Oxidative Medicine and Cellular Longevity

Oxidative Medicine and Cellular Longevity / 2018 / Article

Research Article | Open Access

Volume 2018 |Article ID 8017073 | 12 pages | https://doi.org/10.1155/2018/8017073

Flutamide Induces Hepatic Cell Death and Mitochondrial Dysfunction via Inhibition of Nrf2-Mediated Heme Oxygenase-1

Academic Editor: Shao-Yu Chen
Received23 Feb 2018
Revised22 Apr 2018
Accepted10 May 2018
Published02 Jul 2018

Abstract

Flutamide is a widely used nonsteroidal antiandrogen for prostate cancer therapy, but its clinical application is restricted by the concurrent liver injury. Increasing evidence suggests that flutamide-induced liver injury is associated with oxidative stress, though the precise mechanism is poorly understood. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor regulating endogenous antioxidants including heme oxygenase-1 (HO-1). This study was designed to delineate the role of Nrf2/HO-1 in flutamide-induced hepatic cell injury. Our results showed that flutamide concentration dependently induced cytotoxicity, hydrogen peroxide accumulation, and mitochondrial dysfunction as indicated by mitochondrial membrane potential loss and ATP depletion. The protein expression of Nrf2 and HO-1 was induced by flutamide at 12.5 μM but was downregulated by higher concentrations of flutamide. Silencing either Nrf2 or HO-1 was found to aggravate flutamide-induced hydrogen peroxide accumulation and mitochondrial dysfunction as well as inhibition of the Nrf2 pathway. Moreover, preinduction of HO-1 by Copp significantly attenuated flutamide-induced oxidative stress and mitochondrial dysfunction, while inhibition of HO-1 by Snpp aggravated these deleterious effects. These findings suggest that flutamide-induced hepatic cell death and mitochondrial dysfunction is assoicated with inhibition of Nrf2-mediated HO-1. Pharmacologic intervention of Nrf2/HO-1 may provide a promising therapeutic approach in flutamide-induced liver injury.

1. Introduction

Flutamide (Flu, 2-methyl-N-[4-nitro-3-(trifluoromethyl) phenyl] propanamide) is an oral, widely used nonsteroidal antiandrogen approved for the treatment of prostate cancer [1]. Although generally considered safe, flutamide therapy is compromised by the occurrence of hepatotoxicity [2]. Flutamide can induce cholestasis, jaundice, and liver necrosis, which may eventually require a liver transplant. Because of the concurrent hepatotoxicity, flutamide received a black box warning label by the FDA in 1999 [3]. Mitochondrial injury is increasingly proposed as a putative hazard of flutamide though the susceptibility factors which are unknown [4]. Experimental evidence has shown that flutamide can inhibit mitochondrial complex I [5], induce ATP depletion [6], and disrupt mitochondrial membrane potential (MMP) [7] in cultured hepatic cells. Studies using different mouse models have revealed that flutamide impaired glucose homeostasis and triggered mitochondrial dysfunction [8]. These findings suggest that mitochondria play a critical role in flutamide-induced liver injury. However, the mechanisms of flutamide-induced mitochondrial damage and liver injury remain elusive.

Mitochondria are the main sites of oxidative phosphorylation and energy metabolism. Meanwhile, mitochondria are also major intracellular sources of reactive oxygen species (ROS). Mitochondrial ROS can physiologically act as important redox signaling molecules and play an important role in maintaining cellular oxidant/antioxidant balance. However, excessive ROS production can cause mitochondrial damage that may eventually lead to liver diseases [9]. Emerging evidence implicates that flutamide-induced hepatotoxicity is associated with ROS-mediated oxidative stress [10]. During the metabolism of flutamide in the liver, it is oxidatively metabolized by microsomal metabolic enzymes and turned into electrophilic metabolites. Meanwhile, ROS are also produced as by-products. It has been shown that flutamide induces ROS formation and other prooxidant radicals leading to oxidative stress and mitochondrial dysfunction in primary cultured hepatocytes [4].

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a master transcription factor regulating the oxidative stress response [11]. Nrf2 is particularly important during times of severe oxidative stress through its ability to regulate the expression of antioxidant proteins and phase II enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), and glutathione-S-transferases (GST). Recent studies have shown that HO-1 plays a key role in counteracting oxidative stress and mitochondrial damage [12, 13]. HO-1 is a highly inducible antioxidant enzyme that can be induced under a number of conditions, such as oxidative stress, infection, inflammation, and hypoxia [14]. HO-1 converts potentially toxic heme released by mitochondria into the antioxidant biliverdin. Degradation of heme results in the release of iron and production of CO, which functions as a key signal that independently upregulates cellular iron-responsive and antioxidant defense [14]. In addition, CO can bind to the reduced A3 heme of cytochrome C oxidase (COX), which enhances mitochondrial hydrogen peroxide release and contributes to retrograde activation of mitochondrial biogenesis.

