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ARTICLE Molecular mechanism of 17␣-ethinylestradiol cytotoxicity in isolated rat hepatocytes Luke Wan and Peter O'Brien
Abstract: 17␣-Ethinylestradiol (17-EE) is used in formulations of contraceptives and hormone replacement therapy because it is an estradiol derivative. However, it has been associated with an increase in the risk of liver cancers and injury. The carcinogenic properties of 17-EE are similar to that of other estrogens, but the molecular mechanism of liver injury is still unclear. It is important to identify any secondary toxic mechanisms that can be used to prevent or treat the toxicity. The LC50 of 17-EE toward isolated rat hepatocytes was determined to be 150 ± 8 mol/L. Accelerated cytotoxicity mechanism screening (ACMS) techniques using isolated rat hepatocytes showed that CYP1A inhibitors decreased cytotoxicity, whereas tyrosinase increased toxicity; this suggests that the toxic mechanism involved is the oxidation of 17-EE. A hepatocyte inflammation model also increased 17-EEinduced mitochondrial toxicity, as well as the formation of ROS and H2O2. Cytotoxicity was increased when inhibitors of quinone reduction, catechol-O-methylation, glucuronidation, glutathione conjugation, and sulfation were co-incubated with 17-EE. The hepatocytes could be rescued with antioxidants and quinone trapping agents, thereby suggesting a role for quinoid moiety induced oxidative stress in 17-EE induced cytotoxicity. These mechanisms for 17-EE hepatotoxicity could provide a new perspective for the treating 17-EE-induced liver injury. Key words: 17␣-ethinylestradiol (17-EE), hepatotoxicity, quinoid moieties, molecular mechanism, drug metabolism, oxidative stress, reactive oxygen species (ROS). Résumé : Le 17␣-éthinylestradion (17-EE) est utilisé dans la préparation des contraceptifs et dans la thérapie hormonale de remplacement car il est un dérivé de l'estradiol. Toutefois, il a été associé a` un accroissement du risque de développer une maladie ou un cancer du foie. Les propriétés cancérigènes du 17-EE sont similaires a` celles d'autres œstrogènes, mais les mécanismes moléculaires impliqués dans le dommage hépatique sont encore mal connus. Il est important d'identifier tout mécanisme secondaire toxique qui peut être utilisé pour traiter ou prévenir la toxicité. La CL50 du 17-EE envers les hépatocytes de rats isolés a été déterminée a` 150 ± 8 mol/L. Un criblage ACMS (accelerated cytotoxicity mechanisme screening) utilisé sur les hépatocytes de rat isolés a montré que les inhibiteurs de CYP1A diminuaient la cytotoxicité alors que la tyrosinase l'accroissait; cela suggère que le mécanisme de toxicité impliquait l'oxydation du 17-EE. Un modèle d'inflammation hépatique accroissait aussi la toxicité mitochondriale induite par le 17-EE ainsi que la formation de ERO et de H2O2. La cytotoxicité était accrue lorsque des inhibiteurs de la réduction de la quinone, de catéchol-O-méthylation, de glucuronidation, de conjugaison du glutathion et de sulfation étaient incubés conjointement au 17-EE. Les hépatocytes pouvaient être rescapés par des antioxydants et des piégeurs de quinone, suggérant ainsi que le stress oxydant induit par le groupe quinoïde joue un rôle dans la cytotoxicité induite par le 17-EE. Ces mécanismes d'hépatotoxicité du 17-EE pourraient ouvrir une nouvelle perspective de traitement des dommages hépatiques induits par le 17-EE. [Traduit par la Rédaction] Mots-clés : 17␣-éthinylestradion (17-EE), hépatotoxicité, groupe quinoïde, mécanisme moléculaire, métabolisme des médicaments, stress oxydant, espèce réactive d'oxygène.
Introduction 17␣-ethinylestradiol (17-EE) is used in contraceptives and hormone replacement therapies, but can increase the risk of cancers, including liver cancer (Palmer et al. 1989). Previous studies have shown that 17-EE is a strong tumor promoter with an ability to stimulate liver DNA synthesis (Yager et al. 1994). It has also been found to increase reactive oxygen species (ROS) such as superoxide radicals, which cause chromosomal aberrations and other genotoxic damage (Chen et al. 1999). The genotoxic mechanism of 17-EE has been proposed to involve the formation of o-quinones that can bind to DNA and undergo redox cycling to generate superoxide radicals (Siddique et al. 2005). This is similar to findings using other estrogens that can be oxidized to catechol estrogen quinones thereby increasing the risk of breast and uterine cancers
(Cavalieri et al. 1997; Cavalieri et al. 2006; Bolton and Thatcher 2008). 17-EE has also been shown to cause intrahepatic cholestasis in experimental animals, which can lead to hepatotoxicity (Crocenzi et al. 2001). The exact mechanism for cholestasis is still unclear, but evidence suggests that it is multifactoral in nature, possibly involving a 17-EE glucuronide metabolite or activation of the estrogen receptor signaling pathways (Stieger et al. 2000; Sánchez Pozzi et al. 2003; Yamamoto et al. 2006). There is a build-up of bile salts in hepatocytes during cholestasis, and the bile salts are harmful to hepatocytes (i) owing to their ability to increase oxidative stress, and (ii) their detergent activity, which can disrupt the plasma membrane (Attili et al. 1986; Sokol et al. 1993; Yerushalmi et al. 2001). Cholestatic hepatotoxicity is often associated with hepatic inflammation, which could suggest a necrotic cell-mediated immune
Received 24 July 2013. Accepted 7 October 2013. L. Wan. Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada. P. O'Brien. Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, ON M5S 3M2, Canada. Corresponding author: Peter O'Brien (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 92: 21–26 (2014) dx.doi.org/10.1139/cjpp-2013-0267
Published at www.nrcresearchpress.com/cjpp on 9 October 2013.
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Can. J. Physiol. Pharmacol. Vol. 92, 2014
response (Jaeschke et al. 2002; Kaplowitz 2005). An overactive immune reaction could lead to an increased level of oxidizers such as myeloperoxidases and hypochlorite in the liver, and this in turn could increase the formation of 17-EE oxidative metabolites such as the o-quinones (O'Brien 2000). We have recently developed a hepatocyte inflammation model in which hepatocytes were exposed to a nontoxic peroxidase–H2O2 treatment to mimic an inflammatory response. Activated inflammatory cells release peroxidase and H2O2, formed by NADPH oxidase, and together they can disrupt the normal metabolism of endogenous and exogenous compounds (Babior 2000; Tafazoli and O'Brien 2005). It is known that 17-EE causes hepatotoxicity through cholestasis, but that cannot explain all of the different hepatic toxicological symptoms associated with 17-EE. Some researchers have observed that 17-EE inhibits rat liver growth, a possible indicator of cytotoxicity (Yager et al. 1994). The exact mechanism of the cytotoxicity is unclear, but could be related to redox-cycling-induced oxidative stress. Therefore, it is important to identify any secondary cytotoxic mechanisms and detoxification pathways such that they can be the targets for prevention and treatment. One objective of our research was to determine the molecular basis of 17-EE cytotoxicity associated with inflammation. In this study, metabolizing enzyme modulators were used to determine the relevant activation and detoxification pathways of 17-EE (O'Brien and Siraki 2005). It was found that cytochrome P450, especially the CYP1A isozyme, was responsible for oxidizing 17-EE to form a toxic metabolite. Extrahepatic enzymes such as tyrosinase and peroxidase also generate an increase in oxidized metabolites. The toxic metabolite was suggested to be a quinoid moiety, as cytotoxicity was decreased by o-quinone traps. Detoxification of the 17-EE quinoid metabolite was performed by reduction, glucuronidation, conjugation by glutathione, methylation, and sulfation, in decreasing order of importance.
