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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 and 2-hydroxyflutamide exposure on superoxide levels (A) and NAD+/NADH ratio (B) in HepG2 cells (2 h). Serial concentrations of compounds were used up to 500 µM. Statistical significance compared with vehicle control; flutamide; * P <.05; ** P <.01; *** P <.001, 2-hydroxyflutamide; +P <. 05;++P < .01; +++P < .001. mitoSOX results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.
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kfw126-F9: The effect of flutamide and 2-hydroxyflutamide exposure on superoxide levels (A) and NAD+/NADH ratio (B) in HepG2 cells (2 h). Serial concentrations of compounds were used up to 500 µM. Statistical significance compared with vehicle control; flutamide; * P <.05; ** P <.01; *** P <.001, 2-hydroxyflutamide; +P <. 05;++P < .01; +++P < .001. mitoSOX results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.

Mentions: Downstream impacts of mitochondrial dysfunction, particularly complex I inhibition, include increased generation of reactive oxygen species and depletion of cellular NAD+. Both flutamide and 2-hydroxyflutamide significantly increased cellular superoxide at 30 µM and above, the same concentration at which significant reductions in complex I-mediated maximal respiration were observed (Figures 8A and 9A). A significant increase in superoxide was also seen in galactose media at the same compound concentrations (Supplementary Figure S2). Both compounds also significantly decreased the NAD+/NADH ratio (Figure 9B), though not at concentrations below 100 µM.FIG. 9


Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells
The effect of flutamide and 2-hydroxyflutamide exposure on superoxide levels (A) and NAD+/NADH ratio (B) in HepG2 cells (2 h). Serial concentrations of compounds were used up to 500 µM. Statistical significance compared with vehicle control; flutamide; * P <.05; ** P <.01; *** P <.001, 2-hydroxyflutamide; +P <. 05;++P < .01; +++P < .001. mitoSOX results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036617&req=5

kfw126-F9: The effect of flutamide and 2-hydroxyflutamide exposure on superoxide levels (A) and NAD+/NADH ratio (B) in HepG2 cells (2 h). Serial concentrations of compounds were used up to 500 µM. Statistical significance compared with vehicle control; flutamide; * P <.05; ** P <.01; *** P <.001, 2-hydroxyflutamide; +P <. 05;++P < .01; +++P < .001. mitoSOX results were normalized to µg protein per well. Data are presented as mean ± SEM of n = 3 experiments.
Mentions: Downstream impacts of mitochondrial dysfunction, particularly complex I inhibition, include increased generation of reactive oxygen species and depletion of cellular NAD+. Both flutamide and 2-hydroxyflutamide significantly increased cellular superoxide at 30 µM and above, the same concentration at which significant reductions in complex I-mediated maximal respiration were observed (Figures 8A and 9A). A significant increase in superoxide was also seen in galactose media at the same compound concentrations (Supplementary Figure S2). Both compounds also significantly decreased the NAD+/NADH ratio (Figure 9B), though not at concentrations below 100 µM.FIG. 9

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