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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.


The effect of flutamide, 2-hydroxyflutamide and bicalutamide on mitochondrial OCR in HepG2 cells. Serial concentrations of compounds were used up to 500 µM. A and B, Changes in basal and maximal respiration respectively. Changes in proton leak, spare respiratory capacity and ATP-linked OCR induced by flutamide (C) 2-hydroxyflutamide (D) and bicalutamide (E). Statistical significance compared with vehicle control; (A) and (B) flutamide; *P < .05; ** P <.01; *** P < .001, 2-hydroxyflutamide; +P <.05;  ++P <.01; +++P <.001, and bicalutamide; ^ P <.05; ^^ P <.01; ^^^ P <.001. (C–E) * P < .05; ** P <.01; *** P <.001. All results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.
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kfw126-F6: The effect of flutamide, 2-hydroxyflutamide and bicalutamide on mitochondrial OCR in HepG2 cells. Serial concentrations of compounds were used up to 500 µM. A and B, Changes in basal and maximal respiration respectively. Changes in proton leak, spare respiratory capacity and ATP-linked OCR induced by flutamide (C) 2-hydroxyflutamide (D) and bicalutamide (E). Statistical significance compared with vehicle control; (A) and (B) flutamide; *P < .05; ** P <.01; *** P < .001, 2-hydroxyflutamide; +P <.05;  ++P <.01; +++P <.001, and bicalutamide; ^ P <.05; ^^ P <.01; ^^^ P <.001. (C–E) * P < .05; ** P <.01; *** P <.001. All results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.

Mentions: Following the identification of mitochondrial liabilities in flutamide and 2-hydroxyflutamide, the effect of these compounds on mitochondrial OCR was assessed using a mitochondrial stress test (Figure 2). Both flutamide and 2-hydroxyflutamide induced a significant decrease in basal and maximal respiration whereas bicalutamide only caused a significant decrease in maximal respiration (Figures 6A and B). Both flutamide and 2-hydroxyflutamide (Figures 6C and D) also caused a significant increase in proton leak and a significant decrease in spare respiratory capacity and ATP-linked OCR as a percentage of maximal respiration, changes which were not observed upon bicalutamide-treatment (Figures 6E).FIG. 6


Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells
The effect of flutamide, 2-hydroxyflutamide and bicalutamide on mitochondrial OCR in HepG2 cells. Serial concentrations of compounds were used up to 500 µM. A and B, Changes in basal and maximal respiration respectively. Changes in proton leak, spare respiratory capacity and ATP-linked OCR induced by flutamide (C) 2-hydroxyflutamide (D) and bicalutamide (E). Statistical significance compared with vehicle control; (A) and (B) flutamide; *P < .05; ** P <.01; *** P < .001, 2-hydroxyflutamide; +P <.05;  ++P <.01; +++P <.001, and bicalutamide; ^ P <.05; ^^ P <.01; ^^^ P <.001. (C–E) * P < .05; ** P <.01; *** P <.001. All results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036617&req=5

kfw126-F6: The effect of flutamide, 2-hydroxyflutamide and bicalutamide on mitochondrial OCR in HepG2 cells. Serial concentrations of compounds were used up to 500 µM. A and B, Changes in basal and maximal respiration respectively. Changes in proton leak, spare respiratory capacity and ATP-linked OCR induced by flutamide (C) 2-hydroxyflutamide (D) and bicalutamide (E). Statistical significance compared with vehicle control; (A) and (B) flutamide; *P < .05; ** P <.01; *** P < .001, 2-hydroxyflutamide; +P <.05;  ++P <.01; +++P <.001, and bicalutamide; ^ P <.05; ^^ P <.01; ^^^ P <.001. (C–E) * P < .05; ** P <.01; *** P <.001. All results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.
Mentions: Following the identification of mitochondrial liabilities in flutamide and 2-hydroxyflutamide, the effect of these compounds on mitochondrial OCR was assessed using a mitochondrial stress test (Figure 2). Both flutamide and 2-hydroxyflutamide induced a significant decrease in basal and maximal respiration whereas bicalutamide only caused a significant decrease in maximal respiration (Figures 6A and B). Both flutamide and 2-hydroxyflutamide (Figures 6C and D) also caused a significant increase in proton leak and a significant decrease in spare respiratory capacity and ATP-linked OCR as a percentage of maximal respiration, changes which were not observed upon bicalutamide-treatment (Figures 6E).FIG. 6

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.