30 min prior to the addition of other agents. Glutathione (GSH)depleted hepatocytes were obtained by preincubating the hepatocytes with 200 mol/L 1-bromoheptane for 30 min (Khan and O'Brien 1991). Bromoheptane rapidly conjugates hepatocyte GSH catalysed by GSH transferase without affecting hepatocyte viability (even at a 10-fold higher dose). Glucuronide-inhibited hepatocytes were obtained by incubating cells with 500 mol/L borneol for 15 min prior to the addition of other agents (Gregus et al. 1983; Watkins and Klaassen 1983). The inflammatory model of horseradish peroxidase (HRP) (0.5 mol/L) and H2O2-generating system (10 mmol/L glucose; 0.5 U/mL glucose oxidase) were nontoxic to hepatocytes. Peroxidase was preincubated with hepatocytes for 15 min prior to the addition of other agents. Peroxidase activity was inhibited by 50 mol/L 6-propyl-2-thiouracil (PTU) (Lee et al. 1990) by incubating them with hepatocytes for 15 min along with HRP, prior to the start of the experiment. The concentrations of inhibitors/modulators used did not affect hepatocyte viability.
Materials and methods
Mitochondrial membrane potential (MMP) assay Uptake of the cationic fluorescent dye rhodamine 123 has been used for the estimation of mitochondrial membrane potential, as mentioned by Andersson et al. (1987). Aliquots (500 L) of the cell suspension were separated from the incubation medium by centrifugation at 5000g for 1 min. The cell pellet was resuspended in 2 mL of fresh incubation medium containing 1.5 mol/L rhodamine 123, and incubated at 37 °C in a thermostatic bath for 10 min with gentle shaking. Hepatocytes were then separated by centrifugation and the amount of rhodamine 123 remaining in the incubation medium was measured using a SpectraMax Gemini XS fluorometer set at 490 nm excitation and 520 nm emission wavelengths. The capacity of mitochondria to take up the rhodamine 123 was calculated as the difference in fluorescence intensity between control and treated cells (Andersson et al. 1987).
Chemicals Type II collagenase was purchased from Worthington Biochemical Corporation (Lakewood, New Jersey, USA). N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid (HEPES) was purchased from Boehringer– Mannheim (Montreal, Quebec, Canada). 17-Ethinylestradiol and all other chemicals were obtained from Sigma–Aldrich (Oakville, Ontario, Canada). Animal treatment and hepatocyte preparation Male Sprague–Dawley rats weighing 275–300 g (Charles River Laboratories) were used for experiments, which were carried out in accordance with the Guide to the Care and Use of Experimental Animals (CCAC 1993). Hepatocytes were isolated from rats by collagenase perfusion of the liver, as described by Moldeus et al. (1978). Isolated hepatocytes (106 cells/mL, 10 mL) were suspended in Krebs–Henseleit buffer (pH 7.4) containing 12.5 mmol/L HEPES in continually rotating 50 mL round-bottomed flasks, under an atmosphere of 95% O2 and 5% CO2, in a water bath of 37 °C for 30 min prior to the addition of chemicals. Cell viability Hepatocyte viability was assessed microscopically by plasma membrane disruption as determined by the trypan blue (0.1% w/v) exclusion test (Moldeus et al. 1978). Hepatocyte viability was determined every 30 min during a 3 h incubation period, and they were 80%–90% viable before use. Preparation of metabolizing enzyme-inhibited hepatocytes CYP-inhibited hepatocytes were prepared by adding the nonspecific suicide inhibitor of CYP450s, 1-aminobenzotriazole (100 mol/L, Balani et al. 2002) to hepatocytes 1 h prior to the addition of other agents. All other CYP modulators were preincubated for
Reactive oxygen species (ROS) formation Hepatocyte ROS generation was determined through the use of dichloro-dihydro-fluorescein diacetate (DCFH-DA). DCFH-DA permeated the hepatocytes and was deacetylated by intracellular esterases to form nonfluorescent dichlorofluorescin. Dichlorofluorescin reacts with ROS to form the highly fluorescent dichlorofluorescein that effluxes the cell once the plasma membrane is permeabilized (LeBel et al. 2002). Aliquots (1 mL) were taken from the hepatocyte suspension at 90 min after incubation with 17-EE and modulators, as described. These samples were then centrifuged for 1 min at 5000g. The pellet was resuspended in 1 mL of Krebs–Henseleit medium containing 1.6 mol/L DCFH-DA, and incubated at 37 °C for 10 min. The fluorescent intensity of dichlorofluorescein was measured using a SPECTRAmax Gemini XS spectrofluorometer set at 490 nm excitation and 520 nm emission wavelengths (Shangari et al. 2006).
H2O2 measurements H2O2 was measured in hepatocytes by taking samples at 90 and 180 min and adding FOX 1 reagent (ferrous oxidation of xylenol orange). The FOX 1 reagent consisted of 25 mmol/L sulfuric acid, 250 mol/L ferrous ammonium sulfate, 100 mol/L xylenol orange, and 100 mmol/L sorbitol. At the above time points, 50 L of the hepatocyte suspension (106 cells/mL) were added to 950 L FOX 1 reagent and incubated for 30 min at room temperature. Samples were spectrophotometrically analyzed at 560 nm using a SPECTRAmax Plus 384 spectrophotometer. The extinction coefficient 2.35 × 105 mol−1·cm−1 was used to quantify the molar concentration of H2O2 through the Beer–Lambert law (Khan and O'Brien 1991). Statistical analyses Data from 3 independent experiments were analyzed and are expressed as the mean ± SE. Statistical analyses were performed Published by NRC Research Press
Wan and O'Brien
using a one-way ANOVA and Tukey's post-hoc test to assess significance. Results for P < 0.05 were considered statistically significant.
