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Propylparaben-induced disruption of energy metabolism in human HepG2 cell line leads to increased synthesis of superoxide anions and apoptosis
Toxicology in Vitro 31 (2016) 30–34
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Propylparaben-induced disruption of energy metabolism in human HepG2 cell line leads to increased synthesis of superoxide anions and apoptosis S. Szeląg a, A. Zabłocka a, K. Trzeciak a, A. Drozd a, I. Baranowska-Bosiacka b, A. Kolasa c, M. Goschorska b, D. Chlubek b, I. Gutowska a,⁎ a b c
Department of Biochemistry and Human Nutrition, Pomeranian Medical University, Broniewskiego 24 Str., Szczecin, Poland Department of Biochemistry, Pomeranian Medical University, Powstańców Wlkp 72 Str., Szczecin, Poland Department of Histology and Embryology, Pomeranian Medical University, Powstańców Wlkp 72 Str., Szczecin, Poland
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Article history: Received 18 July 2015 Received in revised form 12 November 2015 Accepted 19 November 2015 Available online 23 November 2015 Keywords: Apoptosis ATP HepG2 Liver Propylparaben ROS
a b s t r a c t The effect of propylparaben (in final concentrations 0.4 ng/ml, 2.3 ng/ml and 4.6 ng/ml) on the energy metabolism of HepG2 hepatocytes, superoxide anion synthesis, apoptosis and necrosis is described. Propylparaben can be toxic to liver cells due to the increased production of superoxide anions, which can contribute to a reduced concentration of superoxide dismutase in vivo and impairment of the body's antioxidant mechanisms. Finally, a further reduction in the mitochondrial membrane potential and uncoupling of the respiratory chain resulting in a reduction in ATP concentration as a result of mitochondrial damage may lead to cell death by apoptosis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Propylparaben (PP) is one of the esters of p-hydroxybenzoic acid, commonly used as a preservative in cosmetics and medicines, and also in food in some countries (Soni et al., 2005). It is one of the most commonly used parabens (PBs) in commercially available cosmetic products (Andersen, 2008; Soni et al., 2001). In the European Union, maximum permissible levels of p-hydroxybenzoic acid esters (including PP) in cosmetic formulations are 0.4% for single esters and 0.8% for several compounds used simultaneously (EC-1223/2009; SCCS/1514/13). Until recently, PP was also used in food products in the EU but it has been removed from the list of permitted food preservatives due to reports of possible adverse effects on the endocrine system. This ban does not include other PBs which are considered safe by the EU and the FDA (Błędzka et al., 2014; EC-52/2006). In other countries, for example in the USA, PP is still approved for use in foods, including baked goods, fruit juices, frozen food, spices, processed vegetables and pickled cucumbers (Soni et al., 2001).
⁎ Corresponding author at: Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24 street, 71–460 Szczecin, Poland. E-mail address: [email protected] (I. Gutowska).
http://dx.doi.org/10.1016/j.tiv.2015.11.011 0887-2333/© 2015 Elsevier Ltd. All rights reserved.