Increasing evidence suggests that the Nrf2 pathway is critically involved in drug-induced liver injury [15]. It is implicated that many drug-induced hepatotoxicity is attributed to the inhibition of the Nrf2 pathway while its activation was found to provide effective protection against liver injury [1618]. However, the precise role of HO-1 in flutamide-induced hepatotoxicity is poorly understood. According to the TT21C report entitled “Toxicity Testing in the Twenty First Century: Vision and Strategy” released by the USA National Research Council (NRC, 2007), toxicity pathway-based drug safety assessments have been proposed as a central part in toxicity testing in the 21st century. As depicted by that report which was regarded as a milestone in toxicology, toxicity testing strategy is undergoing a tremendous transformation from traditional animal tests to in vitro approaches and other nonanimal alternatives that primarily and preferably use human-originated cells/cell lines. In compliance with this report, we utilized HepG2 cells to explore the role of the Nrf2/HO-1 pathway in flutamide-induced toxicity. We demonstrated that flutamide-induced hepatic cell death, oxidative stress, and mitochondrial dysfunction are through the inhibition of Nrf2-mediated HO-1 induction. Preinduction of HO-1 protected against flutamide-induced hepatic mitochondrial dysfunction. In contrast, inhibition of HO-1 exacerbated flutamide-induced hepatotoxicity. These findings highlight an important role of HO-1 in flutamide-induced liver injury and suggest that Nrf2/HO-1 may be a promising therapeutic target for preventing and treating drug-induced liver injury.

2. Materials and Methods

2.1. Cell Culture and Drug Treatments

HepG2 cells were obtained from Shanghai Cell Line Bank (Shanghai, China) and routinely cultured in MEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell cultures were grown at 37°C in humidified incubators containing an atmosphere of 5% CO2. All cells used in our experiments were up to passage 20. Cells were treated with flutamide (Sigma-Aldrich, USA) for 24 h at various concentrations as indicated. In some experiments, cells were pretreated with or without 20 μM tin protoporphyrin (Snpp, Sigma-Aldrich) or 10 μM cobalt protoporphyrin (Copp, Sigma-Aldrich) for 1 h followed by treatment with 50 μM flutamide.

2.2. Evaluation of Cytotoxicity

Cytotoxicity was evaluated by the determination of cell viability and lactate dehydrogenase (LDH) leakage. Cell viability was assessed by using a commercial cell counting Kit-8 (CCK8, Dojindo Molecular Technologies, Japan) according to the manufacturer’s instruction. Briefly, immediately after drug treatment, cells were incubated with CCK-8 working solution at 37°C for 2 h. Subsequently, the absorbance at 450 nm was monitored using a microplate reader (Multiskan MK3, Thermo Fisher Scientific, USA). Data were normalized to the values from control cultures without drug treatment which were considered 100% survival.

LDH release into the medium was measured to estimate the extent of cell damage. At the end of drug treatment, supernatants of culture media were collected for LDH assay. The activity of LDH was measured by recording the absorbance at 490 nm using a cytotoxicity LDH assay kit (Beyotime Biotechnology, China).

2.3. Determination of Hydrogen Peroxide Content

Hydrogen peroxide was measured using a hydrogen peroxide assay kit based on ferrous oxidation of xylenol orange assay according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, China). Cells were freshly collected and lysed immediately after drug treatments. Cell lysate was centrifuged at 12,000 at 4°C for 5 min. The supernatant was collected and incubated with a hydrogen peroxide detecting reagent at room temperature for 30 min. Absorbance at 560 nm was then monitored by a microplate reader.

2.4. Detection of Mitochondrial Membrane Potential

Mitochondrial membrane potential was indicated by a MitoTracker® probe (Invitrogen) which contains a mildly thiol-reactive chloromethyl moiety for labeling mitochondria. After completion of drug treatment, cells were incubated with staining solution containing 100 nM MitoTracker probe in the dark at 37°C for 30 min. Thereafter, cells were washed at least thrice with prewarmed PBS to completely remove extra probe. The fluorescence intensity of MitoTracker probe was measured using a FACS Calibur flow cytometer (Becton Dickinson, USA).

2.5. Determination of ATP

Cellular ATP content was determined by ATP colorimetric assay (BioVision) by utilizing the phosphorylation of glycerol to generate a product that is quantified by colorimetric methods. Samples were collected and processed according to the manufacturer’s instruction. In brief, cells were lysed in an ATP assay buffer, followed by deproteinizing using a deproteinization sample preparation kit (BioVision). The samples were then mixed with ATP assay buffer, along with reaction mix and an ATP probe. The reaction system was incubated in the dark at room temperature for 30 min. Thereafter, absorbance at 570 nm was monitored by a microplate reader.

2.6. siRNA Transfection

Nrf2 knockdown cell model and HO-1 knockdown cell model were established by transfection with specific siRNA as we previously reported [19]. In brief, cells were plated in 6-well plates and transiently transfected with 70 nM of small interfering oligonucleotide (siRNA) against Nrf2 or HO-1 (Santa Cruz Biotechnology, USA) or control nonspecific oligonucleotide (ConsiRNA) using lipid-based transfection system (Lipofectamine 3000, Thermo Fisher Scientific) for 5 h. Thereafter, cells were allowed to recover in fresh media for 24 h according to the manufacturer’s protocol. The efficiency of Nrf2 or HO-1 knockdown was confirmed by the detection of the mRNA and protein level quantified by qPCR and Western blot, respectively.