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Results Implication for metabolism of 17-EE to a quinoid metabolite The LC50 of 17-EE at 2 h using isolated rat hepatocytes was determined to be 150 ± 8 mol/L. It was the concentration that increased the cytotoxicity by half between the starting viability and 100% hepatocyte death. 17-EE exhibited its cytotoxic effect quickly during the first 120 min (Fig. 1). 17-EE also decreased hepatocyte endogenous ROS and H2O2 levels (Table 1) as well as mitochondrial membrane potential (Fig. 1). Cytotoxicity was significantly decreased to approximately control hepatocyte levels with 100 mol/L 1-aminobenzotriazole (ABT), a nonspecific P450 irreversible inhibitor. Quinoline, a CYP1A inhibitor, at 50 mol/L decreased cytotoxicity to approximately control levels. However, 25 mol/L ketoconazole, a CYP3A inhibitor, increased cytotoxicity. Tyrosinase (10 units/mL) and peroxidase–H2O2, both of which can oxidize phenol and form o-quinones, increased 17-EE induced cytotoxicity accompanied by a small increase in endogenous H2O2 and ROS. Inhibition of peroxidase with 50 mol/L 6-n-propylthiouracil (PTU) partially rescued hepatocytes from cytotoxicity caused by peroxidase-metabolized 17-EE. The addition of ethylenediamine (2 mmol/L) or o-phenylenediamine (100 mol/L), which are o-quinone traps (Jellinck and Irwin 1963; Bolt and Kappus 1974), prevented cytotoxicity. Ethylenediamine decreased cytotoxicity, even in the presence of peroxidase–H2O2. Furthermore, preincubation of hepatocytes with 200 mol/L 1-bromoheptane to deplete GSH markedly increased cell death across all time points. GSH-depleted hepatocytes also significantly increased endogenous H2O2 levels in the hepatocytes. Detoxification of 17-EE metabolite by a phase II drug metabolizing enzyme The addition of 20 mol/L dicoumarol, a NAD(P)H:quinone oxidoreductase (NQO) inhibitor, greatly increased 17-EE cytotoxicity with no effect on ROS, MMP, or H2O2 content (Table 2). Inhibition of hepatocyte sulfation with 25 mol/L 2,6-dichloro-4-nitrophenol (DNCP) increased 17-EE cytotoxicity but it did not affect ROS, MMP, or H2O2 content. Simultaneous treatment of 17-EE with 700 mol/L borneol and 100 mol/L dopamine, competitive inhibitors for UDPglucuronosyltransferase (UGT) and catechol-O-methyl transferase (COMT), increased cytotoxicity and significantly decreased the MMP compared with the control hepatocytes, indicating mitochondrial toxicity. Inhibitors of phase 2 drug metabolism, therefore, markedly increased 17-EE induced cytotoxicity at 2 h, suggesting that reduction, glucuronidation, conjugation by glutathione, sulfation, and catechol-O-methylation were involved in the detoxification of 17-EE, in decreasing order of importance. Modulation of 17-EE-induced cytotoxicity by pro-oxidants and antioxidants As shown in Table 3, 17-EE cytotoxicity was increased by the pro-oxidants Cu2+ and Fe2+–HQ. Cu2+ and Fe2+–HQ both significantly increased hepatocyte H2O2 content. Antioxidants such as 1 mmol/L Trolox and 100 mol/L butylated hydroxy toluene (BHT) inhibited 17-EE induced cytotoxicity and decreased H2O2 and ROS levels. Addition of 200 units/mL catalase to the hepatocytes also decreased 17-EE cytotoxicity as well as ROS and H2O2, and this suggests that catalase removes extracellular H2O2 by decreasing the intracellular–extracellular H2O2 equilibrium.
Discussion In this study, 17-EE demonstrated strong ROS scavenging activity similar to other estrogens (Sugioka et al. 1987; Ayres et al. 1998). As shown in Fig. 1, 17-EE cytotoxicity increased rapidly between
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Fig. 1. (A) Concentration and time dependence of 17-EE cytotoxicity towards isolated rat hepatocytes. (B) Mitochondrial membrane potential of 17-EE at 30 and 90 min. 17-EE, 17␣-Ethinylestradiol. The mean ± SE for 3 separate experiments are presented; *, P < 0.05 compared with the control hepatocytes.
100 and 200 mol/L. At 100 mol/L, 17-EE exhibited very little cytotoxic effect at any of the time points, but with 200 mol/L the cytotoxicity increased to almost 100% after 2 h. This suggested that there is a critical threshold before 17-EE cytotoxicity can be observed. This study investigated the involvement of several important drug metabolizing enzymes in the toxicological profile of 17-EE. Using ACMS techniques, it was determined which inhibitors of drug metabolizing enzymes had the greatest effect on 17-EE cytotoxicity. The oxidative metabolism of 17-EE catalyzed by CYPs to hydroxy 17-EE has been well characterized, but none of the metabolites have yet been directly associated with cytotoxicity (Jefcoate et al. 2000). 17-EE cytotoxicity was decreased by nonspecific CYP inhibition, suggesting that a 17-EE oxidative metabolite was responsible for the cytotoxicity. Using quinoline as a competitive inhibitor of CYP1A, it was found that 17-EE cytotoxicity was decreased to a level identical to that of nonspecific CYP inhibition. This demonstrated that oxidation by CYP1A was the major pathway leading to 17-EE cytotoxicity. Inhibition of CYP3A by ketoconazole showed an increase in cytotoxicity, which suggested that oxidation by CYP3A acted as a competitive pathway for 17-EE that did not lead to cytotoxicity (Fig. 2). Previous literature has shown that CYP3A oxidized estrogens at the 4-C position of estradiol more likely than CYP1A, which mainly oxidized at the 2-C position (Lee et al. 2003). As shown in Table 1, addition of the o-quinone traps ethylenediamine and o-phenylenediamine decreased 17-EE cytotoxicity and suggested the formation of a 17-EE o-quinone metabolite. 17-EE o-quinones could also be conjugated and detoxified with Published by NRC Research Press
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Table 1. Modulating 17-EE-induced cytotoxicity and oxidative stress by using phase I and quinone metabolism enzyme inhibitors in isolated rat hepatocytes.
Cytotoxicity (% trypan blue uptake)
ROS
Mitochondrial membrane potential (%)
H2O2 (nmol/106 cells)
Treatment (compound added)
60 min
120 min
180 min
90 min
90 min
90 min
Control hepatocytes + HRP+H2O2-generating system 125 mol/L 17-EE + 100 mol/L ABT + 50 mol/L quinoline + 25 mol/L ketoconazole + 10 units/mL tyrosinase + HRP+H2O2-generating system + 50 mol/L propylthiouracil + 2 mmol/L ethylenediamine 150 mol/L 17-EE + 2 mmol/L ethylenediamine + 100 mol/L o-PD + 200 mol/L 1-bromoheptane
20±1 23±2 37±3a 20±2b 23±2b 36±3a 44±3a 46±4a 40±3a 43±4a 49±4a,b 40±2a 44±4a 91±6a,d
22±1 25±3 40±3a 23±1b 25±3b 52±5a 49±4a 88±7a,b 58±5a,c 67±5a,c 56±5a,b 42±2a,d 46±6a,d 97±3a,d
26±2 27±1 43±2a 30±2b 29±2b 63±5a,b 60±6a,b 97±3a,b 79±6a,c 84±8a,c 67±5a,b 45±4a,d 52±7a,d 100a,d
89±4 Interference* 35±6a 32±4a 31±2a 32±5a 40±3a Interference* Interference* Interference* 28±3a 30±5a 32±7a 63±4a,d
100 96±4 91±3a 93±2a 94±5 90±3a 92±3 95±4 97±3 96±3 89±4a 95±2 93±3 93±3
4.5±0.3 7.8±0.4a 4.2±0.1 4.4±0.3 4.1±0.2 5.0±0.4 5.2±0.3 11.3±1.1a,b 5.7±0.6a,c 6.0±1.0b,c 4.9±0.2 4.1±0.3 4.6±0.3 7.4±0.5a,d
Note: All modulating chemicals were nontoxic concentrations and did not have any significant effect on cytotoxicity when tested alone. ROS, reactive oxygen species in relative units; 17-EE, 17␣-ethinylestradiol; ABT, 1- aminobenzotriazole; o-PD, o-phenylenediamine. Data are the mean ± SE for 3 separate experiments; *, due to the oxidation of 2=7=-dichlorofluorescin by peroxidase; a, P < 0.05 compared with the control hepatocytes; b, P < 0.05 compared with 125 mol/L 17-EE; c, P < 0.05 compared with 125 mol/L 17-EE + HRP/H2O2-generating system; d, P < 0.05 compared with 150 mol/L 17-EE.
Table 2. Modulating 17-EE-induced cytotoxicity and oxidative stress by using Phase II metabolism enzyme inhibitors in isolated rat hepatocytes.
