalc | % Radioactivitye | % Acidf | % Substrateg | ||
| Fresh Gland as Source | ||||||
| Whole homogenate | 3.51 ± 0.37 | 4.04 ±0.36 | 4.46 ±0.39 | 87.9±4.6 | 88.8±1.3 | 4.01 ± 2.37 |
| Blankh | 0.56 | 0.66 | 0.02 | |||
| Mitochondria | 4.18 ±0.80 | 5.63 ± 1.23 | 4.17 ±0.28 | 89.2± 1.8 | 92.2±0.6 | 1.84 ±0.97 |
| Blank | 0.55 | 0.61 | 0.01 | |||
| Cytosol | 0.43 ± 0.26 | 0.43 ±0.26 | 0.65 ±0.11 | 89.0 ±2.7 | 91.7 ±4.6 | 4.33 ± 2.74 |
| Blank | 0.70 | 0.70 | 0.02 | |||
| Microsome | 0.26 ± 0.09 | 0.29 ± 0.13 | 0.17 ± 0.04 | 93.3±2.0 | 82.3±1.3 | 1.64 ± 1.44 |
| Blank | 0.60 | 0.75 | 0.01 | |||
| Frozen Gland as Source | ||||||
| Whole homogenate | 3.14 ±0.24 | 3.23 ± 0.25 | 3.46 ± 0.21 | 89.9±0.8 | 97.1 ±2.4 | 1.98 ± 1.76 |
| Blank | 0.03 | 0.03 | 0.01 | |||
| Mitochondria | 0.80 ± 0.10 | 0.82 ±0.12 | 0.89 ± 0.07 | 91.8±2.5 | 87.8 ± 7.5 | 4.44 ± 1.76 |
| Blank | 0.02 | 0.02 | 0.01 | |||
| Cytosol | 1.19 ± 0.10 | 1.34 ±0.14 | 1.29 ± 0.13 | 94.5± 3.7 | 91.2± 9.8 | 6.38 ± 6.08 |
| Blank | 0.03 | 0.03 | 0.04 | |||
a Protein values for these fractions are as follows: homogenate, 13.0, 12.5; mitochondria, 6.07, 6.36; cytosol, 10.6, 8.91; and microsomes, 5.68 mg/mL for fresh and frozen glands, respectively. b Percentage of radioactivity applied to HPLC analysis which cochromatographed with reference 1-(2-chlorophenyl)-1-(4-iodophenyl)acetic acid. All percentages in this table are the mean and standard deviations of three determinations, and where applicable, their blank values have been substracted. ” Percentage of the total radioactivity applied to HPLC analysis separated as metabolic peaks. dPercentage of the radioactivity of the original substrate associated with bound fractions. e Percentage of the radioactivity associated with the bound fraction that was recovered after hydrolysis prior to HPLC analysis. ‘Percentage of hydrolysate activity applied to HPLC analysis which cochromatographed with the reference acid metabolite. 9 Percentage of hydrolysate activity applied to HPLC analysis recovered as original substrate. ” Enzyme preparations had been placed in boiling water prior to incubation.
dried residue from the lipid sample was dissolved in 10 ÆL of the ethanol standard solution and 5 AL was spotted for TLC analysis.
Mild Hydrolysis-Lipid (0.13 umol) or protein samples (0.2 mg) were hydrolyzed in 1 mL of 1 N hydroxylamine (pH 7.5, 0.5% SDS) at room temperature under at atmosphere of nitrogen for 18 h. The reaction mixtures were acidified with concentrated HCl and extracted with 5 x 5 mL of ether. The combined extracts were dried under nitrogen prior to HPLC and TLC analysis.
Analysis-HPLC-A reverse-phase column (5 mm i.d. x 10 cm NOVA-PAK C-18 Radial Pak Cartridge, Waters) with a radial compressor (Waters RCM-100) protected with a guard cartridge (C- 18, 10 um spherical, 3 cm × 4.6 mm RP-18, MCP, Brownlee Labs) was used for metabolism studies. The solvent system employed in the separation of metabolites was methanol-water-acetic acid (50: 50:0.2) at a flow rate of 1.2 mL/min for 40 min. A gradual change over 10 min to acetonitrile-water-acetic acid 55:45:0.2 was used to measure [14C]-o,p’-DDD or to 65:35:0.2 to measure the unmetabolized iodo substrate.
After the peaks of o,p’-DDD or its iodo derivative appeared, the ratio of acetonitrile-water-acetic acid was changed to 75:25:0.2 at the same flow rate to detect unsaturated metabolites. One-minute fractions of 125I-labeled compounds were collected in small vials and detected directly in a y-counter. The fractions of [14C]-o,p’-DDD were collected in 8-mL scintillation vials, Ecolite (5.0 mL) was added to each fraction, and radioactivity was quantified by liquid scintillation counting.