2.7. Western Blotting Analysis

Cells were lysed with ice-cold RIPA buffer (Applygen Technologies) containing protease and phosphatase inhibitors (Applygen Technologies). Protein samples were collected and resolved by 8% or 12% SDS-PAGE and were then transferred to polyvinylidene difluoride membranes (PVDF) (Millipore, USA). Membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween 20 (TBS-T) for 4 h and incubated with primary antibodies at 4°C overnight, followed by 1 h incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at room temperature. The blots were detected using ECL detection system (Applygen Technologies) and recorded by chemiluminescence imaging analysis. Images were analyzed using ImageJ software (National Institutes of Health, USA).

2.8. Statistical Analysis

All values were expressed as the mean ± SD from 3 independent experiments. Statistical analyses were performed by one-way ANOVA followed by Dunnett’s test. Data were analyzed and presented with PASW Statistics 18.0 software (SPSS Inc., USA). A value < 0.05 was considered statistically significant.

3. Results

3.1. Characterization of the Cytotoxicity of Flutamide in HepG2 Cells

The cytotoxicity of flutamide in HepG2 cells was evaluated by cell viability and LDH leakage. Cells were exposed to flutamide for 24 h at various concentrations ranging from 0 to 200 μM. Compared to cells in the control group, cells treated with flutamide at concentrations higher than 25 μM showed significant and concentration-dependent decreases in cell viability with an LC50 (lethal concentration required to cause 50% reduction in cell viability) of 133 μM (Figure 1(a)). The activity of LDH in media was also concentration dependently increased by flutamide. In line with cell viability assay, a significant increase in LDH activity was found in cells treated with flutamide at concentrations higher than 25 μM (Figure 1(b)).

3.2. Flutamide-Induced ROS Accumulation and Mitochondrial Dysfunction

Excessive ROS production has been implicated as an important causative factor for flutamide-induced hepatotoxicity [20]. Hydrogen peroxide is one of the main types of ROS that can directly attack cellular component, such as lipid, protein, and DNA, leading to oxidative damage [21]. As shown in Figure 2(a), flutamide increased hydrogen peroxide levels by a concentration-dependent manner. Compared with cells in the control group, hydrogen peroxide contents were significantly increased in cells treated with flutamide at concentrations higher than 12.5 μM. For cells treated with flutamide at 100 μM, the hydrogen peroxide content was increased by 2.4-fold compared with cells in the control group.

Mitochondrial function was evaluated by the determination of mitochondrial membrane potential and ATP production. As shown in Figure 2(b), flutamide was found to concentration dependently decrease mitochondrial membrane potential. Significant mitochondrial membrane potential loss was found in the cells treated with flutamide at a concentration over 12.5 μM compared to cells in the control group. Similar results were found in ATP assays. As shown in Figure 2(c), 28.7%, 40.5%, 48.0%, and 51.2% reductions in ATP levels were found, respectively, for cells treated with flutamide at 25, 50, 75, and 100 μM compared to cells in the control group.

3.3. Flutamide-Perturbed Nrf2/HO-1 Pathway

To investigate the effect of flutamide on the Nrf2/HO-1 pathway, the protein expressions of Nrf2, HO-1, and superoxide dismutase-2 (SOD2) were analyzed with Western blot (Figure 3(a)). Protein levels of Nrf2 (Figure 3(b)) and HO-1 (Figure 3(c)) were slightly increased by flutamide at 12.5 μM but were significantly decreased by higher concentrations of flutamide. Compared to the cells in the control group, the protein levels of Nrf2 were increased by 13.7% and decreased by 11.0%, 28.7%, 42.1, and 44.0% for cells treated with flutamide at 12.5, 25, 50, 75, and 100 μM, respectively. Remarkable changes were also found in HO-1 protein expression, which was increased by 24.3% but decreased by 9.0%, 63.7%, 80.6%, and 84.3%, respectively, for cells treated with flutamide at 12.5, 25, 50, 75, and 100 μM, respectively (Figure 3(d)). In contrast, the protein expression of SOD2 was inhibited by flutamide at all concentrations tested with decreases ranging from 12.9% to 33.1%.

3.4. Knockdown of Nrf2/HO-1 Aggravated Flutamide-Induced Oxidative Stress, Mitochondrial Dysfunction, and Inhibition of Nrf2/HO-1 Pathway

To evaluate the role of the Nrf2/HO-1 pathway in flutamide-induced hepatotoxicity, Nrf2 knockdown and HO-1 knockdown cell models were established. HepG2 cells were treated with Nrf2 or HO-1 siRNA at a concentration at which no obvious cytotoxicity was elicited. The efficiency of Nrf2 and HO-1 knockdown was confirmed by RT-PCR and Western blot to detect mRNA and protein levels, respectively. The efficiency of Nrf2 and HO-1 knockdown efficiency at protein level were approximately 50% and 40% (data not shown).

