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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 complex V assay trace. Complex V assays consisted of cells in a solution containing substrates for complex IV before a series of compound injections into the cell culture microplate. FCCP (OXPHOS uncoupler) (Trace A) or MAS buffer (Traces B and C) was first injected, followed by 2 cycles of measurements. Flutamide, 2-hydroxyflutamide, oligomycin (positive control), or vehicle control was then injected into both the uncoupled (FCCP-treated) and coupled (MAS-treated) cells. Change in complex V activity was defined as the reduction in OCR of coupled cells upon flutamide/2-hydroxyflutamide injection minus the change in OCR of uncoupled cells, as a % of vehicle control. Injections for traces shown; A, FCCP, 250 μM 2-hydroxyflutamide; B, MAS, 250 μM 2-hydroxyflutamide; C, MAS, Oligomycin. Each measurement cycle was a total of 3 min.
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kfw126-F4: Representative complex V assay trace. Complex V assays consisted of cells in a solution containing substrates for complex IV before a series of compound injections into the cell culture microplate. FCCP (OXPHOS uncoupler) (Trace A) or MAS buffer (Traces B and C) was first injected, followed by 2 cycles of measurements. Flutamide, 2-hydroxyflutamide, oligomycin (positive control), or vehicle control was then injected into both the uncoupled (FCCP-treated) and coupled (MAS-treated) cells. Change in complex V activity was defined as the reduction in OCR of coupled cells upon flutamide/2-hydroxyflutamide injection minus the change in OCR of uncoupled cells, as a % of vehicle control. Injections for traces shown; A, FCCP, 250 μM 2-hydroxyflutamide; B, MAS, 250 μM 2-hydroxyflutamide; C, MAS, Oligomycin. Each measurement cycle was a total of 3 min.

Mentions: Culture medium was replaced with MAS buffer containing constituents to stimulate oxygen consumption via complex IV as this was not significantly affected by either compound in the in situ respiratory complex assay (ADP; 4.6 mM, ascorbic acid; 20 mM, TMPD; 0.5 mM, antimycin A; 2 µM, BSA; 30 µM, PMP; 1 nM). The assay consisted of a basal OCR measurement of 2 cycles of mix (30 s), wait (30 s), and measure (2 min) followed by MAS or FCCP injection (0.5 µM) and 2 measurement cycles. MAS-injected cells remain coupled whereas FCCP-injected cells become uncoupled meaning Complex V (ATP synthase) inhibition should not result in a change in OCR. Either flutamide, 2-hydroxyflutamide (10–250 µM) or oligomycin (positive control; 1 µM) was then injected into both the uncoupled and coupled cells, followed by a final 2 measurement cycles (Figure 4). Change in complex V activity was defined as the difference in flutamide/2-hydroxyflutamide-induced OCR change between coupled (MAS injection) and uncoupled (FCCP injection) cells compared with vehicle control.FIG. 4


Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells
Representative complex V assay trace. Complex V assays consisted of cells in a solution containing substrates for complex IV before a series of compound injections into the cell culture microplate. FCCP (OXPHOS uncoupler) (Trace A) or MAS buffer (Traces B and C) was first injected, followed by 2 cycles of measurements. Flutamide, 2-hydroxyflutamide, oligomycin (positive control), or vehicle control was then injected into both the uncoupled (FCCP-treated) and coupled (MAS-treated) cells. Change in complex V activity was defined as the reduction in OCR of coupled cells upon flutamide/2-hydroxyflutamide injection minus the change in OCR of uncoupled cells, as a % of vehicle control. Injections for traces shown; A, FCCP, 250 μM 2-hydroxyflutamide; B, MAS, 250 μM 2-hydroxyflutamide; C, MAS, Oligomycin. Each measurement cycle was a total of 3 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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kfw126-F4: Representative complex V assay trace. Complex V assays consisted of cells in a solution containing substrates for complex IV before a series of compound injections into the cell culture microplate. FCCP (OXPHOS uncoupler) (Trace A) or MAS buffer (Traces B and C) was first injected, followed by 2 cycles of measurements. Flutamide, 2-hydroxyflutamide, oligomycin (positive control), or vehicle control was then injected into both the uncoupled (FCCP-treated) and coupled (MAS-treated) cells. Change in complex V activity was defined as the reduction in OCR of coupled cells upon flutamide/2-hydroxyflutamide injection minus the change in OCR of uncoupled cells, as a % of vehicle control. Injections for traces shown; A, FCCP, 250 μM 2-hydroxyflutamide; B, MAS, 250 μM 2-hydroxyflutamide; C, MAS, Oligomycin. Each measurement cycle was a total of 3 min.
Mentions: Culture medium was replaced with MAS buffer containing constituents to stimulate oxygen consumption via complex IV as this was not significantly affected by either compound in the in situ respiratory complex assay (ADP; 4.6 mM, ascorbic acid; 20 mM, TMPD; 0.5 mM, antimycin A; 2 µM, BSA; 30 µM, PMP; 1 nM). The assay consisted of a basal OCR measurement of 2 cycles of mix (30 s), wait (30 s), and measure (2 min) followed by MAS or FCCP injection (0.5 µM) and 2 measurement cycles. MAS-injected cells remain coupled whereas FCCP-injected cells become uncoupled meaning Complex V (ATP synthase) inhibition should not result in a change in OCR. Either flutamide, 2-hydroxyflutamide (10–250 µM) or oligomycin (positive control; 1 µM) was then injected into both the uncoupled and coupled cells, followed by a final 2 measurement cycles (Figure 4). Change in complex V activity was defined as the difference in flutamide/2-hydroxyflutamide-induced OCR change between coupled (MAS injection) and uncoupled (FCCP injection) cells compared with vehicle control.FIG. 4

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.