TLC-TLC plates (3 cm ~ 10 cm × 0.25 mm silica gel 60 F254 on glass) were developed to 7 cm by a solvent system of hexane-acetic acid-n-butanol (85:10:5). Reference substrate and iodo acid were detected via fluorescent quenching under shortwave UV light, and radioactivity was quantified by y-radiation counting of 1- or 1.5-cm bands of silica scraped from the plates. This method was used to determine the purity of 1-(2-chlorophenyl)-1-(4-iodophenyl)acetic acid and to separate the hydrolysates from lipids fractions.
Results and Discussion
A dog treated in vivo with the iodo analog of mitotane, 1-(2- chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, by the pro- cedure we previously described for mitotane20 had a similar fall in serum cortisol and rise in plasma ACTH level and adrenolytic activity as described for mitotane. Also, this iodo
compound has been shown to have a similar distribution of metabolites following incubation with dog, rat, or bovine adrenal mitochondrial preparations.7 Thus, because of the much higher specific activity obtainable for the 125I-labeled compound over that for [14C]mitotane, the majority of the present study was conducted with the 125I analog of DDD before comparison to [14C]mitotane.
In confirmation of the previous reports for mitotane, 6,25 the distribution of metabolic activity in the subcellular fractions prepared from freshly isolated glands (Table 1) showed the mitochondria to be the subcellular fraction primarily respon- sible for the metabolism of the 125I analog with little contribu- tion from microsomes or cytosol. However, as we have observed for mitotane, this was not the case for subcellular fractions prepared from previously frozen glands, where the predominance of activity in mitochondria was lost and the cytosol activity was greatly increased. As has been suggested for the presence of cholesterol side-chain cleavage activity in the cytosol of some bovine adrenal cortex preparations, 26 activity in the cytosol could be due to the dissociation of enzymes from the mitochondria with their subsequent isola- tion in the cytosol. As it has been demonstrated that freezing and thawing will damage mitochondria,27 this procedure could contribute to such dissociation. As noted in Table 1, however, there was little difference between the extent of metabolism in whole homogenates prepared from fresh and previously frozen dog adrenal glands. Therefore, since it was not usually possible to work with preparations from fresh glands, espe- cially when pooled glands from several animals were involved, adrenal glands were frozen and stored at -80 ℃ before being used to prepare cortex whole homogenates.
The extent of bound radioactivity after incubations with adrenal cortical whole homogenates from dogs was examined using a procedure based upon that used for o,p’-DDD binding to mouse lung and liver macromolecular fractions.21 As noted in Table 2, the majority of binding was to the protein fraction, with about one-sixth of the total bound activity appearing in the phospholipid fraction while no binding to DNA was detected in these adrenal homogenates. Both bound fractions
| Incubationsª | % Transformation | % Binding | |||
|---|---|---|---|---|---|
| Acidb | Totalc | Proteinsd | Phospholipidsd | DNA | |
| Homogenate | 4.53 ±0.34 | 4.58 ± 0.39 | 5.53 ± 0.39 | 1.10 ± 0.28 | None detected |
| Blanks | 0.03 | 0.03 | 0.01 | 0.03 | |
| Fraction | 6 N HCI, 24 h, 110 ℃ | 1 N NH2OH (pH 7.5), 18 h, room temp | ||||
|---|---|---|---|---|---|---|
| % Radioactivity® Recovered | % Acid Metabolitef Released | % Substrateg Recovered | % Radioactivitye Recovered | % Acid Metabolite™ Released | % Substrateg Recovered | |
| Proteins | 89.9 ±5.4 | 87.8 ± 2.4 | 4.74 ±4.14 | 24.8 ± 2.8 | 83.3±4.8 | 4.30 ±2.30 |
| Phospholipids | 96.7 ±2.6 | 87.9±6.0 | 7.60 ± 7.00 | 69.7±1.5 | 79.1 ± 10.6 | 14.7±7.9 |
a Dog adrenal cortex whole homogenates prepared from frozen glands were used. Footnotes b-g are the same as in Table 1.
