Ibuprofen is one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) and is the main ingredients of major over-the-counter (OTC) drugs, such as Advil® and Motrin®. The number of prescription for ibuprofen is over 1.5 million in the US in 2015, with the number steadily increasing (Agency for Healthcare Research and Quality (AHRQ), 2016). Not only is ibuprofen consumed by prescription, but it is also commonly purchased over the counter. Therefore, consumption of ibuprofen is expected to be larger than the figures aforementioned. Although ibuprofen is commonly known as a “safe” OTC drug, it can cause several drug-induced adverse effects, such as liver toxicity, kidney toxicity, and stomach bleeding.
In fact, NSAIDs are one of the most notable causes of drug-induced liver injuries, with about 3 to 23 per 100,000 patient years (Aithal and Day, 2007), and as a result, three different NSAIDS, bromfenac, ibufenac, and benoxaprofen, had been removed from the UK and/or US markets because of their hepatotoxic side effects (Goldkind and Laine, 2006). These adverse effects of NSAIDs have been witnessed in children (Cardile
Meanwhile, alcohol is one most important factor which aggravates the liver toxicity of NSAIDs. It has been reported that acetaminophen, another NSAID, is contra-indicated for the patients drank alcohol due to the increased liver toxicity for the patients with alcoholism (Slattery
Our study sought to examine the impact of liver toxicity of ibuprofen when administered together with alcohol. More specifically, we will use 2D and 3D cultured hepatoma cell line, HepG2. The 3D cell cultures, because of their distinct shape, demonstrate behaviors that strongly resemble
Ethanol and ibuprofen were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of best quality available.
HepG2 cells (human hepatocellular carcinoma) were purchased from ATCC (Manassas, VA, USA). As previously described (Sooklert
HepG2 spheroids were cultivated on 96-well ultra-low-attachment plates (Corning Co., Corning, NY, USA). Briefly, 1×104 cells/well were seeded in 100 μL of media and cultured for 14 days with media changes every 2-3 days. The formation of spheroids was examined under a microscope. After culturing, ibuprofen was added to a concentration indicated from a stock solution in dimethyl sulfoxide (final concentration 0.1%) with or without ethanol, and incubation was continued for 72 h. For histology, spheroids were washed in Dulbecco’s phosphate-buffered saline (DPBS) and collected in a tube. After removing DBPS, 100 μL of 2% agarose was added, and the samples were centrifuged (2 s, 150×
WST-1 (4-[3-(iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) (Roche, Indianapolis, IN, USA) solution was used to evaluate cell viability (Joo
After the treatment, the media was removed and cells were washed twice with phosphate-buffered saline (PBS). HepG2 cells were observed and photographed under a phase-contrast optical microscope with the magnification of 200 (×200, ECLIPSE TS100, Nikon, Tokyo, Japan). For individual cell tracking, cells were culture on a microgrid slide chamber (μ-slide 8 well Grid-500, Ibidi Co., Fitchburg, WI, USA) and observed under the microscope.
Production of ROS was measured by 2′,7′-dichlorofluorescein diacetate (DCF-DA, Eugene, OR, USA)-enhanced fluorescence assay as described previously (Kim
HepG2 cells treated for 24 h were collected and washed once with PBS, and the total ribonucleic acid (RNA) was extracted with TRIzol reagent (Invitrogen, CARLSBAD, CA, USA) according to the manufacturer’s protocol. RNA precipitates were dissolved in RNase free DEPC treated water (usb, USA). The concentration of RNA was determined with NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).