Until recently, PBs mainly owed their popularity to their physical and chemical properties, including a broad spectrum of antimicrobial activity and relatively low toxicity (Błędzka et al., 2014; Aubert et al., 2012). However, a growing number of reports have indicated the possible negative effects of chronic exposure to these compounds. Their estrogenic activity in vitro was demonstrated as early as 1998 (Routledge et al., 1998), which although markedly weaker than the natural action of estradiol, started a discussion on the safety of PB use. Moreover, studies on male rats have shown that exposure to PBs is associated with a dose-dependent decrease in the concentration of testosterone in the blood of animals and a disruption in the activity of the reproductive system (Oishi, 2002). Other negative effects of PBs have been observed in liver cells. PBs have been shown to increase the permeability of the mitochondrial membrane (Nakagawa and Moore, 1999), which leads to a swelling of these organelles, a decrease in intracellular ATP and consequently to the death of hepatocytes. Other studies have shown increased lipid peroxidation and free radical reactions in experimental animals treated with butylparaben, another preservative from the paraben group (Shah and Verma, 2011). Humans are exposed to PBs on a daily basis via ingestion, skin contact and inhalation (Andersen, 2008). The widespread use of PBs in manufacturing has led to global environmental pollution with these compounds. Their presence in the air, surface water, biota and soil (Błędzka et al., 2014) is a considerable source of human exposure to
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parabens, in addition to exposure from personal care products, food preservatives and pharmaceuticals. Parabens applied to the skin are absorbed and metabolized by esterases located in the skin (Abbas et al., 2010; Jewell et al., 2007; Ozaki et al., 2013). The same esterasebased mechanism takes place during biotransformation of the PBs delivered into the body via the oral route (Boberg et al., 2010). The liver, an organ involved in countless biochemical transformations including the metabolism of xenobiotics, is highly exposed to their harmful effects and the damage to this organ has serious consequences for the entire body. Based on the premises on the possible adverse effect of PBs on liver cells, we decided to examine the effect of one of these compounds, namely PP, on the energy metabolism of HepG2 hepatocytes, superoxide anion synthesis, and apoptosis and necrosis occurring in these cells.
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2.4. Imaging of mitochondrial ROS generation Mitochondrial superoxide level was visualized by fluorescent marker MitoSOX Red (Invitrogen). This fluorescence dye is highly selective for the detection of superoxide in living cell mitochondria. The reagent is oxidized by superoxide but not by other ROS or reactive nitrogen species, and exhibits red fluorescence (excitation 510 nm, emission 580 nm). MitoSOX Red stock solution (50 mM in DMSO) was diluted in the incubation medium (BME) to a final concentration of 2.5 μg/ml. The cells were incubated in this solution for 10 min (humidified 95% air/CO2 atmosphere at 37 °C). Next, cells were washed with BME at room temperature and were examined under the fluorescent microscope. 2.5. Measurement of denine nucleotides concentration
2. Material and methods 2.1. Reagents and sources HepG2 was obtained from LGC Standards (USA). EMEM medium, glutamine, dimethyl sulfoxide (DMSO), trypsine and antibiotics (penicillin and streptomycin) were from Sigma-Aldrich (Poznan, Poland). Fetal bovine serum (FBS) was from Gibco (Gibco, Paisley, UK). Phosphate buffered saline (PBS) was from PAP Laboratories (Vienna, Austria). Propylparaben and deionized water were from Sigma-Aldrich. MicroBCA KIT for protein measurement was from ThermoOrion. An Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from BD Pharmingen. MitoSOX™ Red was from Invitrogen (Oregon, USA). All buffer chemicals and solvents used as mobile phases for HPLC measurements were of HPLC grade and were purchased from Sigma Aldrich (St Louis,MO, USA) or Merck. Double-distilled water was obtained from a Milli-Q Water System (Millipore, Billerica, MA, USA). All buffers used for HPLC analysis were filtered through 0.22 μm nylon filters (Agilent). 2.2. Cell cultures The study was conducted on hepatocytes HepG2, which were cultured (37 °C in 5% CO2) in EMEM medium supplemented with 10% fetal bovine serum (without fatty acid), penicillin (100 U/mL), streptomycin (100 mg/mL) and propylparaben in final concentrations 0,4 ng/ml, 2,3 ng/ml and 4,6 ng/ml (Ye et al., 2008). Control cells were incubated with solvent used to make an appropriate solution of propylparaben (DMSO) to find out influence of solvent on examined processes. Propylparaben concentrations were selected on the basis of results of its concentration in human serum (Ye et al., 2008). After 48 h the cells were collected and a pellet was obtained by centrifugation (250 g for 5 min). Protein concentration was measured by using MicroBCA Protein Assay Kit (Thermo Scientific, Pierce Biotechnology, USA) and ELISA. 2.3. Fluorescent microscopy: imaging of apoptosis and necrosis processes Hepatocytes, in the number of 5 × 105 were incubated with propylparaben solutions on microscope slides according to the aforementioned procedure. After the end of cultivation, the cells were rinsed with PBS. Next, the cells were suspended in a binding buffer and stained with 1 ng/mL Annexin V-FITC and 5 ng/mL propidium iodide for 30 min in the dark. Cells that are viable are Annexin V-FITC and PI negative; cells that are in early apoptosis are Annexin V-FITC positive and PI negative; and cells that are in late apoptosis or already dead are both Annexin V-FITC and PI positive (Vermes et al., 1995). A dual-pass FITC/rhodamine filter set was applied (Olimpus FluoView SV100).