| Tissue Sourcesª | % Transformation | % Apparent Protein Bindingd | Hydrolysis of Protein Fractions | |||
|---|---|---|---|---|---|---|
| Acidb | Totale | % Radioactivitye | % Acidf | % Substrateg | ||
| Bovine | 8.96 ±0.24 | 9.46 ± 0.51 | 7.22 ± 0.26 | 89.9 ±5.4 | 93.7 ±5.0 | 4.53 ± 3.36 |
| Blankh | 0.44 | 0.44 | 0.13 | |||
| Dog | 4.53 ± 0.34 | 4.58 ±0.39 | 5.53 ± 0.39 | 89.0 ± 3.6 | 87.8 ± 2.4 | 4.74 ±4.14 |
| Blank | 0.03 | 0.03 | 0.01 | |||
| Rat | 1.18 ±0.38 | 1.31 ± 0.46 | 0.24 ± 0.04 | 81.6 ±5.8 | 81.1 ±6.5 | 6.37 ± 3.15 |
| Blank | 0.88 | 0.88 | 0.06 | |||
| Human | 0.23 ± 0.04 | 0.25 ± 0.04 | 0.21 ± 0.04 | 84.2 ±4.7 | 82.9 ± 8.7 | 10.0± 10.8 |
| Blank | <0.01 | <0.01 | <0.01 | |||
| Human Tumors | ||||||
| Benign | 0.21 ± 0.06 | 0.25 ± 0.08 | 0.19 ± 0.01 | 83.5 ± 4.8 | 86.4 ± 3.4 | 11.9 ±6.6 |
| Blank | 0.02 | 0.02 | <0.01 | |||
| Malignant | 0.14 ± 0.01 | 0.18 ±0.02 | 0.14 ±0.02 | 86.9 ± 2.2 | 76.9 ±7.2 | 14.2 ± 9.3 |
| Blank | 0.14 | 0.14 | 0.06 | |||
a Adrenal whole homogenates were prepared from pooled cortices of 4 bovine, 20 dog, 40 rat, 3 human glands, 1 benign tumor, and 1 malignant tumor. Footnotes b-h are the same as in Table 1.
were hydrolyzed under mild (1 N NH2OH) and strong (6 N HCI) conditions (Table 2). Analogous to chloramphenicol binding studies,28,29 the release of radioactivity under the mild conditions could be indicative of the hydrolysis of ester and thioester bonds while the increase in hydrolysis products under the strong acid conditions would be primarily indicative of hydrolysis of amide bonds. There was a greater proportion of activity released under the mild conditions from ester bonds in the phospholipid fraction than from the proteins. The increase in radioactivity under the strong hydrolysis condi- tions for the phospholipid fraction could be explained by additional hydrolysis of esters as well as hydrolysis of amide bonds. In this last regard, such amide bonds would have to be with amines, such as those amines associated with biologi- cal membranes, that would be present in the phosopholipid fraction, as we did not detect proteins in this fraction prior to hydrolysis.
As previously noted,9 the extent of metabolism and binding were significantly different when adrenal homogenates from different sources were incubated under the same conditions (Table 3). Previous literature reports for transformations of [14C]-o,p’-DDD by homogenates of the cortex from human normal adrenals are in conflict. While Martz and Straw9 demonstrated such transformations and binding, Papadopou- los et al.30 did not detect any metabolism of o,p’-DDD. In our studies of whole homogenates from human normal adrenal cortex and adrenal tumors, transformations and binding for the 125I analog of mitotane were low in comparison to the other species studied. Nevertheless, even in the case of incubations of homogenates from a malignant tumor, they were at least
twice that of the blank value. The fact that transformations are low in human adrenal preparations and that Papadopou- los et al.30 used mitochondrial fractions prepared from frozen glands, where we have shown (Table 1) mitochondrial activity could be further reduced, might account for their inability to detect mitotane transformations.
The 125I analog of mitotane was also used (Table 4) as a substrate in connection with studies being conducted in our laboratories of cultured human adrenal cancer cells from the NCI H295 cell line.19 Metabolism of the 125I analog to the [125]]DDA analog and binding was observed after 1 day of incubation, but lengthening the incubation up to a total of 7 days did not lead to significant increases in metabolite formation or binding. These cells metabolized the 125I analog of mitotane in a manner qualitatively similar to that for the adrenal cortex and tumor homogenates. The acid metabolite was detected in both the cells and the medium while the reminder of radioactivity was present as unchanged 125I analog. The presence of the acid metabolite in the medium resulted predominantly from metabolic activity of the cells, since only 0.08% of the 125I analog cochromatographed with reference acid when the medium was incubated without cells.