Nrf2 knockdown by itself showed no tangible effect on the hydrogen peroxide level, mitochondrial membrane potential, and ATP content. However, compared to cells treated with ConsiRNA, 50 μM flutamide-induced hydrogen peroxide accumulation, mitochondrial membrane potential loss, and ATP depletion were significantly aggravated in Nrf2 knockdown cells. Flutamide elevated the levels of hydrogen peroxide by 1.7- and 2.9-fold, respectively, for ConsiRNA-treated cells and Nrf2 knockdown cells (Figure 4(a)). The mitochondrial membrane potential was decreased by 14.5% and 42.1%, respectively, for ConsiRNA-treated cells and Nrf2 knockdown cells following flutamide exposure (Figure 4(b)). Similarly, flutamide was found to induce 34.5% and 73.0% reduction in ATP levels for ConsiRNA-treated cells and Nrf2 knockdown cells, respectively, (Figure 4(c)). As expected, the protein expressions of Nrf2, HO-1, and SOD2 were significantly decreased in Nrf2 knockdown cells (Figures 4(d) and 4(e)). Flutamide-induced inhibition of Nrf2 and HO-1 was further aggravated by Nrf2 knockdown. Interestingly, no significant difference was found in the protein expression of SOD2 between ConsiRNA-treated cells and Nrf2 knockdown cells following flutamide exposure.

Knockdown of HO-1 was also found to exacerbate flutamide-induced oxidative stress and mitochondrial function. The hydrogen peroxide level, mitochondrial membrane potential, and ATP level were not affected by HO-1 silencing (Figures 5(a)5(c)). HO-1 knockdown only significantly decreased the expression of HO-1 but did not alter the protein expression of Nrf2 and SOD2 (Figures 5(d) and 5(e)). Compared to cells without drug treatment, the hydrogen peroxide level was increased by 2.1-fold while the mitochondrial membrane potential and ATP content were decreased by 25.9% and 54.5%, respectively, in HO-1 knockdown cells treated with 50 μM. Silencing of HO-1 exacerbated flutamide-induced inhibition of HO-1 protein expression, but no significant difference was found in the protein expression of Nrf2 and SOD2 between ConsiRNA-treated cells and HO-1 knockdown cells following flutamide exposure.

3.5. Effects of HO-1 Interventions on Flutamide-Induced Oxidative Stress and Mitochondrial Dysfunction

Given the prominent alterations of HO-1 following flutamide treatment, we further investigated the effect of HO-1 interventions on flutamide-mediated hepatic injury and Nrf2/HO-1 pathway perturbation. Cells were pretreated with 10 μM Copp (HO-1 inducer) or 20 μM Snpp (HO-1 inhibitor) for 1 h before flutamide (50 μM) treatment. Copp significantly blocked flutamide-induced oxidative stress and mitochondrial dysfunction, while inhibition of HO-1 by Snpp remarkably aggravated these deleterious effects. Compared to cells in the control group without drug treatment, the levels of hydrogen peroxide in cells treated with flutamide, Snpp plus flutamide, and Copp plus flutamide were elevated by 1.9-, 2.1-, and 1.4-fold, respectively (Figures 6(a)6(c)). The mitochondrial membrane potential and ATP content were reduced by 27.5 and 48.6% in cells treated with Snpp plus flutamide. In comparison, cells treated with Copp only showed a 7.0% reduction in mitochondrial membrane potential and 17.8% decrease in ATP content. Results from Western blot analysis showed that Snpp pretreatment significantly potentiated flutamide-induced inhibition of Nrf2 and HO-1 protein expression (Figures 6(d) and 6(e)), while Copp can ameliorate these changes by flutamide (Figures 6(f) and 6(g)). Neither Snpp nor Copp has an obvious effect on flutamide-induced inhibition of SOD2 expression.

4. Discussion

Flutamide is an antiandrogen drug that is widely used for the treatment of prostate cancer. However, the therapeutic effects of flutamide have been overshadowed by reports of liver dysfunction in 1–10% of its users [2, 22]. This flutamide-induced liver injury is not acute but delayed [3, 23]. In most cases, flutamide caused liver dysfu1nction after a latency period of approximately 16 weeks [24]. Our results demonstrated that 24 h treatment of HepG2 cells with flutamide concentration dependently induced reduction of cell viability and LDH leakage. Flutamide did not elicit significant effects on these cytotoxicity indices at the concentration of 12.5 and 25 μM. Thus, the observed adverse effect level (NOAEL) of flutamide-induced cytotoxicity in HepG2 cells is 25 μM. Flutamide-induced mitochondrial damage was evaluated by the determination of mitochondrial membrane potential and ATP production. Our results showed that flutamide concentration dependently decreased mitochondrial membrane potential and reduced the APT level. Significant mitochondrial toxic effects were found at the concentration of 25 μM at which no significant cytotoxicity was observed induced, suggesting mitochondria as a sensitive target of flutamide-induced hepatocyte toxicity.

Mitochondria are thought to play a critical role in the development and pathogenesis of drug-induced liver injury, not only due to their role as the main source of endogenous ROS but also due to the role as the target of ROS attack. It has been shown that the accumulation of ROS in hepatic cells is an essential step in flutamide hepatotoxicity [4]. It is implicated that flutamide can promote ROS generation, especially in the mitochondria, by multiple mechanisms [10]. For instance, flutamide has been shown to inhibit mitochondrial complex I leading to superoxide production through the reduction of molecular oxygen by the NADH/ubiquinone oxidoreductase [25]. Flutamide can also produce many reactive oxidants during its metabolism in the liver. In the present study, flutamide was found to increase the level of hydrogen peroxide in a concentration-dependent manner; however, the specific source of the observed flutamide-elicited ROS accumulation still needs further investigation.