Relative expression levels of mRNAs were measured by quantitative real-time PCR. Total RNA, extracted from HepG2 cells, was used to synthesize cDNA using a pre-master mix with oligo dT (Bioepis, Seoul, Korea). Each reaction was performed using Power SYBR Green PCR master mix in a StepOnePlusTM Real-time PCR machine (Applied Biosystems, Warrington, UK). The sequence of primers were as follows: forward superoxide dismutase (SOD), 5’- ATGGACCAGTGAAGGTGTGG-3’, reverse SOD 5’-GCCCACCGTGTTTTCTGGAT-3’; forward catalase (CAT), 5’-CTCCGGAACAACAGCCTTCT-3’, reverse CAT5’-ATAGAATGCCCGCACCTGAG-3’; forward glutathione peroxidase (GPX3), 5’-AGAAGTCGAAGATGGACTGCC-3’, reverse GPX3 5’-CAAAGAGGACGTATTTGCCAGC-3’; forward cytochrome P450 family 2 subfamily E member 1 (CYP2E1), 5’-TTGAAGCCTCTCGTTGACCC, reverse CYP2E1 5’-TCATGAGCGGGGAATGACAC-3’; forward glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5’-GCATCCTGGGCTACACTGAG-3’, reverse GAPDH 5’- AAGTGGTCGTTGAGGGCAAT-3’. Cycling parameters were 52°C for 2 min, 95°C for 10 min, 40 cycles of 95°C 15 s and 52°C 1 min. Semi-quantitative RT-PCR was performed using electrophoresis through a 1.5% agarose gel with eco dye (EcoDye DNA staining solution, Biofact, Daejeon, Korea).
Hepg2 cells were washed after 24-h exposure to Ibuprofen 0.8 mM and EtOH 200 mM. Then, cells were homogenized in RIPA buffer (Sigma-Aldrich) containing 1% protease inhibitor cocktail and a phosphatase inhibitor cocktail. The homogenate was centrifuged (12,000 rpm, 10 min) and the supernatant was collected. The protein concentration was measured and an aliquot (15.6 μg protein) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the fractionated proteins were then transferred to a nitrocellulose membrane. For immunoblotting, the following primary antibodies were used: rabbit anti- SOD antibody (1:300; Bioss, MA, USA), rabbit anti- Catalase (1:200; Bioss), goat anti- Glutathione Peroxidase 3/GPx-3 antibody (1:500 dilution; abcam, Cambridge, UK) and rabbit anti- Cytochrome P450 2E1 antibody (1:200 dilution; abcam) after incubation with HRP-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA), the immunoreactive bands were visualized using ECL Western blotting detection reagents (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and an LAS. Band intensity was measured using the ImageJ program (NIH). Rabbit anti- GAPDH Polyclonal Antibody (1:150 dilution; Bioss), was used as a control for immunoblotting.
Data are presented as the mean ± SE unless indicated otherwise. Difference between groups was examined using student
To evaluate whether ibuprofen can potentiate the hepatotoxicity of ethanol (EtOH), HepG2, human hepatocellular carcinoma cell system was cultured in and seeded to 96 well plates. Then, HepG2 cells were exposed to ibuprofen (0, 0.4, 0.8 and 2 mM) with or without ethanol (EtOH 200 mM or 700 mM) for 24 h and measured for cell viability using WST-1 assay.
Therapeutic blood concentration of ibuprofen is ~0.25 mM (Janssen and Venema, 1985) and EtOH reaches up to >20 mM when extremely drunken (Grant
Cells under cytotoxic conditions show damaged pattern such as floating from the culture dish or rounding by loss of attachment. We examined the morphology of HepG2 cells after exposure to ibuprofen with or without ethanol under a microscope. In accordance with the increased hepatotoxicity of combined treatment of ibuprofen and EtOH as measured with WST-1 assay, cell morphology observed under a light microscope, showed floating and rounding appearance, characteristic features of cell death, when exposed to both ibuprofen and EtOH (Fig. 1). This pattern appeared more evident when the exposed individual cells were tracked under a microscope with a microgrid slide chamber as shown in Fig. 2. While non-treated cells showed normal cell division and attachment onto the dish surface, cells treated with EtOH alone or combination of ibuprofen and EtOH showed rounding and deformation. Cells exposed to both ibuprofen and EtOH showed the severest changes.
2D cultured cells have limited value in predicting organ responses to chemicals since the cells are growing in stretched appearance over plastic culture ware. To overcome this, 3D spheroid-cultured HepG2 cells were employed. 3D spheroid has a multi-cell layered structure and exhibits natural cell shapes which is in spheroid or aggregated forms. 3D spheroids were also advantageous in examining longer time-frame of exposure since they can be cultured for a longer time than 2D culture. To examine the synergy of combined exposure to ibuprofen and EtOH in hepatotoxicity, HepG2 spheroids were exposed for 72 h. As shown in Fig. 3A, synergistic hepatotoxicity of ibuprofen and EtOH could be observed as evidenced by decreased cell viability. Notably, much lower concentrations of ibuprofen were determined to be hepatotoxic in 3D spheroids than observed in 2D culture (0.2 mM in spheroid vs 0.8 mM in 2D).