Strong acid hydrolysis was undertaken for all the bound fractions listed in Tables 1-4. The percentage of the radio- activity recovered from the hydrolysates of these fractions by ether extraction was uniformly high. Also, the same HPLC system that was used in our transformation studies showed that the only metabolite present in the ether extracts cochro- matographed with the acidic metabolite, 1-(2-chlorophenyl)- 1-(4-iodophenyl)acetic acid. Again, the percentage of the total
| Results | Cultured Times | ||
|---|---|---|---|
| 1 day | 4 days | 7 days | |
| % Transformation to acida | |||
| Medium | 0.22 ± 0.05 | 0.22 ± 0.05 | 0.41 ± 0.07 |
| Cell | 0.048 ± 0.002 | 0.040 ± 0.012 | 0.033 ± 0.005 |
| % Bound to cellular proteinsb | 0.10 ± 0.01 | 0.10 ± 0.01 | 0.14 ±0.02 |
| % Radioactivity releasedc | 91.5 ±2.2 | 94.1 ±2.4 | 93.0 ± 6.8 |
| % Acid metabolite releasedd | 74.6 ± 10.0 | 72.1± 14.6 | 85.3 ±2.8 |
| % Substrate recoverede | 13.1 ± 12.6 | 23.1 ± 18.6 | 9.78 ±4.25 |
a Radioactive metaboliite corresponding with reference 1-(2-chlorophenyl)-1- (4-iodophenyl)acetic acid separated by HPLC. Expressed as the mean and its standard deviation (N = 3) of the percentage of total radioactivity applied to HPLC analysis. The mean + SD (N = 3) of total substrate incubated. ºEach value represents the percentage of total radioactivity recovered from 6 N HCI hydrolysis of bound proteins and is the average of three determinations. d Percentage of radioactivity applied to HPLC analysis after 6 N HCI hydrolysis that cochromatographed with reference 1-(2-chlorophenyl)-1-(4-iodophenyl)acetic acid. Expressed as the mean and its standard deviation (N = 3). º Substrate recovered after hydrolysis in 6 N HCI and expressed as the mean and its standard deviation (N = 3) of the percentage of total radioactivity applied to HPLC analysis.
activity recovered as the acetic acid derivative was uniformly high. The only other peak in the chromatographs of the total recovered radioactivity corresponded to traces of the original substrate as shown in Tables 1-4. That is, even after extensive washing of the bound fractions, a small amount of the original substrate was still carried through the isolation procedure. This, of course, had the greatest effect in decreas- ing the percent of acid metabolite in the HPLC analysis of those experiments where binding was low such as with the human samples (Table 3) and for the cell cultures (Table 4).
While it has previously been shown7 that the metabolism of [14C]mitotane and the 125I analog of mitotane are similar with dog, rat, and bovine adrenal incubation, it was also important to confirm that the binding results with the 125I analog were predictive of the results for mitotane, per se. Therefore, binding studies were also conducted with 14C- labeled mitotane but with the limitation of much lower specific activity (83 „Ci/mmol). In an attempt to obtain maximal binding, this set of comparison data was conducted in whole homogenates of the cortex prepared from fresh bovine adrenal glands. In addition to the detection of a 7.76 ± 0.88% (N = 3) yield of o,p’-DDA, there was 4.57 ± 0.89% (N = 3) apparent protein binding with a recovery of 92.1% of this activity after acid hydrolysis from which only o,p’-DDA (90.6%) and o,p’- DDD (2.64%) were detected upon HPLC analysis. These data are comparable to the bovine 125I incubations summarized in Table 3.
The consistently high recovery of either 125I or 14C in the hydrolysate of the protein fractions and the fact that the only radiolabeled product was the acidic metabolite rule out aliphatic epoxide and arene oxide intermediates as major pathways for mitotane binding in the adrenal cortex. Such intermediates upon reaction with bionucleophiles would form stable covalent bonds which would not be cleaved under our hydrolysis conditions to yield the acid metabolite. These results, however, are consistent in all the test systems examined (Tables 1-4) with an acyl chloride being the reactive intermediate for both DDA formation and covalent binding to adrenal cortical bionucleophiles. That is, a high correlation between DDA formation and binding together with the recovery of the bound activity as DDA is evident in these tables.
While the relationship between covalent binding and the adrenolytic effect of mitotane is still to be established, it is of
interest that the lack of adrenolytic activity of such mitotane- related compounds as the insecticides methoxychloro, p,p’-, and o,p’-DDT31,32 and the B-methyl derivative of mitotane, 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloropropane,20 is also consistent with the proposed acyl chlorides route of metabolic activation. These inactive compounds all lack hydrogen at the chlorinated aliphatic carbon of mitotane so that B-hydroxylation and subsequent acyl chloride formation could not occur. This structure-activity information together with the metabolic evidence for an acyl chloride active intermediate leads to the conclusion that, in the search for a more active drug to treat adrenocortical carcinoma, the dihalogenated methyl moiety of mitotane needs to be retained.