Excessive ROS generation can disrupt mitochondrial membrane potential, induce mitochondrial dysfunction, and compromise the capacity of the antioxidant defense system [26]. The mitochondria have been indicated as important targets of flutamide-mediated adverse effects in the liver. It has been shown that flutamide can alter mitochondrial morphology and profoundly downregulate genes associated with fatty acid β-oxidation and upregulate genes related to antioxidant defense in hepatocytes [6]. The loss of ATP was also found to be a critical event in the cytotoxicity of flutamide caused by its ability to target complex I of the electron transport chain and impair oxidative phosphorylation [6]. Consistent with the observation of increased hydrogen peroxide accumulation, our results demonstrated that flutamide induced mitochondrial membrane potential loss and reduction of ATP.

Nrf2 is ubiquitously expressed throughout human tissues, with high expression in detoxification organs, especially the liver. Nrf2 serves as a major regulator of the cellular antioxidant defense pathway by which hepatic cells combat oxidative stress [27, 28]. During oxidative stress, Nrf2 activation is initiated to transcriptionally regulate its target genes to detoxify and eliminate potentially harmful exogenous chemicals and their metabolites [29, 30]. Growing evidence has suggested that the activation of the Nrf2 pathway is a key mechanism underlying the protective effects of many pharmacological agents [31, 32]. In contrast, the inhibition of the Nrf2 pathway has been implicated as a critical cause leading to the adverse effects of several chemicals [33]. Nrf2 knockout mice and Nrf2-deficient cells have been found to be more sensitive to oxidative injury [3436]. In the present study, the protein expression of Nrf2 was slightly increased by flutamide at a lower concentration (12.5 μM) but was significantly inhibited by higher concentrations (≥50 μM). Knockdown of Nrf2 was found to significantly sensitize the cells to flutamide-induced ROS accumulation and mitochondrial dysfunction. The changes of Nrf2 were mirrored by HO-1 and SOD2, though the magnitude of SOD2 alternation was less than Nrf2 and HO-1. These findings suggest that the Nrf2 pathway was activated by flutamide at a low dose to counteract oxidative stress. However, cells exposed to flutamide at a higher dose might undergo saturated Nrf2 pathway activation, thus failing to combat increased ROS generation, consequently leading to inhibition of this pathway and mitochondrial dysfunction.

HO-1 is implicated as one of the most important downstream antioxidant targets of Nrf2 protecting against oxidative stress damage [3739]. Increasing evidence suggests that HO-1 is particularly important in regulating mitochondrial function and energy metabolism as well as oxidative stress [40, 41]. HO-1 represents a prime antioxidant defense mechanism against mitochondrial oxidative stress through several ways [4244]. The induction of HO-1 leads to increased cellular CO production, which generates a redox signal for the induction of mitochondrial biogenesis [45]. HO-1 has also been implicated in the regulation of mitophagy and mitochondrial quality control cycle [46]. Our results demonstrated that HO-1 was induced by flutamide at a low concentration but was inhibited by high concentrations. Compared to the changes of Nrf2 and SOD2, the alteration of HO-1 was much more prominent, indicating HO-1 as a sensitive marker of liver injury following flutamide treatment. A recent study by Teppner et al. showed that the gene expression of HO-1 was increased but no obvious cytotoxic effects were induced in in vitro cultured rat hepatocytes treated with flutamide at 50 and 100 μM, which are similar to the concentrations used in the present study [47]. This discrepancy with our results might involve several causes such as different cell models and experimental parameters. In addition, HO-1 silence significantly augmented flutamide-induced oxidative stress and mitochondrial dysfunction. Preinduction of HO-1 by Copp significantly attenuated flutamide-induced ROS accumulation and mitochondrial dysfunction, while inhibition of HO-1 by Snpp remarkably aggravated the deleterious effects. Taken together, these findings suggested that HO-1 was particularly important in flutamide-induced hepatotoxicity (Figure 7).

In conclusion, our results suggested that flutamide-induced hepatotoxicity involves, at least in part, the inhibition of the Nrf2/HO-1 pathway and highlighted an important role of HO-1 in flutamide-induced hepatic mitochondrial injury. The induction of HO-1 prevented flutamide-induced hepatic mitochondrial dysfunction while inhibition of HO-1 exacerbated the adverse effects. These findings provide new evidence to understand the mechanism of flutamide-induced liver injury. Given that oxidative stress and mitochondrial dysfunction underlie the liver injury induced by various drugs, the results obtained from the present study suggest the Nrf2 pathway, particularly HO-1, as a potential therapeutic target for patients with drug-induced adverse effects in the liver.

Data Availability

The authors are willing to share the detailed/raw data in private with interested researchers.

