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Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells

View Article: PubMed Central - PubMed

ABSTRACT

The androgen receptor antagonist, flutamide, is strongly associated with idiosyncratic drug-induced liver injury (DILI). Following administration, flutamide undergoes extensive first-pass metabolism to its primary metabolite, 2-hydroxyflutamide. Flutamide is a known mitochondrial toxicant; however there has been limited investigation into the potential mitochondrial toxicity of 2-hydroxyflutamide and its contribution to flutamide-induced liver injury. In this study we have used the acute glucose or galactose-conditioning of HepG2 cells to compare the mitochondrial toxicity of flutamide, 2-hydroxyflutamide and the structurally-related, non-hepatotoxic androgen receptor antagonist, bicalutamide. Compound-induced changes in mitochondrial oxygen consumption rate were assessed using Seahorse technology. Permeabilization of cells and delivery of specific substrates and inhibitors of the various respiratory complexes provided more detailed information on the origin of mitochondrial perturbations. These analyses were supported by assessment of downstream impacts including changes in cellular NAD+/NADH ratio. Bicalutamide was not found to be a mitochondrial toxicant, yet flutamide and 2-hydroxyflutamide significantly reduced basal and maximal respiration. Both flutamide and 2-hydroxyflutamide significantly reduced respiratory complex I-linked respiration, though 2-hydroxyflutamide also significantly decreased complex II and V-linked respiration; liabilities not demonstrated by the parent compound. This study has identified for the first time, the additional mitochondrial liabilities of the major metabolite, 2-hydroxyflutamide compared with its parent drug, flutamide. Given the rapid production of this metabolite upon administration of flutamide, but not bicalutamide, we propose that the additional mitochondrial toxicity of 2-hydroxyflutamide may fundamentally contribute to the idiosyncratic DILI seen in flutamide-treated, but not bicalutamide-treated patients.

No MeSH data available.


Representative in situ respiratory complex assay trace. In situ respiratory complex assays consisted of cells in a solution containing substrates for complex I and flutamide/2-hydroxyflutamide or vehicle control (shown) prior to 3 cycles of measurements and a series of compound injections into the cell culture microplate. Injections consisted of rotenone (complex I inhibitor), succinate (complex II substrate), antimycin A (complex III inhibitor), and TMPD/ascorbate (complex IV substrates) with 2 cycles of measurements following each. This series of manipulations enabled the calculation of complex I (A), II (B), and IV (C) activity. Each measurement cycle was a total of 3 min.
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kfw126-F3: Representative in situ respiratory complex assay trace. In situ respiratory complex assays consisted of cells in a solution containing substrates for complex I and flutamide/2-hydroxyflutamide or vehicle control (shown) prior to 3 cycles of measurements and a series of compound injections into the cell culture microplate. Injections consisted of rotenone (complex I inhibitor), succinate (complex II substrate), antimycin A (complex III inhibitor), and TMPD/ascorbate (complex IV substrates) with 2 cycles of measurements following each. This series of manipulations enabled the calculation of complex I (A), II (B), and IV (C) activity. Each measurement cycle was a total of 3 min.

Mentions: Following a basal measurement of 3 cycles of mix (30 s), wait (30 s), and measure (2 min), sequential injections of A: rotenone (2 µM), B: succinate + rotenone (20 mM, 2 µM, respectively), C: antimycin A (2 µM) and D: ascorbic acid + N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) + antimycin A (20 mM, 0.5 mM, and 2 µM, respectively) were performed with a 2 cycle interval between each, allowing measurement of changes in complexes I (A), II (B), and IV (C and D) activity respectively (Figure 3). MAS buffer, all constituents and compound injections were used at pH 7.2.FIG. 3


Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells
Representative in situ respiratory complex assay trace. In situ respiratory complex assays consisted of cells in a solution containing substrates for complex I and flutamide/2-hydroxyflutamide or vehicle control (shown) prior to 3 cycles of measurements and a series of compound injections into the cell culture microplate. Injections consisted of rotenone (complex I inhibitor), succinate (complex II substrate), antimycin A (complex III inhibitor), and TMPD/ascorbate (complex IV substrates) with 2 cycles of measurements following each. This series of manipulations enabled the calculation of complex I (A), II (B), and IV (C) activity. Each measurement cycle was a total of 3 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036617&req=5

kfw126-F3: Representative in situ respiratory complex assay trace. In situ respiratory complex assays consisted of cells in a solution containing substrates for complex I and flutamide/2-hydroxyflutamide or vehicle control (shown) prior to 3 cycles of measurements and a series of compound injections into the cell culture microplate. Injections consisted of rotenone (complex I inhibitor), succinate (complex II substrate), antimycin A (complex III inhibitor), and TMPD/ascorbate (complex IV substrates) with 2 cycles of measurements following each. This series of manipulations enabled the calculation of complex I (A), II (B), and IV (C) activity. Each measurement cycle was a total of 3 min.
Mentions: Following a basal measurement of 3 cycles of mix (30 s), wait (30 s), and measure (2 min), sequential injections of A: rotenone (2 µM), B: succinate + rotenone (20 mM, 2 µM, respectively), C: antimycin A (2 µM) and D: ascorbic acid + N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) + antimycin A (20 mM, 0.5 mM, and 2 µM, respectively) were performed with a 2 cycle interval between each, allowing measurement of changes in complexes I (A), II (B), and IV (C and D) activity respectively (Figure 3). MAS buffer, all constituents and compound injections were used at pH 7.2.FIG. 3

View Article: PubMed Central - PubMed

ABSTRACT

The androgen receptor antagonist, flutamide, is strongly associated with idiosyncratic drug-induced liver injury (DILI). Following administration, flutamide undergoes extensive first-pass metabolism to its primary metabolite, 2-hydroxyflutamide. Flutamide is a known mitochondrial toxicant; however there has been limited investigation into the potential mitochondrial toxicity of 2-hydroxyflutamide and its contribution to flutamide-induced liver injury. In this study we have used the acute glucose or galactose-conditioning of HepG2 cells to compare the mitochondrial toxicity of flutamide, 2-hydroxyflutamide and the structurally-related, non-hepatotoxic androgen receptor antagonist, bicalutamide. Compound-induced changes in mitochondrial oxygen consumption rate were assessed using Seahorse technology. Permeabilization of cells and delivery of specific substrates and inhibitors of the various respiratory complexes provided more detailed information on the origin of mitochondrial perturbations. These analyses were supported by assessment of downstream impacts including changes in cellular NAD+/NADH ratio. Bicalutamide was not found to be a mitochondrial toxicant, yet flutamide and 2-hydroxyflutamide significantly reduced basal and maximal respiration. Both flutamide and 2-hydroxyflutamide significantly reduced respiratory complex I-linked respiration, though 2-hydroxyflutamide also significantly decreased complex II and V-linked respiration; liabilities not demonstrated by the parent compound. This study has identified for the first time, the additional mitochondrial liabilities of the major metabolite, 2-hydroxyflutamide compared with its parent drug, flutamide. Given the rapid production of this metabolite upon administration of flutamide, but not bicalutamide, we propose that the additional mitochondrial toxicity of 2-hydroxyflutamide may fundamentally contribute to the idiosyncratic DILI seen in flutamide-treated, but not bicalutamide-treated patients.

No MeSH data available.