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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.

No MeSH data available.


Representative control mitochondrial stress test trace. Mitochondrial stress test assays consisted of a series of compound injections into the cell culture microplate. Flutamide/2-hydroxyflutamide or vehicle control (shown) was first injected, followed by 9 measurement cycles. Remaining injections consisted of oligomycin (ATP synthase inhibitor), FCCP (OXPHOS uncoupler), and rotenone/antimycin A (complex I and III inhibitors, respectively) with each followed by 3 measurement cycles. This series of manipulations enabled the calculation of parameters: basal, ATP-linked, maximum, and non-mitochondrial OCR, as well as proton leak. Each measurement cycle was a total of 6 min.
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kfw126-F2: Representative control mitochondrial stress test trace. Mitochondrial stress test assays consisted of a series of compound injections into the cell culture microplate. Flutamide/2-hydroxyflutamide or vehicle control (shown) was first injected, followed by 9 measurement cycles. Remaining injections consisted of oligomycin (ATP synthase inhibitor), FCCP (OXPHOS uncoupler), and rotenone/antimycin A (complex I and III inhibitors, respectively) with each followed by 3 measurement cycles. This series of manipulations enabled the calculation of parameters: basal, ATP-linked, maximum, and non-mitochondrial OCR, as well as proton leak. Each measurement cycle was a total of 6 min.

Mentions: Cells were incubated for 1 h (37°C, 0% CO2) before culture medium was replaced by 175 µl of unbuffered Seahorse XF Base medium supplemented with glucose (25 mM), L-glutamine (2 mM), sodium pyruvate (1 mM), pre-warmed to 37 °C (pH 7.4). Prior to measurement of OCR, the Seahorse XFe96 instrument gently mixed the assay medium in each well for 10 min to enable the oxygen partial pressure to reach equilibrium. The OCR was then measured 3 times to establish a baseline rate prior to the acute injection of flutamide, 2-hydroxyflutamide or bicalutamide (7.8–500 µM). There were 9 OCR measurement cycles following compound injection and each measurement cycle consisted of a 3 min mix and 3 min measure. Following this compound incubation (54 min), a mitochondrial stress test was performed consisting of sequential injections of oligomycin (1 µM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 µM) and antimycin A/rotenone (1 µM each) (all compound concentrations were optimized to generate the maximum effect in the absence of toxicity). After stress test compound injections there were 3 measurement cycles before the injection of the next stress test compound. This enabled the calculation of basal respiration (OCR prior to oligomycin injection—non-mitochondrial OCR), proton leak (OCR after oligomycin injection—non-mitochondrial OCR), ATP-linked OCR (basal respiration—proton leak—non-mitochondrial OCR), maximal respiration (first injection after FCCP injection—non-mitochondrial OCR) and spare respiratory capacity (maximal respiration—basal respiration) (Figure 2).FIG. 2


Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells
Representative control mitochondrial stress test trace. Mitochondrial stress test assays consisted of a series of compound injections into the cell culture microplate. Flutamide/2-hydroxyflutamide or vehicle control (shown) was first injected, followed by 9 measurement cycles. Remaining injections consisted of oligomycin (ATP synthase inhibitor), FCCP (OXPHOS uncoupler), and rotenone/antimycin A (complex I and III inhibitors, respectively) with each followed by 3 measurement cycles. This series of manipulations enabled the calculation of parameters: basal, ATP-linked, maximum, and non-mitochondrial OCR, as well as proton leak. Each measurement cycle was a total of 6 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036617&req=5

kfw126-F2: Representative control mitochondrial stress test trace. Mitochondrial stress test assays consisted of a series of compound injections into the cell culture microplate. Flutamide/2-hydroxyflutamide or vehicle control (shown) was first injected, followed by 9 measurement cycles. Remaining injections consisted of oligomycin (ATP synthase inhibitor), FCCP (OXPHOS uncoupler), and rotenone/antimycin A (complex I and III inhibitors, respectively) with each followed by 3 measurement cycles. This series of manipulations enabled the calculation of parameters: basal, ATP-linked, maximum, and non-mitochondrial OCR, as well as proton leak. Each measurement cycle was a total of 6 min.
Mentions: Cells were incubated for 1 h (37°C, 0% CO2) before culture medium was replaced by 175 µl of unbuffered Seahorse XF Base medium supplemented with glucose (25 mM), L-glutamine (2 mM), sodium pyruvate (1 mM), pre-warmed to 37 °C (pH 7.4). Prior to measurement of OCR, the Seahorse XFe96 instrument gently mixed the assay medium in each well for 10 min to enable the oxygen partial pressure to reach equilibrium. The OCR was then measured 3 times to establish a baseline rate prior to the acute injection of flutamide, 2-hydroxyflutamide or bicalutamide (7.8–500 µM). There were 9 OCR measurement cycles following compound injection and each measurement cycle consisted of a 3 min mix and 3 min measure. Following this compound incubation (54 min), a mitochondrial stress test was performed consisting of sequential injections of oligomycin (1 µM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 µM) and antimycin A/rotenone (1 µM each) (all compound concentrations were optimized to generate the maximum effect in the absence of toxicity). After stress test compound injections there were 3 measurement cycles before the injection of the next stress test compound. This enabled the calculation of basal respiration (OCR prior to oligomycin injection—non-mitochondrial OCR), proton leak (OCR after oligomycin injection—non-mitochondrial OCR), ATP-linked OCR (basal respiration—proton leak—non-mitochondrial OCR), maximal respiration (first injection after FCCP injection—non-mitochondrial OCR) and spare respiratory capacity (maximal respiration—basal respiration) (Figure 2).FIG. 2

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.