Additional Points

Highlights. Flutamide induces cell death, oxidative stress, and mitochondrial dysfunction. The Nrf2/HO-1 pathway was perturbed by flutamide. Knockdown of Nrf2/HO-1 aggravates flutamide-induced hepatic cell injury. Induction of HO-1 prevents hepatic oxidative stress and mitochondrial dysfunction. Inhibition of HO-1 exacerbates flutamide-induced hepatotoxicity.

Conflicts of Interest

The authors declare no potential conflict of interests with the data presented in this article.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81430090), Beijing Nova Program (Z171100001117103), AMMS Innovative Foundation (2017CXJJ13), and Unilever International Collaborative Project (MA-2015-00410).

References

  1. P. Kang, D. Dalvie, E. Smith, S. Zhou, and A. Deese, “Identification of a novel glutathione conjugate of flutamide in incubations with human liver microsomes,” Drug Metabolism and Disposition, vol. 35, no. 7, pp. 1081–1088, 2007. View at: Publisher Site | Google Scholar
  2. A. Osculati and C. Castiglioni, “Fatal liver complications with flutamide,” Lancet, vol. 367, no. 9517, pp. 1140-1141, 2006. View at: Publisher Site | Google Scholar
  3. Z. Thole, G. Manso, E. Salgueiro, P. Revuelta, and A. Hidalgo, “Hepatotoxicity induced by antiandrogens: a review of the literature,” Urologia Internationalis, vol. 73, no. 4, pp. 289–295, 2004. View at: Publisher Site | Google Scholar
  4. R. Kashimshetty, V. G. Desai, V. M. Kale et al., “Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity,” Toxicology and Applied Pharmacology, vol. 238, no. 2, pp. 150–159, 2009. View at: Publisher Site | Google Scholar
  5. J. Hynes, L. D. Marroquin, V. I. Ogurtsov et al., “Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes,” Toxicological Sciences, vol. 92, no. 1, pp. 186–200, 2006. View at: Publisher Site | Google Scholar
  6. K. J. Coe, Y. Jia, H. K. Ho et al., “Comparison of the cytotoxicity of the nitroaromatic drug flutamide to its cyano analogue in the hepatocyte cell line TAMH: evidence for complex I inhibition and mitochondrial dysfunction using toxicogenomic screening,” Chemical Research in Toxicology, vol. 20, no. 9, pp. 1277–1290, 2007. View at: Publisher Site | Google Scholar
  7. A. Al Maruf and P. O'Brien, “Flutamide-induced cytotoxicity and oxidative stress in an in vitro rat hepatocyte system,” Oxidative Medicine and Cellular Longevity, vol. 2014, Article ID 398285, 9 pages, 2014. View at: Publisher Site | Google Scholar
  8. L. Choucha Snouber, A. Bunescu, M. Naudot et al., “Metabolomics-on-a-chip of hepatotoxicity induced by anticancer drug flutamide and Its active metabolite hydroxyflutamide using HepG2/C3a microfluidic biochips,” Toxicological Sciences, vol. 132, no. 1, pp. 8–20, 2013. View at: Publisher Site | Google Scholar
  9. N. Rayamajhi, S.-K. Kim, H. Go et al., “Quercetin induces mitochondrial biogenesis through activation of HO-1 in HepG2 cells,” Oxidative Medicine and Cellular Longevity, vol. 2013, Article ID 154279, 10 pages, 2013. View at: Publisher Site | Google Scholar
  10. M. Teppner, F. Boss, B. Ernst, and A. Pahler, “Application of lipid peroxidation products as biomarkers for flutamide-induced oxidative stress in vitro,” Toxicology Letters, vol. 238, no. 3, pp. 53–59, 2015. View at: Publisher Site | Google Scholar
  11. R. N. Jadeja, K. K. Upadhyay, R. V. Devkar, and S. Khurana, “Naturally occurring Nrf2 activators: potential in treatment of liver injury,” Oxidative Medicine and Cellular Longevity, vol. 2016, Article ID 3453926, 13 pages, 2016. View at: Publisher Site | Google Scholar
  12. C. Gorrini, I. S. Harris, and T. W. Mak, “Modulation of oxidative stress as an anticancer strategy,” Nature Reviews Drug Discovery, vol. 12, no. 12, pp. 931–947, 2013. View at: Publisher Site | Google Scholar
  13. K. Shukla, H. Sonowal, A. Saxena, K. V. Ramana, and S. K. Srivastava, “Aldose reductase inhibitor, fidarestat regulates mitochondrial biogenesis via Nrf2/HO-1/AMPK pathway in colon cancer cells,” Cancer Letters, vol. 411, pp. 57–63, 2017. View at: Publisher Site | Google Scholar
  14. R. Gozzelino, V. Jeney, and M. P. Soares, “Mechanisms of cell protection by heme oxygenase-1,” Annual Review of Pharmacology and Toxicology, vol. 50, no. 1, pp. 323–354, 2010. View at: Publisher Site | Google Scholar