Isolation of adenine nucleotides from cells was performed according Baranowska-Bosiacka et al. (2009). The HPLC separations were performed on an Agilent Technologies 1260 liquid chromatograph, consisting of model G1379B degasser, a model G1312B bin pump, a model G1316A column oven and a model G1315CDAD VL +. Samples were injected using a model G1329B. An Agilent ChemStation software (Agilent Technologies, Cheadle, UK), were used for instrument control and data acquisition and analysis. The separation was completed on a Thermo Scientific Hypersil BDS C18 column 100 × 4.6 mm 3 μm (cat no. 28103–104630). The temperature of column oven was set at 20 °C. A dual mobile phase gradient was used to achieve appropriate separation of all analytes of interest. Mobile phase A contained 150 mM KH2PO4/K2HPO4, 150 mM KCl pH 6.0. Mobile phase B had the same final concentrations as mobile phase A, except for the addition of 15% acetonitrile (v/v). The elution was performed with a linear gradient. The flow rate was 1.0 mL/min. The sample injection volume was 20 mL. The DAD detector monitored peaks by adsorption at 254 nm. 2.6. Statistical analysis The obtained results were analyzed statistically using the software package Statistica 10 (Statsoft, Poland). Arithmetical mean and the standard deviation (SD) were found for each of the studied parameters. The distribution of results for individual variables was obtained with the Shapiro–Wilk W test. As most of the distributions deviated from the normal Gauss distribution, non-parametric tests were used for further analyses. For related samples, significance was first checked with Friedmann's analysis of variance, and significant results were subjected to the Wilcoxon matched-pair test. The level of significance was p ≤ 0.05. 3. Results 3.1. Apoptosis and necrosis Immunohistochemical analysis using Annexin V and propidium iodide was performed for the qualitative determination of apoptosis or necrosis of hepatocytes treated with PP. Only a small number of cells underwent necrosis while the majority of cells were subject to apoptosis. Comparative analysis of confocal microscope images showed that the cell death was caused by the presence of PP in the culture medium, while the intensity of the process increased together with the increase in PP level (Fig. 1A). 3.2. Superoxide anion production Application of propylparaben in a hepatocyte culture resulted in an increased production of superoxide anions by the treated cells. The images show an increase in the intensity of red fluorescence that reflects the process of O− 2 production, which rose together with PP
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Fig. 1. Imaging of A) apoptosis by Annexin V-FITC (n = 3) and B) ROS production by MitoSOX Red (n = 3) both with fluorescence microscopy of human hepatocytes (HepG2) after a 48 h incubation with increasing concentrations of propylparaben.
concentration. The most intense production of O− 2 was observed at a concentration of PP at 4.6 ng/ml (Fig. 1B). 3.3. Analysis of adenine nucleotide levels Analysis of results obtained by high performance liquid chromatography (HPLC) suggests that an increase in PP is accompanied by a decrease in cellular ATP. In cells treated with the lowest PP level, i.e. 0.4 ng/ml, the average ATP level was 28.28 nM/mg protein. This level was 11% higher than in control cells, where the average of seven replicates was 25.56 nM/mg protein. However, statistical analysis did not confirm a statistical significance compared to the control, as p N 0.05. In samples where PP level was 2.3 ng/ml, the decrease in ATP was statistically significant compared to the control and the average ATP level fell to 21.13 nM/mg protein, 17% lower than for the control. For samples with PP at 4.6 ng/ml, average cellular ATP was only 3.19 nM/mg protein, i.e. about 88% lower than in cells cultured without the addition of PP. The statistical significance of this result was observed at p ≤ 0.02 (Fig. 2A).