3D spheroid can be further observed for cellular morphology through histology. Spheroids were sectioned, stained with hematoxylin and eosin and observed under a light microscope. As shown in Fig. 3B, spheroids exposed to ibuprofen and EtOH showed loss of cell number and cytosol, and the accumulation of nucleus (pyknosis), indicative of necrosis and apoptosis.
To examine the mechanism underlying the synergistic hepatotoxicity of ibuprofen and EtOH, the generation of hydrogen peroxide, as an indicator of oxidative stress, was measured with a fluorescent dye, DCF-DA. As shown in Fig. 4, combined exposure to ibuprofen and EtOH resulted in the increased generation of hydrogen peroxide suggestive of potentiation of oxidative stress. Increased oxidative stress induces augmentation of antioxidant system in cells. Indeed, antioxidant systems such as glutathione peroxidase (GPX) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) were augmented as evidenced by increased mRNA expression of these enzymes in qPCR measurement and Western blot analysis (Fig. 5, 6).
Drug-induced liver toxicity is affected by several factors including the ingested amount, alcohol consumption, co-treated medications, diets, and health conditions. Accordingly, individual variation is considerable in the outcome of drug-induced liver toxicity. Frequently, chronic alcoholics are known to be susceptible to drug-induced liver toxicity (Prescott, 2000; Riordan and Williams, 2002). NSAIDs are a major culprit for drug-induced liver toxicity, consisting almost 10% of whole cases (Bessone, 2010) and showing stronger associations with liver injury than other drug classes (Garcia Rodriguez
Ibuprofen is known to be relatively safer than other NSAIDs. Despite the heavy use of ibuprofen around the world, low incidence of adverse events indicates an excellent safety. Indeed, there are no reports demonstrating the association of ibuprofen with liver diseases (Boureau
Alcohol is one of most frequent causes for liver diseases, including fatty liver, liver fibrosis and liver cancer. Mechanism underlying alcohol-induced hepatotoxicity is mostly ascribable to the generation of reactive oxygen species (ROS) during alcohol metabolism (Lieber, 1990). Oxidative stress is generated from ROS produced intrinsically and extrinsically, which ultimately disrupts cellular antioxidant capacity (Toyokuni
Indeed, ROS appears to mediate the toxicity of ibuprofen and EtOH as evidenced by increased ROS generation and expression of endogenous antioxidant systems, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). SOD is one of the most effective antioxidant enzymes, which catalyzes the transformation of O2− (superoxide anion) to O (molecular oxygen) or H2O2 (hydrogen peroxide) (Flora, 2009). CAT and GPx remove H2O2 produced by SOD using NADP+ and glutathione, respectively (Ha
Moreover, CYP2E1, which is accountable for microsomal oxidation of alcohol can metabolize xenobiotics into toxic metabolites, contributing to the synergistic hepatotoxicity of alcohol and xenobiotics like industrial solvents and prescription drugs, over-the-counter analgesics and chemical carcinogens. CYP2E1 produces a large amount of ROS during the metabolism of ethanol, which plays a pivotal role in alcohol-induced oxidative stress in liver (Bang
Collectively, this study has demonstrated that ibuprofen and EtOH can induce synergistic hepatotoxicity, providing an important line of evidence for caution against the use of ibuprofen in alcoholic patients. By introducing 3D spheroids, this study has shown that prolonged exposure to ibuprofen and EtOH at realistic condition can induce hepatotoxicity at much lower concentrations, which would be important to predict their toxicity in chronic alcoholics. Oxidative stress appeared to be key in mediating the synergistic hepatotoxicity of ibuprofen and EtOH, which suggests the utility of antioxidant dietary supplement in preventing liver toxicity from them, although the further confirmatory study is necessary.
This work was supported by the National Research Foundation of Korea (2018R1D1A1B07042919 and NRF-2020R1I1A1A01067636) and the Korea government (MSIT) (2018R1A5A2025286). Authors are grateful to Eunice Bae Lee for her support in the preparation of manuscript.
All authors declare no conflict of interest regarding this work.