Cytotoxicity (% trypan blue uptake)
ROS
Mitochondrial membrane potential (%)
Treatment (compound added)
60 min
120 min
180 min
90 min
90 min
90 min
Control hepatocytes 75 mol/L 17-EE + 700 mol/L borneol + 20 mol/L dicoumarol + 25 mol/L DCNP + 200 mol/L 1-bromoheptane + 100 mol/L dopamine
20±1 23±2 52±4a,b 44±5a,b 38±3a,b 44±3a,b 39±3a,b
22±1 26±3 58±4a,b 84±7a,b 41±4a,b 51±3a,b 47±3a,b
26±2 30±3 62±6a,b 98±2a,b 47±4a,b 59±2a,b 51±2a,b
91±4 55±4a 62±4a 52±4a 58±4a 60±4a 58±4a
100 92±4 87±3a 94±3 90±5a 96±2 89±3a
4.7±0.2 3.1±0.2a 2.8±0.3a 3.3±0.4a 3.5±0.3a 4.2±0.5b 3.4±0.2a
H2O2 (nmol/106 cells)
Note: All modulating chemicals were nontoxic concentrations and did not have any significant effect on cytotoxicity when tested alone. ROS, reactive oxygen species in relative units; 17-EE, 17␣-ethinylestradiol; DCNP, 2,6-dichloro-4-nitrophenol. Data are the mean ± SE for 3 separate experiments; a, P < 0.05 compared with the control hepatocytes; b, P < 0.05 compared with 75 mol/L 17-EE.
Table 3. Modulating 17-EE-induced cytotoxicity and oxidative stress using antioxidant and pro-oxidant conditions.
Cytotoxicity (% trypan blue uptake)
ROS
Mitochondrial membrane potential (%)
H2O2 (nmol/106 cells)
Treatment (compound added)
60 min
120 min
180 min
90 min
90 min
90 min
Control hepatocytes 125 mol/L 17-EE + 10 mol/L CuSO4 + H2O2-generating system + 25 mol/L CuSO4 + 2 mol/L FeSO4/4 mol/L HQ + 1 mmol/L Trolox + 100 mol/L BHT + 200 units/mL catalase
20±1 36±3a 41±4a 80±6a 94±2a,b 38±3a 26±2a,b 28±4a,b 27±3a,b
22±1 43±3a 46±3a 87±4a 98±2a,b 46±5a 34±2a,b 30±2a,b 33±3a,b
26±2 49±5a 55±3a 96±4a 100a,b 54±4a 36±4a,b 33±5b 36±2a,b
88±2 33±5a 37±3a 47±6a,b 42±2a,b 46±3a,b 27±9a 31±5a 30±4a
100 92±3 90±2a 84±5a 86±4a 87±3a 93±4 90±5a 94±2
4.4±0.2 3.9±0.1 4.5±0.3 6.0±0.5a,b 5.1±0.4a,b 4.8±0.3b 3.2±0.4a 3.5±0.2a 3.4±0.2a
Note: All modulating chemicals were nontoxic concentrations and did not have any significant effect on cytotoxicity when tested alone. ROS, reactive oxygen species in relative units; 17-EE, 17␣-ethinylestradiol; HQ, 8-hydroxyquinoline. Data are the mean ± SE for 3 separate experiments; a, P < 0.05 compared with the control hepatocytes; b, P < 0.05 compared with 125 mol/L 17-EE.
GSH, catalyzed by GSH transferase, which explains the increase in toxicity after the depletion of cellular GSH by 1-bromoheptane. The addition of tyrosinase and peroxidase/H2O2 also increased toxicity and provided evidence for the cytotoxicity of 17-EE oxidative metabolites. Peroxidase–H2O2 greatly increased the potency of 17-EE cytotoxicity as 125 mol/L 17-EE in the presence of
peroxidase–H2O2 showed a similar cytotoxicity to 200 mol/L 17-EE alone. Tyrosinase is an oxidase that catalyzes both the hydroxylation of phenolic groups to catechols and the conversion of catechols to o-quinones, and has been shown to mediate 17-EE binding to cellular protein (Bolt and Kappus 1974). On the other hand, peroxidase–H2O2 can form phenoxyl radicals and perform Published by NRC Research Press
Wan and O'Brien
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Fig. 2. Proposed activation and detoxification pathways of 17␣-ethinylestradiol in isolated rat hepatocytes.
the 2-step oxidation of catechol estrogens to semiquinone radicals and o-quinones (Sipe et al. 1994; Cavalieri et al. 2006). The major detoxification pathway of the 17-EE o-quinone metabolite was found to result from reduction catalyzed by the quinone reductase NQO. Inhibition of hepatocyte NQO by dicoumarol was found to be the most effective drug metabolizing inhibitor that increased 17-EE cytotoxicity. This also suggests that NQO detoxifies o-quinones in the molecular cytotoxic mechanism of 17-EE (Table 2). The second largest increase in cytotoxicity was observed with borneol, an inhibitor of UGT2B that inhibits the glucuronidation of 17-EE and hydroxyestradiols. DCNP, a sulfotransferase (SULT) inhibitor, was the least effective at increasing 17-EE cytotoxicity. Specific COMT inhibitors were not used, but dopamine acting as a competing substrate also increased cytotoxicity. All 3 conjugation pathways have been described but it is not yet clear until now whether they are involved in 17-EE detoxification or activation (Raxworthy and Gulliver 1982; Ebner et al. 1993; Schrag et al. 2004). Even though 17-EE is a ROS scavenger (Ruiz-Larrea et al. 2000), the cytotoxic mechanism of 17-EE likely implied oxidative stress caused by Cu2+- or Fe2+-catalyzed auto-oxidation of hydroxy 17-EE metabolites to form o-quinones and increase hepatocyte H2O2 levels (Table 3). Pro-oxidant conditions therefore increased 17-EE cytotoxicity, while antioxidants decreased cytotoxicity. Cu2+ also has previously been shown to generate free radicals from estrogen catechols, thereby supporting the findings of this study (Seacat et al. 1997). Oxidative stress as the cytotoxic mechanism could explain the presence of a critical 17-EE concentration that was required to observe significant cell death. As an estrogen analogue, 17-EE was able to scavenge ROS at low concentrations, but at higher concentrations, its metabolism to an o-quinone would undergo redox cycling and could overwhelm the antioxidant cellular defenses. The hepatocytes were equipped to handle oxidative stress up to a specific threshold, at which point, once the protective mechanisms were exhausted, toxicity commenced. The prevention of 17-EE cytotoxicity by the antioxidants BHT or Trolox (a vitamin E analogue) was a strong piece of evidence for oxidative stress induced cytotoxicity. Vitamin E has also been used to pre-
vent cell death caused by 2-methoxyestradiol-induced hydrogen peroxide production (Mergny et al. 2003). It has been shown that 17-EE hepatocyte mitochondrial toxicity involved the collapse of the membrane potential. It was likely to have occurred before cytotoxicity ensued and contributed to the 17-EE molecular cytotoxic mechanism. Other studies have shown that 17-estradiol or 2-methoxyestradiol also decreased the cellular mitochondrial membrane potential causing cytochrome c release, caspase 9 activation, and apoptotic cell death (Mergny et al. 2003; Mishra and Shaha 2005). Even though previous studies used spermatogenic and sarcoma cells, the mitochondrial toxicity of estrogens can be translated to hepatocytes as well. In conclusion, these results suggest that the cytotoxicity of 17-EE manifests through the formation of an o-quinone metabolite, which is a product of 17-EE oxidation catalyzed by either CYP1A or tyrosinase or peroxidase–H2O2 and may be a mitochondrial toxin. The mechanism of cytotoxicity was also found to be related to oxidative stress and mitochondrial dysfunction. Enzymes catalyzing reduction, glucuronidation, conjugation by glutathione, methylation, and sulfation all played a role in the detoxification of 17-EE, likely in this order of effectiveness. Conflict of interest The authors declare that there are no conflicts of interest associated with this study.
Acknowledgements This research was funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.