In contrast to ATP, other adenine nucleotides in the analyzed hepatocytes showed levels inversely dependent on the dose of PP. At the lowest PP concentration (0.4 ng/ml) ADP rose to 9.13 nM/mg, 38% higher than the control. The increase was statistically significant (p ≤ 0.02). At the next PP level, 2.3 ng/ml, average cellular ADP was 8.97 nM/mg; 55% higher than the control – also statistically significant (p ≤ 0.02). At the highest PP concentration tested (4.6 ng/ml), the concentration of ADP was 6.50 nM/mg protein, 13% higher than the concentration measured for the control. This increase, however, was not statistically significant (Fig. 2B). AMP was another adenine nucleotide analyzed in this study. At a PP level of 0.4 ng/ml the average AMP level was 3.95 nM/mg, a 17% increase relative to control and statistically insignificant (p N 0.05). At a PP level of 2.3 ng/ml, AMP reached 4.07 nM/mg protein, 20% higher than the control, again not statistically significant. In the case of cells cultured in the presence of PP at 4.6 ng/ml, the concentration of AMP was 14.03 nM/mg, a 314% increase compared to control cells. This value achieved statistical significance (p ≤ 0.05) (Fig. 2C).
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Fig. 2. Effect of increasing concentrations of propylparaben on the levels of ATP (A), ADP (B) and AMP(C) in human hepatocytes (HepG2) after 48 h of incubation. *p-Value ≤ 0.05; **p-value ≤ 0.02, significant difference vs control (n = 7).
Fig. 2A, B and C show the correlation between the concentrations of adenosine tri-, di- and monophosphates in the tested HepG2 cell line and the concentration of PP added to the cultures. 4. Discussion Nowadays, contact with preservatives from the paraben group is almost inevitable. Their ubiquitous occurrence is reflected in their detectable presence in human urine and blood (Calafat et al., 2010; Frederiksen et al., 2011; Kang et al., 2013). Therefore, it is necessary to know the exact molecular activity of PBs and determine the effects of chronic exposure to these compounds. In connection with previous reports on the negative activity of PBs on liver cells, we decided to evaluate the effect of propylparaben on the synthesis of adenine nucleotides and superoxide anion, and apoptosis and necrosis occurring in HepG2 line cells treated with this compound. Immunohistochemical evaluation of superoxide anion synthesis showed an increase dependent on PP concentration. The increased
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oxidative stress, a consequence of the enhanced production of O− 2 , may cause a number of irregularities at the molecular level, and thus may have negative consequences for the whole body (Afonso et al., 2007; Emerit et al., 2004; Granot and Kohen, 2004). The superoxide anion is able to penetrate cell membranes and is durable enough to circulate in the cellular environment and activate a cascade of free radical reactions, thus interacting with organelles distant from the site of its origin (Bartosz, 2003). On the other hand oxidative species, particularly HO(*), were the most important reactive oxygen species mediating photocatalytic degradation of PPB, and PPB degradation was found to be significantly affected by pH because it was controlled by the radical reaction mechanism. The estrogenic activity decreased as PPB was degraded, while the acute toxicity at three trophic levels first increased slowly and then decreased rapidly as the total organic carbon decreased during photocatalytic degradation (Fang et al., 2013). In 2011, Shah and Verma (Shah and Verma, 2011) observed that mouse liver homogenates which received different concentrations of butylparaben, had reduced concentrations of antioxidants and elevated levels of malondialdehyde (MDA), one of the major products of lipid peroxidation. The results indicated an increase of oxidative stress caused by reactive oxygen species after exposure to butylparaben. Similar conclusions were reached by Popa et al. (2011), who studied the concentration of MDA and 2,3-dihydroxybenzoic acid (2,3-DHBA) in the blood serum of rats exposed to methylparaben. Our analysis performed with a propyl ester of p-hydroxybenzoic acid shows that this compound is also capable of promoting increased oxidative stress by the production of O− 2 in liver cells in vitro. In addition, in the exposed animals observed by Shah and Verma (2011), a decrease in the concentration of superoxide dismutase (SOD) (an enzyme that catalyzes dismutation of superoxide anion (Johnson and Giulivi, 2005) could