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Adrenal proteins bound by a reactive intermediate of mitotane
W Cai1, R E Counsell, D E Schteingart
1College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Purpose:
Mitotane (o,p'-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have shown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride intermediate. It was the objective of this study to compare the electrophoresis separation patterns of such adducts following activation of mitotane by various adrenocortical sources.
Methods:
With the use of a 125I-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, gel electrophoresis patterns were obtained for homogenates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-450scc and adrenodoxin.
Results:
The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immunoblotting analyses indicated that the band at 49.5 kDa corresponded in molecular weight to that of adrenal cytochrome P-450scc, but the band at 11.5 kDa did not correspond to adrenodoxin.
Conclusions:
The similarity of the results with canine and bovine adrenal preparations to that of human material offers useful systems for studying mitotane and its analogs. This should aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.
Insights
This study compared mitotane (o,p'-DDD) protein adducts in various adrenal tissues. Canine and bovine models effectively mimic human responses, aiding adrenocortical carcinoma treatment research.
Area of Science:
- Biochemistry
- Endocrinology
- Pharmacology
Background:
- Mitotane (o,p'-DDD) is the sole adrenolytic drug for adrenocortical carcinoma.
- Mitotane forms covalent protein adducts via a reactive acyl chloride intermediate in adrenal tissue.
Purpose of the Study:
- To compare electrophoresis separation patterns of mitotane-induced protein adducts.
- To investigate these patterns using various adrenocortical sources.
Main Methods:
- Utilized a 125I-labeled mitotane analog for gel electrophoresis.
- Analyzed homogenates from bovine, canine, and human adrenal preparations.
- Employed Western immunoblotting to identify adducts of cytochrome P-450scc and adrenodoxin.
Main Results:
- Electrophoresis revealed consistent patterns across all preparations, with prominent bands at 49.5 kDa and 11.5 kDa.
- Western immunoblotting identified the 49.5 kDa band as cytochrome P-450scc adducts.
- The 11.5 kDa band did not correspond to adrenodoxin.
Conclusions:
- Canine and bovine adrenal models provide valuable systems for studying mitotane.
- These models can advance understanding of mitotane's mechanism of action.
- Findings may guide the development of new adrenocortical carcinoma treatments.