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ARTICLE Molecular mechanism of 17␣-ethinylestradiol cytotoxicity in isolated rat hepatocytes Luke Wan and Peter O'Brien
Abstract: 17␣-Ethinylestradiol (17-EE) is used in formulations of contraceptives and hormone replacement therapy because it is an estradiol derivative. However, it has been associated with an increase in the risk of liver cancers and injury. The carcinogenic properties of 17-EE are similar to that of other estrogens, but the molecular mechanism of liver injury is still unclear. It is important to identify any secondary toxic mechanisms that can be used to prevent or treat the toxicity. The LC50 of 17-EE toward isolated rat hepatocytes was determined to be 150 ± 8 mol/L. Accelerated cytotoxicity mechanism screening (ACMS) techniques using isolated rat hepatocytes showed that CYP1A inhibitors decreased cytotoxicity, whereas tyrosinase increased toxicity; this suggests that the toxic mechanism involved is the oxidation of 17-EE. A hepatocyte inflammation model also increased 17-EEinduced mitochondrial toxicity, as well as the formation of ROS and H2O2. Cytotoxicity was increased when inhibitors of quinone reduction, catechol-O-methylation, glucuronidation, glutathione conjugation, and sulfation were co-incubated with 17-EE. The hepatocytes could be rescued with antioxidants and quinone trapping agents, thereby suggesting a role for quinoid moiety induced oxidative stress in 17-EE induced cytotoxicity. These mechanisms for 17-EE hepatotoxicity could provide a new perspective for the treating 17-EE-induced liver injury. Key words: 17␣-ethinylestradiol (17-EE), hepatotoxicity, quinoid moieties, molecular mechanism, drug metabolism, oxidative stress, reactive oxygen species (ROS). Résumé : Le 17␣-éthinylestradion (17-EE) est utilisé dans la préparation des contraceptifs et dans la thérapie hormonale de remplacement car il est un dérivé de l'estradiol. Toutefois, il a été associé a` un accroissement du risque de développer une maladie ou un cancer du foie. Les propriétés cancérigènes du 17-EE sont similaires a` celles d'autres œstrogènes, mais les mécanismes moléculaires impliqués dans le dommage hépatique sont encore mal connus. Il est important d'identifier tout mécanisme secondaire toxique qui peut être utilisé pour traiter ou prévenir la toxicité. La CL50 du 17-EE envers les hépatocytes de rats isolés a été déterminée a` 150 ± 8 mol/L. Un criblage ACMS (accelerated cytotoxicity mechanisme screening) utilisé sur les hépatocytes de rat isolés a montré que les inhibiteurs de CYP1A diminuaient la cytotoxicité alors que la tyrosinase l'accroissait; cela suggère que le mécanisme de toxicité impliquait l'oxydation du 17-EE. Un modèle d'inflammation hépatique accroissait aussi la toxicité mitochondriale induite par le 17-EE ainsi que la formation de ERO et de H2O2. La cytotoxicité était accrue lorsque des inhibiteurs de la réduction de la quinone, de catéchol-O-méthylation, de glucuronidation, de conjugaison du glutathion et de sulfation étaient incubés conjointement au 17-EE. Les hépatocytes pouvaient être rescapés par des antioxydants et des piégeurs de quinone, suggérant ainsi que le stress oxydant induit par le groupe quinoïde joue un rôle dans la cytotoxicité induite par le 17-EE. Ces mécanismes d'hépatotoxicité du 17-EE pourraient ouvrir une nouvelle perspective de traitement des dommages hépatiques induits par le 17-EE. [Traduit par la Rédaction] Mots-clés : 17␣-éthinylestradion (17-EE), hépatotoxicité, groupe quinoïde, mécanisme moléculaire, métabolisme des médicaments, stress oxydant, espèce réactive d'oxygène.
Introduction 17␣-ethinylestradiol (17-EE) is used in contraceptives and hormone replacement therapies, but can increase the risk of cancers, including liver cancer (Palmer et al. 1989). Previous studies have shown that 17-EE is a strong tumor promoter with an ability to stimulate liver DNA synthesis (Yager et al. 1994). It has also been found to increase reactive oxygen species (ROS) such as superoxide radicals, which cause chromosomal aberrations and other genotoxic damage (Chen et al. 1999). The genotoxic mechanism of 17-EE has been proposed to involve the formation of o-quinones that can bind to DNA and undergo redox cycling to generate superoxide radicals (Siddique et al. 2005). This is similar to findings using other estrogens that can be oxidized to catechol estrogen quinones thereby increasing the risk of breast and uterine cancers
(Cavalieri et al. 1997; Cavalieri et al. 2006; Bolton and Thatcher 2008). 17-EE has also been shown to cause intrahepatic cholestasis in experimental animals, which can lead to hepatotoxicity (Crocenzi et al. 2001). The exact mechanism for cholestasis is still unclear, but evidence suggests that it is multifactoral in nature, possibly involving a 17-EE glucuronide metabolite or activation of the estrogen receptor signaling pathways (Stieger et al. 2000; Sánchez Pozzi et al. 2003; Yamamoto et al. 2006). There is a build-up of bile salts in hepatocytes during cholestasis, and the bile salts are harmful to hepatocytes (i) owing to their ability to increase oxidative stress, and (ii) their detergent activity, which can disrupt the plasma membrane (Attili et al. 1986; Sokol et al. 1993; Yerushalmi et al. 2001). Cholestatic hepatotoxicity is often associated with hepatic inflammation, which could suggest a necrotic cell-mediated immune
Received 24 July 2013. Accepted 7 October 2013. L. Wan. Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada. P. O'Brien. Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, ON M5S 3M2, Canada. Corresponding author: Peter O'Brien (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 92: 21–26 (2014) dx.doi.org/10.1139/cjpp-2013-0267
Published at www.nrcresearchpress.com/cjpp on 9 October 2013.
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Can. J. Physiol. Pharmacol. Vol. 92, 2014
response (Jaeschke et al. 2002; Kaplowitz 2005). An overactive immune reaction could lead to an increased level of oxidizers such as myeloperoxidases and hypochlorite in the liver, and this in turn could increase the formation of 17-EE oxidative metabolites such as the o-quinones (O'Brien 2000). We have recently developed a hepatocyte inflammation model in which hepatocytes were exposed to a nontoxic peroxidase–H2O2 treatment to mimic an inflammatory response. Activated inflammatory cells release peroxidase and H2O2, formed by NADPH oxidase, and together they can disrupt the normal metabolism of endogenous and exogenous compounds (Babior 2000; Tafazoli and O'Brien 2005). It is known that 17-EE causes hepatotoxicity through cholestasis, but that cannot explain all of the different hepatic toxicological symptoms associated with 17-EE. Some researchers have observed that 17-EE inhibits rat liver growth, a possible indicator of cytotoxicity (Yager et al. 1994). The exact mechanism of the cytotoxicity is unclear, but could be related to redox-cycling-induced oxidative stress. Therefore, it is important to identify any secondary cytotoxic mechanisms and detoxification pathways such that they can be the targets for prevention and treatment. One objective of our research was to determine the molecular basis of 17-EE cytotoxicity associated with inflammation. In this study, metabolizing enzyme modulators were used to determine the relevant activation and detoxification pathways of 17-EE (O'Brien and Siraki 2005). It was found that cytochrome P450, especially the CYP1A isozyme, was responsible for oxidizing 17-EE to form a toxic metabolite. Extrahepatic enzymes such as tyrosinase and peroxidase also generate an increase in oxidized metabolites. The toxic metabolite was suggested to be a quinoid moiety, as cytotoxicity was decreased by o-quinone traps. Detoxification of the 17-EE quinoid metabolite was performed by reduction, glucuronidation, conjugation by glutathione, methylation, and sulfation, in decreasing order of importance.