be linked with an excessive production of O− 2 , observed in our experiment. This is evidence of impaired antioxidant mechanisms in cells exposed to PBs. The excess of free radicals, uncontrollable by the cell defense mechanisms, may contribute to damage to the mitochondria and the uncoupling of the respiratory chain (Paradies et al., 2002). As can be observed in the graphs, PP used at a concentration of 4.6 ng/ml caused a significant decrease in ATP in the HepG2 cell line. In addition, a corresponding increase in ADP and AMP might suggest the initiation of cellular defense mechanisms against energy loss, although not reflected in the compensation of high-energy particle deficiency. The results confirm recent research by Nakagawa and Moldéus, 1998, who observed a decrease in cell viability, reduction in the mitochondrial membrane potential, as well as reduced total concentration of adenine nucleotides and ATP in freshly isolated rat hepatocytes incubated with various PBs. However, compared to our analysis, the concentrations of PBs used by Nakagawa and Moldéus, (1998) were very high (0.5–2.0 mM), which corresponds to 90–360 mg/l (Błędzka et al., 2014), which proves that they may adversely affect the energy status of liver cells even at lower concentrations, i.e. in the ranges detected in the blood of people exposed to PBs (Ye et al., 2008; Frederiksen et al., 2011; Sandanger et al., 2011). However, it should be borne in mind that the actual exposure of this organ to this xenobiotic in vivo is much smaller than is the case in cells cultured in vitro. The reason for this is the greater mass of the organ and the greater number of cells in the liver than in a cell culture. Both phenomena demonstrated that intensification of oxidative stress and a reduction in energy reserves in the HepG2 cell line can lead to lower cell viability. Our study confirms the results obtained in other studies (Nakagawa and Moldéus, 1998; Pérez Martín et al., 2010; Martín et al., 2014), and determines how cell death occurs. Analysis of confocal microscopy images indicates that the hepatocytes treated with various concentrations of PP, were subject mainly to apoptosis, at 4.6 ng/ml it concerned most cells in the culture. Necrosis was observed at each PP level but included only a small number of cultured cells; this, however, may have depended not on the presence of PP in the culture medium but on the specificity of the cell line
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(http://www.hepg2.com/). The tested hepatocytes are susceptible to the formation of cell aggregates in the culture, which may cut off access to the necessary nutrients from cells located within the aggregate, and results in an environment rich in toxic by-products of cellular metabolism, which may in turn trigger necrosis. This does not change the fact that the HepG2 cell culture in the presence of PP leads to a PP concentration-dependent reduction in cell viability, and this process occurs primarily via apoptosis. Probably apoptosis process noted in PP-treated hepatocytes is caused by induction of DNA double-strand breaks and oxidative damage underlies the cytostatic effect what was observed by Pérez Martín et al. (2010) in treated Vero cells. Additional the authors noticed that at the lowest concentration tested, changes in cell-proliferation rates rather than in cell viability were observed. A significant and dose-dependent decline in the percentage of mitotic cells was observed, mainly due to cell-cycle arrest at the G0/G1 phase (Pérez Martín et al., 2010; Martín et al., 2014).These cytotoxic effects on cells are greater when in experiments simultaneous were used propylparaben and butylated hydroxyanisole (Martin et al., 2014). The findings indicated that using mixtures of parabens it potentiate their single pro-oxidant effect what could change their safety assessment. The obtained results allow a conclusion that propylparaben, one of the two most commonly used parabens in manufacturing, can be toxic to liver cells. The toxicity of this compound is associated with the increased production of superoxide anions, which can contribute to a reduced concentration of superoxide dismutase in vivo and impairment of the body's antioxidant mechanisms (Kang et al., 2013; Nakagawa and Moldéus, 1998). A further reduction in the potential of mitochondrial membranes (Nakagawa and Moldéus, 1998) and uncoupling of the respiratory chain resulting in a reduction in ATP concentration, may consequently lead to cell death via apoptosis induced by mitochondrial damage. In