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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 the activity of mitochondrial respiratory complexes I (A), II (B), III (C), and V (D) in permeabilized HepG2 cells. Compounds were used at 10–250 µM. Statistical significance compared with vehicle control; * P < .05; ** P <.01; *** P <.001. Complex I, II, and III activity were defined as complex I, II, or III-stimulated maximal respiration, respectively, compared with vehicle control. Complex V activity was defined as the difference in compound-induced OCR change between coupled and uncoupled cells relative to vehicle control. All results were normalized to µg protein per well. Data are presented as mean + SEM of n = 3 experiments.
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kfw126-F8: The effect of flutamide and 2-hydroxyflutamide exposure on the activity of mitochondrial respiratory complexes I (A), II (B), III (C), and V (D) in permeabilized HepG2 cells. Compounds were used at 10–250 µM. Statistical significance compared with vehicle control; * P < .05; ** P <.01; *** P <.001. Complex I, II, and III activity were defined as complex I, II, or III-stimulated maximal respiration, respectively, compared with vehicle control. Complex V activity was defined as the difference in compound-induced OCR change between coupled and uncoupled cells relative to vehicle control. All results were normalized to µg protein per well. Data are presented as mean + SEM of n = 3 experiments.

Mentions: Identification of a significant reduction in complex I and II activity following flutamide/2-hydroxyflutamide treatment prompted the specific investigation of compound-induced changes in maximal respiration driven by these complexes. Cells were permeabilized in a solution containing substrates specific for the complex of interest before being treated with flutamide or 2-hydroxyflutamide and a mitochondrial stress test. A significant reduction in complex I activity was induced by both compounds at 30 µM and above (Figure 8A). In agreement with in situ respiratory complex assay results, only 2-hydroxyflutamide and not flutamide induced a significant reduction in complex II activity (Figure 8B). Assessment of complex V activity by comparison of compound-induced OCR change in uncoupled and coupled cells showed a significant reduction in complex V activity following treatment with 2-hydroxyflutamide at 125 µM and above, though this was not the case for flutamide (Figure 8D). Assessment of complex III activity showed no significant inhibition induced by either compound (Figure 8C).FIG. 8


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 the activity of mitochondrial respiratory complexes I (A), II (B), III (C), and V (D) in permeabilized HepG2 cells. Compounds were used at 10–250 µM. Statistical significance compared with vehicle control; * P < .05; ** P <.01; *** P <.001. Complex I, II, and III activity were defined as complex I, II, or III-stimulated maximal respiration, respectively, compared with vehicle control. Complex V activity was defined as the difference in compound-induced OCR change between coupled and uncoupled cells relative to vehicle control. All 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-F8: The effect of flutamide and 2-hydroxyflutamide exposure on the activity of mitochondrial respiratory complexes I (A), II (B), III (C), and V (D) in permeabilized HepG2 cells. Compounds were used at 10–250 µM. Statistical significance compared with vehicle control; * P < .05; ** P <.01; *** P <.001. Complex I, II, and III activity were defined as complex I, II, or III-stimulated maximal respiration, respectively, compared with vehicle control. Complex V activity was defined as the difference in compound-induced OCR change between coupled and uncoupled cells relative to vehicle control. All results were normalized to µg protein per well. Data are presented as mean + SEM of n = 3 experiments.
Mentions: Identification of a significant reduction in complex I and II activity following flutamide/2-hydroxyflutamide treatment prompted the specific investigation of compound-induced changes in maximal respiration driven by these complexes. Cells were permeabilized in a solution containing substrates specific for the complex of interest before being treated with flutamide or 2-hydroxyflutamide and a mitochondrial stress test. A significant reduction in complex I activity was induced by both compounds at 30 µM and above (Figure 8A). In agreement with in situ respiratory complex assay results, only 2-hydroxyflutamide and not flutamide induced a significant reduction in complex II activity (Figure 8B). Assessment of complex V activity by comparison of compound-induced OCR change in uncoupled and coupled cells showed a significant reduction in complex V activity following treatment with 2-hydroxyflutamide at 125 µM and above, though this was not the case for flutamide (Figure 8D). Assessment of complex III activity showed no significant inhibition induced by either compound (Figure 8C).FIG. 8

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