30 min prior to the addition of other agents. Glutathione (GSH)depleted hepatocytes were obtained by preincubating the hepatocytes with 200 mol/L 1-bromoheptane for 30 min (Khan and O'Brien 1991). Bromoheptane rapidly conjugates hepatocyte GSH catalysed by GSH transferase without affecting hepatocyte viability (even at a 10-fold higher dose). Glucuronide-inhibited hepatocytes were obtained by incubating cells with 500 mol/L borneol for 15 min prior to the addition of other agents (Gregus et al. 1983; Watkins and Klaassen 1983). The inflammatory model of horseradish peroxidase (HRP) (0.5 mol/L) and H2O2-generating system (10 mmol/L glucose; 0.5 U/mL glucose oxidase) were nontoxic to hepatocytes. Peroxidase was preincubated with hepatocytes for 15 min prior to the addition of other agents. Peroxidase activity was inhibited by 50 mol/L 6-propyl-2-thiouracil (PTU) (Lee et al. 1990) by incubating them with hepatocytes for 15 min along with HRP, prior to the start of the experiment. The concentrations of inhibitors/modulators used did not affect hepatocyte viability.
Materials and methods
Mitochondrial membrane potential (MMP) assay Uptake of the cationic fluorescent dye rhodamine 123 has been used for the estimation of mitochondrial membrane potential, as mentioned by Andersson et al. (1987). Aliquots (500 L) of the cell suspension were separated from the incubation medium by centrifugation at 5000g for 1 min. The cell pellet was resuspended in 2 mL of fresh incubation medium containing 1.5 mol/L rhodamine 123, and incubated at 37 °C in a thermostatic bath for 10 min with gentle shaking. Hepatocytes were then separated by centrifugation and the amount of rhodamine 123 remaining in the incubation medium was measured using a SpectraMax Gemini XS fluorometer set at 490 nm excitation and 520 nm emission wavelengths. The capacity of mitochondria to take up the rhodamine 123 was calculated as the difference in fluorescence intensity between control and treated cells (Andersson et al. 1987).
Chemicals Type II collagenase was purchased from Worthington Biochemical Corporation (Lakewood, New Jersey, USA). N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid (HEPES) was purchased from Boehringer– Mannheim (Montreal, Quebec, Canada). 17-Ethinylestradiol and all other chemicals were obtained from Sigma–Aldrich (Oakville, Ontario, Canada). Animal treatment and hepatocyte preparation Male Sprague–Dawley rats weighing 275–300 g (Charles River Laboratories) were used for experiments, which were carried out in accordance with the Guide to the Care and Use of Experimental Animals (CCAC 1993). Hepatocytes were isolated from rats by collagenase perfusion of the liver, as described by Moldeus et al. (1978). Isolated hepatocytes (106 cells/mL, 10 mL) were suspended in Krebs–Henseleit buffer (pH 7.4) containing 12.5 mmol/L HEPES in continually rotating 50 mL round-bottomed flasks, under an atmosphere of 95% O2 and 5% CO2, in a water bath of 37 °C for 30 min prior to the addition of chemicals. Cell viability Hepatocyte viability was assessed microscopically by plasma membrane disruption as determined by the trypan blue (0.1% w/v) exclusion test (Moldeus et al. 1978). Hepatocyte viability was determined every 30 min during a 3 h incubation period, and they were 80%–90% viable before use. Preparation of metabolizing enzyme-inhibited hepatocytes CYP-inhibited hepatocytes were prepared by adding the nonspecific suicide inhibitor of CYP450s, 1-aminobenzotriazole (100 mol/L, Balani et al. 2002) to hepatocytes 1 h prior to the addition of other agents. All other CYP modulators were preincubated for
Reactive oxygen species (ROS) formation Hepatocyte ROS generation was determined through the use of dichloro-dihydro-fluorescein diacetate (DCFH-DA). DCFH-DA permeated the hepatocytes and was deacetylated by intracellular esterases to form nonfluorescent dichlorofluorescin. Dichlorofluorescin reacts with ROS to form the highly fluorescent dichlorofluorescein that effluxes the cell once the plasma membrane is permeabilized (LeBel et al. 2002). Aliquots (1 mL) were taken from the hepatocyte suspension at 90 min after incubation with 17-EE and modulators, as described. These samples were then centrifuged for 1 min at 5000g. The pellet was resuspended in 1 mL of Krebs–Henseleit medium containing 1.6 mol/L DCFH-DA, and incubated at 37 °C for 10 min. The fluorescent intensity of dichlorofluorescein was measured using a SPECTRAmax Gemini XS spectrofluorometer set at 490 nm excitation and 520 nm emission wavelengths (Shangari et al. 2006).
H2O2 measurements H2O2 was measured in hepatocytes by taking samples at 90 and 180 min and adding FOX 1 reagent (ferrous oxidation of xylenol orange). The FOX 1 reagent consisted of 25 mmol/L sulfuric acid, 250 mol/L ferrous ammonium sulfate, 100 mol/L xylenol orange, and 100 mmol/L sorbitol. At the above time points, 50 L of the hepatocyte suspension (106 cells/mL) were added to 950 L FOX 1 reagent and incubated for 30 min at room temperature. Samples were spectrophotometrically analyzed at 560 nm using a SPECTRAmax Plus 384 spectrophotometer. The extinction coefficient 2.35 × 105 mol−1·cm−1 was used to quantify the molar concentration of H2O2 through the Beer–Lambert law (Khan and O'Brien 1991). Statistical analyses Data from 3 independent experiments were analyzed and are expressed as the mean ± SE. Statistical analyses were performed Published by NRC Research Press
Wan and O'Brien
using a one-way ANOVA and Tukey's post-hoc test to assess significance. Results for P < 0.05 were considered statistically significant.