our study, these adverse effects of PP on the liver cells were best visible at the highest concentration (4.6 ng/ml). This value was twice the highest level recorded by Ye et al. (2008) for the concentration of free PP in human blood. The other measurements of PB concentrations in the blood (Frederiksen et al., 2011; Sandanger et al., 2011) also probably do not reflect the actual scale of exposure to these compounds, as Sandanger et al. (Sandanger et al., 2011) analyzed samples derived only from women, while Frederiksen et al. (2011) studied only men. Therefore, in order to assess actual exposure to the esters of p-hydroxybenzoic acid, large-scale studies are needed to reliably determine the concentrations of these compounds in the blood of people who have regular contact with PBs. In addition, more research is needed on the molecular activity of PBs and the possible effects of exposure to specific concentrations of these xenobiotics. Conflict of interest statement This study was supported by the statutory budget of the Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Poland. Transparency document The Transparency document associated with this article can be found, in online version. References Abbas, S., Greige-Gerges, H., Karam, N., Piet, M.H., Netter, P., Magdalou, J., 2010. Metabolism of parabens (4-hydroxybenzoic acid esters) by hepatic esterases and UDP-glucuronosyltransferases in man. Drug Metab. Pharmacokinet. 25, 568–577. Afonso, V., Champy, R., Mitrovic, D., Collin, P., Lomri, A., 2007. Reactive oxygen species and superoxide dismutases: role in joint diseases. Joint Bone Spine 74, 324–329. Andersen, F.A., 2008. Final amended report on the safety assessment of methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, isobutylparaben, and benzylparaben as used in cosmetic products. Int. J. Toxicol. 27, 1–82.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Propylparaben-induced disruption of energy metabolism in human HepG2 cell line leads to increased synthesis of superoxide anions and apoptosis S. Szeląg a, A. Zabłocka a, K. Trzeciak a, A. Drozd a, I. Baranowska-Bosiacka b, A. Kolasa c, M. Goschorska b, D. Chlubek b, I. Gutowska a,⁎ a b c
Department of Biochemistry and Human Nutrition, Pomeranian Medical University, Broniewskiego 24 Str., Szczecin, Poland Department of Biochemistry, Pomeranian Medical University, Powstańców Wlkp 72 Str., Szczecin, Poland Department of Histology and Embryology, Pomeranian Medical University, Powstańców Wlkp 72 Str., Szczecin, Poland
a r t i c l e
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Article history: Received 18 July 2015 Received in revised form 12 November 2015 Accepted 19 November 2015 Available online 23 November 2015 Keywords: Apoptosis ATP HepG2 Liver Propylparaben ROS
a b s t r a c t The effect of propylparaben (in final concentrations 0.4 ng/ml, 2.3 ng/ml and 4.6 ng/ml) on the energy metabolism of HepG2 hepatocytes, superoxide anion synthesis, apoptosis and necrosis is described. Propylparaben can be toxic to liver cells due to the increased production of superoxide anions, which can contribute to a reduced concentration of superoxide dismutase in vivo and impairment of the body's antioxidant mechanisms. Finally, a further reduction in the mitochondrial membrane potential and uncoupling of the respiratory chain resulting in a reduction in ATP concentration as a result of mitochondrial damage may lead to cell death by apoptosis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Propylparaben (PP) is one of the esters of p-hydroxybenzoic acid, commonly used as a preservative in cosmetics and medicines, and also in food in some countries (Soni et al., 2005). It is one of the most commonly used parabens (PBs) in commercially available cosmetic products (Andersen, 2008; Soni et al., 2001). In the European Union, maximum permissible levels of p-hydroxybenzoic acid esters (including PP) in cosmetic formulations are 0.4% for single esters and 0.8% for several compounds used simultaneously (EC-1223/2009; SCCS/1514/13). Until recently, PP was also used in food products in the EU but it has been removed from the list of permitted food preservatives due to reports of possible adverse effects on the endocrine system. This ban does not include other PBs which are considered safe by the EU and the FDA (Błędzka et al., 2014; EC-52/2006). In other countries, for example in the USA, PP is still approved for use in foods, including baked goods, fruit juices, frozen food, spices, processed vegetables and pickled cucumbers (Soni et al., 2001).