  15. H. Yan, Z. Huang, Q. Bai et al., “Natural product andrographolide alleviated APAP-induced liver fibrosis by activating Nrf2 antioxidant pathway,” Toxicology, vol. 396-397, pp. 1–12, 2018. View at: Publisher Site | Google Scholar
  16. X. Peng, C. Dai, Q. Liu, J. Li, and J. Qiu, “Curcumin attenuates on carbon tetrachloride-induced acute liver injury in mice via modulation of the Nrf2/HO-1 and TGF-β1/Smad3 pathway,” Molecules, vol. 23, no. 1, p. 215, 2018. View at: Publisher Site | Google Scholar
  17. Y.-H. Lee, S. Kim, S. Lee et al., “Antioxidant effect of barley sprout extract via enhancement of nuclear factor-erythroid 2 related factor 2 activity and glutathione synthesis,” Nutrients, vol. 9, no. 11, article 1252, 2017. View at: Publisher Site | Google Scholar
  18. A. M. Mahmoud, W. G. Hozayen, and S. M. Ramadan, “Berberine ameliorates methotrexate-induced liver injury by activating Nrf2/HO-1 pathway and PPARγ, and suppressing oxidative stress and apoptosis in rats,” Biomedicine & Pharmacotherapy, vol. 94, pp. 280–291, 2017. View at: Publisher Site | Google Scholar
  19. W. Zhou, X. Yuan, L. Zhang et al., “Overexpression of HO-1 assisted PM2.5-induced apoptosis failure and autophagy-related cell necrosis,” Ecotoxicology and Environmental Safety, vol. 145, pp. 605–614, 2017. View at: Publisher Site | Google Scholar
  20. E. Navarro, L. Gonzalez-Lafuente, I. Pérez-Liébana et al., “Heme-oxygenase I and PCG-1α regulate mitochondrial biogenesis via microglial activation of Alpha7 nicotinic acetylcholine receptors using PNU282987,” Antioxidants & Redox Signaling, vol. 27, no. 2, pp. 93–105, 2017. View at: Publisher Site | Google Scholar
  21. P. R. Angelova and A. Y. Abramov, “Role of mitochondrial ROS in the brain: from physiology to neurodegeneration,” FEBS Letters, vol. 592, no. 5, pp. 692–702, 2018. View at: Publisher Site | Google Scholar
  22. Y. Nakagawa, M. Koyama, and M. Matsumoto, “Flutamide-induced hepatic disorder and serum concentrations of flutamide and its metabolites in patients with prostate cancer,” Hinyokika Kiyo, vol. 45, no. 12, pp. 821–826, 1999. View at: Google Scholar
  23. G. Manso, Z. Thole, E. Salgueiro, P. Revuelta, and A. Hidalgo, “Spontaneous reporting of hepatotoxicity associated with antiandrogens: data from the Spanish pharmacovigilance system,” Pharmacoepidemiology and Drug Safety, vol. 15, no. 4, pp. 253–259, 2006. View at: Publisher Site | Google Scholar
  24. M. Ohbuchi, M. Miyata, D. Nagai, M. Shimada, K. Yoshinari, and Y. Yamazoe, “Role of enzymatic N-hydroxylation and reduction in flutamide metabolite-induced liver toxicity,” Drug Metabolism and Disposition, vol. 37, no. 1, pp. 97–105, 2009. View at: Publisher Site | Google Scholar
  25. J. F. Turrens, “Mitochondrial formation of reactive oxygen species,” The Journal of Physiology, vol. 552, no. 2, pp. 335–344, 2003. View at: Publisher Site | Google Scholar
  26. Y. Wang, S. H. Guo, X. J. Shang et al., “Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response,” Acta Pharmacologica Sinica, vol. 39, no. 2, pp. 311–327, 2017. View at: Publisher Site | Google Scholar
  27. C. Knörr-Wittmann, A. Hengstermann, S. Gebel, J. Alam, and T. Müller, “Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke,” Free Radical Biology & Medicine, vol. 39, no. 11, pp. 1438–1448, 2005. View at: Publisher Site | Google Scholar
  28. A. A. Turanov, D. Su, and V. N. Gladyshev, “Characterization of alternative cytosolic forms and cellular targets of mouse mitochondrial thioredoxin reductase,” The Journal of Biological Chemistry, vol. 281, no. 32, pp. 22953–22963, 2006. View at: Publisher Site | Google Scholar
  29. Y. J. Lee, H. Y. Jeong, Y. B. Kim et al., “Reactive oxygen species and PI3K/Akt signaling play key roles in the induction of Nrf2-driven heme oxygenase-1 expression in sulforaphane-treated human mesothelioma MSTO-211H cells,” Food and Chemical Toxicology, vol. 50, no. 2, pp. 116–123, 2012. View at: Publisher Site | Google Scholar
  30. J. Wang, L. Yuan, H. Xiao, C. Xiao, Y. Wang, and X. Liu, “Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways,” Apoptosis, vol. 18, no. 6, pp. 751–765, 2013. View at: Publisher Site | Google Scholar
  31. E. Y. Lin, U. Bayarsengee, C. C. Wang, Y. H. Chiang, and C. W. Cheng, “The natural compound 2,3,5,4-tetrahydroxystilbene-2-O-β-dglucoside protects against adriamycin-induced nephropathy through activating the Nrf2-Keap1 antioxidant pathway,” Environmental Toxicology, vol. 33, no. 1, pp. 72–82, 2018. View at: Publisher Site | Google Scholar