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Results Implication for metabolism of 17-EE to a quinoid metabolite The LC50 of 17-EE at 2 h using isolated rat hepatocytes was determined to be 150 ± 8 mol/L. It was the concentration that increased the cytotoxicity by half between the starting viability and 100% hepatocyte death. 17-EE exhibited its cytotoxic effect quickly during the first 120 min (Fig. 1). 17-EE also decreased hepatocyte endogenous ROS and H2O2 levels (Table 1) as well as mitochondrial membrane potential (Fig. 1). Cytotoxicity was significantly decreased to approximately control hepatocyte levels with 100 mol/L 1-aminobenzotriazole (ABT), a nonspecific P450 irreversible inhibitor. Quinoline, a CYP1A inhibitor, at 50 mol/L decreased cytotoxicity to approximately control levels. However, 25 mol/L ketoconazole, a CYP3A inhibitor, increased cytotoxicity. Tyrosinase (10 units/mL) and peroxidase–H2O2, both of which can oxidize phenol and form o-quinones, increased 17-EE induced cytotoxicity accompanied by a small increase in endogenous H2O2 and ROS. Inhibition of peroxidase with 50 mol/L 6-n-propylthiouracil (PTU) partially rescued hepatocytes from cytotoxicity caused by peroxidase-metabolized 17-EE. The addition of ethylenediamine (2 mmol/L) or o-phenylenediamine (100 mol/L), which are o-quinone traps (Jellinck and Irwin 1963; Bolt and Kappus 1974), prevented cytotoxicity. Ethylenediamine decreased cytotoxicity, even in the presence of peroxidase–H2O2. Furthermore, preincubation of hepatocytes with 200 mol/L 1-bromoheptane to deplete GSH markedly increased cell death across all time points. GSH-depleted hepatocytes also significantly increased endogenous H2O2 levels in the hepatocytes. Detoxification of 17-EE metabolite by a phase II drug metabolizing enzyme The addition of 20 mol/L dicoumarol, a NAD(P)H:quinone oxidoreductase (NQO) inhibitor, greatly increased 17-EE cytotoxicity with no effect on ROS, MMP, or H2O2 content (Table 2). Inhibition of hepatocyte sulfation with 25 mol/L 2,6-dichloro-4-nitrophenol (DNCP) increased 17-EE cytotoxicity but it did not affect ROS, MMP, or H2O2 content. Simultaneous treatment of 17-EE with 700 mol/L borneol and 100 mol/L dopamine, competitive inhibitors for UDPglucuronosyltransferase (UGT) and catechol-O-methyl transferase (COMT), increased cytotoxicity and significantly decreased the MMP compared with the control hepatocytes, indicating mitochondrial toxicity. Inhibitors of phase 2 drug metabolism, therefore, markedly increased 17-EE induced cytotoxicity at 2 h, suggesting that reduction, glucuronidation, conjugation by glutathione, sulfation, and catechol-O-methylation were involved in the detoxification of 17-EE, in decreasing order of importance. Modulation of 17-EE-induced cytotoxicity by pro-oxidants and antioxidants As shown in Table 3, 17-EE cytotoxicity was increased by the pro-oxidants Cu2+ and Fe2+–HQ. Cu2+ and Fe2+–HQ both significantly increased hepatocyte H2O2 content. Antioxidants such as 1 mmol/L Trolox and 100 mol/L butylated hydroxy toluene (BHT) inhibited 17-EE induced cytotoxicity and decreased H2O2 and ROS levels. Addition of 200 units/mL catalase to the hepatocytes also decreased 17-EE cytotoxicity as well as ROS and H2O2, and this suggests that catalase removes extracellular H2O2 by decreasing the intracellular–extracellular H2O2 equilibrium.
Discussion In this study, 17-EE demonstrated strong ROS scavenging activity similar to other estrogens (Sugioka et al. 1987; Ayres et al. 1998). As shown in Fig. 1, 17-EE cytotoxicity increased rapidly between
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Fig. 1. (A) Concentration and time dependence of 17-EE cytotoxicity towards isolated rat hepatocytes. (B) Mitochondrial membrane potential of 17-EE at 30 and 90 min. 17-EE, 17␣-Ethinylestradiol. The mean ± SE for 3 separate experiments are presented; *, P < 0.05 compared with the control hepatocytes.
100 and 200 mol/L. At 100 mol/L, 17-EE exhibited very little cytotoxic effect at any of the time points, but with 200 mol/L the cytotoxicity increased to almost 100% after 2 h. This suggested that there is a critical threshold before 17-EE cytotoxicity can be observed. This study investigated the involvement of several important drug metabolizing enzymes in the toxicological profile of 17-EE. Using ACMS techniques, it was determined which inhibitors of drug metabolizing enzymes had the greatest effect on 17-EE cytotoxicity. The oxidative metabolism of 17-EE catalyzed by CYPs to hydroxy 17-EE has been well characterized, but none of the metabolites have yet been directly associated with cytotoxicity (Jefcoate et al. 2000). 17-EE cytotoxicity was decreased by nonspecific CYP inhibition, suggesting that a 17-EE oxidative metabolite was responsible for the cytotoxicity. Using quinoline as a competitive inhibitor of CYP1A, it was found that 17-EE cytotoxicity was decreased to a level identical to that of nonspecific CYP inhibition. This demonstrated that oxidation by CYP1A was the major pathway leading to 17-EE cytotoxicity. Inhibition of CYP3A by ketoconazole showed an increase in cytotoxicity, which suggested that oxidation by CYP3A acted as a competitive pathway for 17-EE that did not lead to cytotoxicity (Fig. 2). Previous literature has shown that CYP3A oxidized estrogens at the 4-C position of estradiol more likely than CYP1A, which mainly oxidized at the 2-C position (Lee et al. 2003). As shown in Table 1, addition of the o-quinone traps ethylenediamine and o-phenylenediamine decreased 17-EE cytotoxicity and suggested the formation of a 17-EE o-quinone metabolite. 17-EE o-quinones could also be conjugated and detoxified with Published by NRC Research Press
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Can. J. Physiol. Pharmacol. Vol. 92, 2014
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Table 1. Modulating 17-EE-induced cytotoxicity and oxidative stress by using phase I and quinone metabolism enzyme inhibitors in isolated rat hepatocytes.
Cytotoxicity (% trypan blue uptake)
ROS
Mitochondrial membrane potential (%)
H2O2 (nmol/106 cells)
Treatment (compound added)
60 min
120 min
180 min
90 min
90 min
90 min
Control hepatocytes + HRP+H2O2-generating system 125 mol/L 17-EE + 100 mol/L ABT + 50 mol/L quinoline + 25 mol/L ketoconazole + 10 units/mL tyrosinase + HRP+H2O2-generating system + 50 mol/L propylthiouracil + 2 mmol/L ethylenediamine 150 mol/L 17-EE + 2 mmol/L ethylenediamine + 100 mol/L o-PD + 200 mol/L 1-bromoheptane
20±1 23±2 37±3a 20±2b 23±2b 36±3a 44±3a 46±4a 40±3a 43±4a 49±4a,b 40±2a 44±4a 91±6a,d
22±1 25±3 40±3a 23±1b 25±3b 52±5a 49±4a 88±7a,b 58±5a,c 67±5a,c 56±5a,b 42±2a,d 46±6a,d 97±3a,d
26±2 27±1 43±2a 30±2b 29±2b 63±5a,b 60±6a,b 97±3a,b 79±6a,c 84±8a,c 67±5a,b 45±4a,d 52±7a,d 100a,d
89±4 Interference* 35±6a 32±4a 31±2a 32±5a 40±3a Interference* Interference* Interference* 28±3a 30±5a 32±7a 63±4a,d
100 96±4 91±3a 93±2a 94±5 90±3a 92±3 95±4 97±3 96±3 89±4a 95±2 93±3 93±3
4.5±0.3 7.8±0.4a 4.2±0.1 4.4±0.3 4.1±0.2 5.0±0.4 5.2±0.3 11.3±1.1a,b 5.7±0.6a,c 6.0±1.0b,c 4.9±0.2 4.1±0.3 4.6±0.3 7.4±0.5a,d
Note: All modulating chemicals were nontoxic concentrations and did not have any significant effect on cytotoxicity when tested alone. ROS, reactive oxygen species in relative units; 17-EE, 17␣-ethinylestradiol; ABT, 1- aminobenzotriazole; o-PD, o-phenylenediamine. Data are the mean ± SE for 3 separate experiments; *, due to the oxidation of 2=7=-dichlorofluorescin by peroxidase; a, P < 0.05 compared with the control hepatocytes; b, P < 0.05 compared with 125 mol/L 17-EE; c, P < 0.05 compared with 125 mol/L 17-EE + HRP/H2O2-generating system; d, P < 0.05 compared with 150 mol/L 17-EE.
Table 2. Modulating 17-EE-induced cytotoxicity and oxidative stress by using Phase II metabolism enzyme inhibitors in isolated rat hepatocytes.
