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

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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.

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The effect of flutamide (A), 2-hydroxyflutamide (B), and bicalutamide (C) exposure on ATP content and cytotoxicity of HepG2 cells (2 h) compared with vehicle control. Serial concentrations of compounds were used up to 300 µM in glucose or galactose media. ATP values are expressed as a percentage of those of the vehicle control, LDH release is expressed as extracellular LDH as a % of total LDH. Statistical significance compared with vehicle control; glucose *P < .05; **P < .01; ***P < .001, galactose +P < .05;  ++P < .01;  +++P < .001, between glucose and galactose ^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-F5: The effect of flutamide (A), 2-hydroxyflutamide (B), and bicalutamide (C) exposure on ATP content and cytotoxicity of HepG2 cells (2 h) compared with vehicle control. Serial concentrations of compounds were used up to 300 µM in glucose or galactose media. ATP values are expressed as a percentage of those of the vehicle control, LDH release is expressed as extracellular LDH as a % of total LDH. Statistical significance compared with vehicle control; glucose *P < .05; **P < .01; ***P < .001, galactose +P < .05;  ++P < .01;  +++P < .001, between glucose and galactose ^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: When the concentration of a compound required to reduce cellular ATP content by 50% (EC50ATP) is compared in glucose-conditioned vs galactose-conditioned cells, a fold difference of ≥ 2 (EC50ATPglu/EC50ATPgal ≥ 2) is considered to indicate that the compound contains a mitochondrial liability (Kamalian et al., 2015; Swiss et al., 2013). None of the compounds induced significant cytotoxicity in HepG2 cells (quantified in this study as a significant increase in LDH release) in either glucose or galactose medium. However, the decrease in ATP content induced by flutamide and 2-hydroxyflutamide was significantly more in the galactose medium, but this was not the case for bicalutamide (Figures 5A–C). This translated into an EC50ATP ratio (glucose vs galactose media) ≥ 2 for flutamide and 2-hydroxyflutamide, whereas the difference in EC50 induced by bicalutamide was ≤ 2 and was not significant (Table 1).FIG. 5


Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells
The effect of flutamide (A), 2-hydroxyflutamide (B), and bicalutamide (C) exposure on ATP content and cytotoxicity of HepG2 cells (2 h) compared with vehicle control. Serial concentrations of compounds were used up to 300 µM in glucose or galactose media. ATP values are expressed as a percentage of those of the vehicle control, LDH release is expressed as extracellular LDH as a % of total LDH. Statistical significance compared with vehicle control; glucose *P < .05; **P < .01; ***P < .001, galactose +P < .05;  ++P < .01;  +++P < .001, between glucose and galactose ^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-F5: The effect of flutamide (A), 2-hydroxyflutamide (B), and bicalutamide (C) exposure on ATP content and cytotoxicity of HepG2 cells (2 h) compared with vehicle control. Serial concentrations of compounds were used up to 300 µM in glucose or galactose media. ATP values are expressed as a percentage of those of the vehicle control, LDH release is expressed as extracellular LDH as a % of total LDH. Statistical significance compared with vehicle control; glucose *P < .05; **P < .01; ***P < .001, galactose +P < .05;  ++P < .01;  +++P < .001, between glucose and galactose ^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: When the concentration of a compound required to reduce cellular ATP content by 50% (EC50ATP) is compared in glucose-conditioned vs galactose-conditioned cells, a fold difference of ≥ 2 (EC50ATPglu/EC50ATPgal ≥ 2) is considered to indicate that the compound contains a mitochondrial liability (Kamalian et al., 2015; Swiss et al., 2013). None of the compounds induced significant cytotoxicity in HepG2 cells (quantified in this study as a significant increase in LDH release) in either glucose or galactose medium. However, the decrease in ATP content induced by flutamide and 2-hydroxyflutamide was significantly more in the galactose medium, but this was not the case for bicalutamide (Figures 5A–C). This translated into an EC50ATP ratio (glucose vs galactose media) ≥ 2 for flutamide and 2-hydroxyflutamide, whereas the difference in EC50 induced by bicalutamide was ≤ 2 and was not significant (Table 1).FIG. 5

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.


Related in: MedlinePlus