  32. G. R. Sharath Babu, T. Anand, N. Ilaiyaraja, F. Khanum, and N. Gopalan, “Pelargonidin modulates Keap1/Nrf2 pathway gene expression and ameliorates citrinin-induced oxidative stress in HepG2 cells,” Frontiers in Pharmacology, vol. 8, p. 868, 2017. View at: Publisher Site | Google Scholar
  33. D. Ren, N. F. Villeneuve, T. Jiang et al., “Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 4, pp. 1433–1438, 2011. View at: Publisher Site | Google Scholar
  34. Y. Wei, J. Gong, Z. Xu et al., “Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A,” Proceedings of the National Academy of Sciences of the United States of America, vol. 112, no. 50, pp. E6927–E6936, 2015. View at: Publisher Site | Google Scholar
  35. U. A. Köhler, S. Kurinna, D. Schwitter et al., “Activated Nrf2 impairs liver regeneration in mice by activation of genes involved in cell-cycle control and apoptosis,” Hepatology, vol. 60, no. 2, pp. 670–678, 2014. View at: Publisher Site | Google Scholar
  36. A. A. Merchant, A. Singh, W. Matsui, and S. Biswal, “The redox-sensitive transcription factor Nrf2 regulates murine hematopoietic stem cell survival independently of ROS levels,” Blood, vol. 118, no. 25, pp. 6572–6579, 2011. View at: Publisher Site | Google Scholar
  37. A. Loboda, A. Jazwa, A. Grochot-Przeczek et al., “Heme oxygenase-1 and the vascular bed: from molecular mechanisms to therapeutic opportunities,” Antioxidants & Redox Signaling, vol. 10, no. 10, pp. 1767–1812, 2008. View at: Publisher Site | Google Scholar
  38. A. Jozkowicz, H. Was, and J. Dulak, “Heme oxygenase-1 in tumors: is it a false friend?” Antioxidants & Redox Signaling, vol. 9, no. 12, pp. 2099–2118, 2007. View at: Publisher Site | Google Scholar
  39. Y. J. Jun, M. Lee, T. Shin, N. Yoon, J. H. Kim, and H. R. Kim, “Eckol enhances heme oxygenase-1 expression through activation of Nrf2/JNK pathway in HepG2 cells,” Molecules, vol. 19, no. 10, pp. 15638–15652, 2014. View at: Publisher Site | Google Scholar
  40. W. Yan, D. Li, T. Chen, G. Tian, P. Zhou, and X. Ju, “Umbilical cord MSCs reverse D-galactose-induced hepatic mitochondrial dysfunction via activation of Nrf2/HO-1 pathway,” Biological & Pharmaceutical Bulletin, vol. 40, no. 8, pp. 1174–1182, 2017. View at: Publisher Site | Google Scholar
  41. T. Kimura and Y. Watanabe, “Tryptophan protects hepatocytes against reactive oxygen species-dependent cell death via multiple pathways including Nrf2-dependent gene induction,” Amino Acids, vol. 48, no. 5, pp. 1263–1274, 2016. View at: Publisher Site | Google Scholar
  42. H. Was, J. Dulak, and A. Jozkowicz, “Heme oxygenase-1 in tumor biology and therapy,” Current Drug Targets, vol. 11, no. 12, pp. 1551–1570, 2010. View at: Publisher Site | Google Scholar
  43. D. Goven, A. Boutten, V. Lecon-Malas, J. Boczkowski, and M. Bonay, “Prolonged cigarette smoke exposure decreases heme oxygenase-1 and alters Nrf2 and Bach1 expression in human macrophages: roles of the MAP kinases ERK(1/2) and JNK,” FEBS Letters, vol. 583, no. 21, pp. 3508–3518, 2009. View at: Publisher Site | Google Scholar
  44. L. Liang, C. Gao, M. Luo et al., “Dihydroquercetin (DHQ) induced HO-1 and NQO1 expression against oxidative stress through the Nrf2-dependent antioxidant pathway,” Journal of Agricultural and Food Chemistry, vol. 61, no. 11, pp. 2755–2761, 2013. View at: Publisher Site | Google Scholar
  45. C. A. Piantadosi and H. B. Suliman, “Redox regulation of mitochondrial biogenesis,” Free Radical Biology & Medicine, vol. 53, no. 11, pp. 2043–2053, 2012. View at: Publisher Site | Google Scholar
  46. T. D. Hull, R. Boddu, L. Guo et al., “Heme oxygenase-1 regulates mitochondrial quality control in the heart,” JCI Insight, vol. 1, no. 2, p. e85817, 2016. View at: Publisher Site | Google Scholar
  47. M. Teppner, F. Boess, B. Ernst, and A. Pahler, “Biomarkers of flutamide-bioactivation and oxidative stress in vitro and in vivo,” Drug Metabolism and Disposition, vol. 44, no. 4, pp. 560–569, 2016. View at: Publisher Site | Google Scholar

Copyright © 2018 Li Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


More related articles

923 Views | 282 Downloads | 2 Citations
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at help@hindawi.com to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19. Sign up here as a reviewer to help fast-track new submissions.