Cytotoxicity (% trypan blue uptake)
ROS
Mitochondrial membrane potential (%)
Treatment (compound added)
60 min
120 min
180 min
90 min
90 min
90 min
Control hepatocytes 75 mol/L 17-EE + 700 mol/L borneol + 20 mol/L dicoumarol + 25 mol/L DCNP + 200 mol/L 1-bromoheptane + 100 mol/L dopamine
20±1 23±2 52±4a,b 44±5a,b 38±3a,b 44±3a,b 39±3a,b
22±1 26±3 58±4a,b 84±7a,b 41±4a,b 51±3a,b 47±3a,b
26±2 30±3 62±6a,b 98±2a,b 47±4a,b 59±2a,b 51±2a,b
91±4 55±4a 62±4a 52±4a 58±4a 60±4a 58±4a
100 92±4 87±3a 94±3 90±5a 96±2 89±3a
4.7±0.2 3.1±0.2a 2.8±0.3a 3.3±0.4a 3.5±0.3a 4.2±0.5b 3.4±0.2a
H2O2 (nmol/106 cells)
Note: All modulating chemicals were nontoxic concentrations and did not have any significant effect on cytotoxicity when tested alone. ROS, reactive oxygen species in relative units; 17-EE, 17␣-ethinylestradiol; DCNP, 2,6-dichloro-4-nitrophenol. Data are the mean ± SE for 3 separate experiments; a, P < 0.05 compared with the control hepatocytes; b, P < 0.05 compared with 75 mol/L 17-EE.
Table 3. Modulating 17-EE-induced cytotoxicity and oxidative stress using antioxidant and pro-oxidant conditions.
Cytotoxicity (% trypan blue uptake)
ROS
Mitochondrial membrane potential (%)
H2O2 (nmol/106 cells)
Treatment (compound added)
60 min
120 min
180 min
90 min
90 min
90 min
Control hepatocytes 125 mol/L 17-EE + 10 mol/L CuSO4 + H2O2-generating system + 25 mol/L CuSO4 + 2 mol/L FeSO4/4 mol/L HQ + 1 mmol/L Trolox + 100 mol/L BHT + 200 units/mL catalase
20±1 36±3a 41±4a 80±6a 94±2a,b 38±3a 26±2a,b 28±4a,b 27±3a,b
22±1 43±3a 46±3a 87±4a 98±2a,b 46±5a 34±2a,b 30±2a,b 33±3a,b
26±2 49±5a 55±3a 96±4a 100a,b 54±4a 36±4a,b 33±5b 36±2a,b
88±2 33±5a 37±3a 47±6a,b 42±2a,b 46±3a,b 27±9a 31±5a 30±4a
100 92±3 90±2a 84±5a 86±4a 87±3a 93±4 90±5a 94±2
4.4±0.2 3.9±0.1 4.5±0.3 6.0±0.5a,b 5.1±0.4a,b 4.8±0.3b 3.2±0.4a 3.5±0.2a 3.4±0.2a
Note: All modulating chemicals were nontoxic concentrations and did not have any significant effect on cytotoxicity when tested alone. ROS, reactive oxygen species in relative units; 17-EE, 17␣-ethinylestradiol; HQ, 8-hydroxyquinoline. Data are the mean ± SE for 3 separate experiments; a, P < 0.05 compared with the control hepatocytes; b, P < 0.05 compared with 125 mol/L 17-EE.
GSH, catalyzed by GSH transferase, which explains the increase in toxicity after the depletion of cellular GSH by 1-bromoheptane. The addition of tyrosinase and peroxidase/H2O2 also increased toxicity and provided evidence for the cytotoxicity of 17-EE oxidative metabolites. Peroxidase–H2O2 greatly increased the potency of 17-EE cytotoxicity as 125 mol/L 17-EE in the presence of
peroxidase–H2O2 showed a similar cytotoxicity to 200 mol/L 17-EE alone. Tyrosinase is an oxidase that catalyzes both the hydroxylation of phenolic groups to catechols and the conversion of catechols to o-quinones, and has been shown to mediate 17-EE binding to cellular protein (Bolt and Kappus 1974). On the other hand, peroxidase–H2O2 can form phenoxyl radicals and perform Published by NRC Research Press
Wan and O'Brien
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Fig. 2. Proposed activation and detoxification pathways of 17␣-ethinylestradiol in isolated rat hepatocytes.
the 2-step oxidation of catechol estrogens to semiquinone radicals and o-quinones (Sipe et al. 1994; Cavalieri et al. 2006). The major detoxification pathway of the 17-EE o-quinone metabolite was found to result from reduction catalyzed by the quinone reductase NQO. Inhibition of hepatocyte NQO by dicoumarol was found to be the most effective drug metabolizing inhibitor that increased 17-EE cytotoxicity. This also suggests that NQO detoxifies o-quinones in the molecular cytotoxic mechanism of 17-EE (Table 2). The second largest increase in cytotoxicity was observed with borneol, an inhibitor of UGT2B that inhibits the glucuronidation of 17-EE and hydroxyestradiols. DCNP, a sulfotransferase (SULT) inhibitor, was the least effective at increasing 17-EE cytotoxicity. Specific COMT inhibitors were not used, but dopamine acting as a competing substrate also increased cytotoxicity. All 3 conjugation pathways have been described but it is not yet clear until now whether they are involved in 17-EE detoxification or activation (Raxworthy and Gulliver 1982; Ebner et al. 1993; Schrag et al. 2004). Even though 17-EE is a ROS scavenger (Ruiz-Larrea et al. 2000), the cytotoxic mechanism of 17-EE likely implied oxidative stress caused by Cu2+- or Fe2+-catalyzed auto-oxidation of hydroxy 17-EE metabolites to form o-quinones and increase hepatocyte H2O2 levels (Table 3). Pro-oxidant conditions therefore increased 17-EE cytotoxicity, while antioxidants decreased cytotoxicity. Cu2+ also has previously been shown to generate free radicals from estrogen catechols, thereby supporting the findings of this study (Seacat et al. 1997). Oxidative stress as the cytotoxic mechanism could explain the presence of a critical 17-EE concentration that was required to observe significant cell death. As an estrogen analogue, 17-EE was able to scavenge ROS at low concentrations, but at higher concentrations, its metabolism to an o-quinone would undergo redox cycling and could overwhelm the antioxidant cellular defenses. The hepatocytes were equipped to handle oxidative stress up to a specific threshold, at which point, once the protective mechanisms were exhausted, toxicity commenced. The prevention of 17-EE cytotoxicity by the antioxidants BHT or Trolox (a vitamin E analogue) was a strong piece of evidence for oxidative stress induced cytotoxicity. Vitamin E has also been used to pre-
vent cell death caused by 2-methoxyestradiol-induced hydrogen peroxide production (Mergny et al. 2003). It has been shown that 17-EE hepatocyte mitochondrial toxicity involved the collapse of the membrane potential. It was likely to have occurred before cytotoxicity ensued and contributed to the 17-EE molecular cytotoxic mechanism. Other studies have shown that 17-estradiol or 2-methoxyestradiol also decreased the cellular mitochondrial membrane potential causing cytochrome c release, caspase 9 activation, and apoptotic cell death (Mergny et al. 2003; Mishra and Shaha 2005). Even though previous studies used spermatogenic and sarcoma cells, the mitochondrial toxicity of estrogens can be translated to hepatocytes as well. In conclusion, these results suggest that the cytotoxicity of 17-EE manifests through the formation of an o-quinone metabolite, which is a product of 17-EE oxidation catalyzed by either CYP1A or tyrosinase or peroxidase–H2O2 and may be a mitochondrial toxin. The mechanism of cytotoxicity was also found to be related to oxidative stress and mitochondrial dysfunction. Enzymes catalyzing reduction, glucuronidation, conjugation by glutathione, methylation, and sulfation all played a role in the detoxification of 17-EE, likely in this order of effectiveness. Conflict of interest The authors declare that there are no conflicts of interest associated with this study.
Acknowledgements This research was funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.
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