References and Notes
1. Hutter, A. M .; Kayhoe, D. E. Am. J. Med. 1966, 41, 581-592.
2. Temple, T. E., Jr .; Jones, D. J., Jr .; Liddle, G. W .; Dexter, R. N. N. Eng. J. Med. 1969, 281, 801-805.
3. Sinsheimer, J. E .; Guilford, J .; Bobrin, L. J .; Schteingart, D. E. J. Pharm. Sci. 1972, 61, 314-316.
4. Reif, V. D .; Sinsheimer, J. E .; Ward, J. C .; Schteingart, D. E. J. Pharm. Sci. 1974, 63, 1703-1736.
5. Reif, V. D .; Sinsheimer, J. E. Drug. Metab. Dispos. 1975, 3, 15- 25.
6. Reif, V. D .; Littleton, B. C .; Sinsheimer, J. E. J. Agric. Food Chem. 1975, 23, 996-999.
7. Pohland, R. C .; Counsell, R. E. Drug. Metab. Dispos. 1985, 13, 113-115.
8. Sinsheimer, J. E .; Freeman, C. J. Drug Metab. Dispos. 1987, 15,267-269.
9. Martz, F .; Straw, J. A. Drug. Metab. Dispos. 1980, 8, 127-130. 10. Gold, B .; Brunk, G. Chem .- Biol. Interact. 1982, 41, 327-339.
11. Gold, B .; Brunk, G. Cancer Res. 1983, 43, 2644-2647.
12. Gold, B .; Brunk, G. Biochem. Pharmacol. 1984, 33, 979-982.
13. Anders, M. W .; Pohl, L. R. In Bioactivation of Foreign Com- pounds; Anders, M. W., Ed., Academic Press: New York, 1985; pp 283-315.
14. Counsell, R. E .; Willett, R. E .; DiGuilio, W. J. Med. Chem. 1967, 10,975-977.
15. Counsell, R. E .; Willette, R. E. J. Pharm. Sci. 55, 1966, 1012- 1015.
16. Weichert, J. P .; Van Dort, M. E .; Groziak, M. R .; Counsell, R. E. Appl. Radiat. Isot. 1986, 37, 907-913.
17. Jenkins, S. S. J. Am. Chem. Soc. 1931, 53, 2341-2343.
18. Miller, G. L. Anal. Chem. 1959, 31, 964.
19. 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 .; LaRocca, R. V. Cancer Res. 1990, 50, 5488-5496.
20. Schteingart, D. E .; Sinsheimer, J. E .; Counsell, R. E .; Abrams, G. D .; Mcclellan, N .; Djanegara, T .; Hines, J .; Ruangwises, N .; Benitez, R .; Wotring, L. L. Cancer. Chemother. Pharmacol. 1993, 31, 459-466.
21. Lund, B .- O .; Bergman, A .; Brandt, I. Chem .- Biol. Interact. 1989, 70, 63-72.
22. Trudinger, P. A. Anal. Biochem. 1970, 36, 225-226.
23. Hellman, B .; Ullberg, S. Cell Tissue Kinet. 1986, 19, 183-194.
24. Gendimenico, G. J .; Bouguin, P. L .; Tramposch, K. M. Anal. Biochem. 1988, 173, 45-48.
25. Martz, F .; Straw, J. A. Drug Metab. Dispos. 1977, 5, 482-486.
26. Warne, P. A .; Greenfield, N. J .; Liberman, S. Natl. Acad. Sci. U.S.A. 1983, 80, 1877-1881.
27. Churchill, P. F .; de Alvare, L. R .; Kimura, T. J. Biol. Chem. 1978, 253, 4924-4929.
28. Pohl, L. R .; Nelson, S. D .; Krishna, G. Biochem. Pharmacol. 1978, 27, 491-496.
29. Halpert, J. Biochem. Pharmacol. 1981, 30, 875-881.
30. Papadopoulos, D .; Seidegard, J .; Rydstrom, J. Cancer Lett. 1984, 22, 23-30.
31. Nelson, A. A .; Woodard, G. Arch. Pathol. 1949, 48, 387-394.
32. Copeland, M. F .; Cranmer, M. F. Toxicol. Appl. Pharmacol. 1974, 27,1-10.
Acknowledgments
This study was supported by grant R01 CA 37794 from the National Cancer Institute, the Millie Schembechler Adrenal Cancer Program, and the In Vitro Drug Evaluation core of the University of Michigan Comprehensive Center via Grant 2P30 CA 46592 from the National Cancer Institute, DHHS. The authors thank Drs. Mohamed Ruyan and Daniel McConnell for the synthesis of the 125I-labeled analog of mitotane.
JS940559V