⁎ Corresponding author at: Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24 street, 71–460 Szczecin, Poland. E-mail address: [email protected] (I. Gutowska).
http://dx.doi.org/10.1016/j.tiv.2015.11.011 0887-2333/© 2015 Elsevier Ltd. All rights reserved.
Until recently, PBs mainly owed their popularity to their physical and chemical properties, including a broad spectrum of antimicrobial activity and relatively low toxicity (Błędzka et al., 2014; Aubert et al., 2012). However, a growing number of reports have indicated the possible negative effects of chronic exposure to these compounds. Their estrogenic activity in vitro was demonstrated as early as 1998 (Routledge et al., 1998), which although markedly weaker than the natural action of estradiol, started a discussion on the safety of PB use. Moreover, studies on male rats have shown that exposure to PBs is associated with a dose-dependent decrease in the concentration of testosterone in the blood of animals and a disruption in the activity of the reproductive system (Oishi, 2002). Other negative effects of PBs have been observed in liver cells. PBs have been shown to increase the permeability of the mitochondrial membrane (Nakagawa and Moore, 1999), which leads to a swelling of these organelles, a decrease in intracellular ATP and consequently to the death of hepatocytes. Other studies have shown increased lipid peroxidation and free radical reactions in experimental animals treated with butylparaben, another preservative from the paraben group (Shah and Verma, 2011). Humans are exposed to PBs on a daily basis via ingestion, skin contact and inhalation (Andersen, 2008). The widespread use of PBs in manufacturing has led to global environmental pollution with these compounds. Their presence in the air, surface water, biota and soil (Błędzka et al., 2014) is a considerable source of human exposure to
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parabens, in addition to exposure from personal care products, food preservatives and pharmaceuticals. Parabens applied to the skin are absorbed and metabolized by esterases located in the skin (Abbas et al., 2010; Jewell et al., 2007; Ozaki et al., 2013). The same esterasebased mechanism takes place during biotransformation of the PBs delivered into the body via the oral route (Boberg et al., 2010). The liver, an organ involved in countless biochemical transformations including the metabolism of xenobiotics, is highly exposed to their harmful effects and the damage to this organ has serious consequences for the entire body. Based on the premises on the possible adverse effect of PBs on liver cells, we decided to examine the effect of one of these compounds, namely PP, on the energy metabolism of HepG2 hepatocytes, superoxide anion synthesis, and apoptosis and necrosis occurring in these cells.
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2.4. Imaging of mitochondrial ROS generation Mitochondrial superoxide level was visualized by fluorescent marker MitoSOX Red (Invitrogen). This fluorescence dye is highly selective for the detection of superoxide in living cell mitochondria. The reagent is oxidized by superoxide but not by other ROS or reactive nitrogen species, and exhibits red fluorescence (excitation 510 nm, emission 580 nm). MitoSOX Red stock solution (50 mM in DMSO) was diluted in the incubation medium (BME) to a final concentration of 2.5 μg/ml. The cells were incubated in this solution for 10 min (humidified 95% air/CO2 atmosphere at 37 °C). Next, cells were washed with BME at room temperature and were examined under the fluorescent microscope. 2.5. Measurement of denine nucleotides concentration
2. Material and methods 2.1. Reagents and sources HepG2 was obtained from LGC Standards (USA). EMEM medium, glutamine, dimethyl sulfoxide (DMSO), trypsine and antibiotics (penicillin and streptomycin) were from Sigma-Aldrich (Poznan, Poland). Fetal bovine serum (FBS) was from Gibco (Gibco, Paisley, UK). Phosphate buffered saline (PBS) was from PAP Laboratories (Vienna, Austria). Propylparaben and deionized water were from Sigma-Aldrich. MicroBCA KIT for protein measurement was from ThermoOrion. An Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from BD Pharmingen. MitoSOX™ Red was from Invitrogen (Oregon, USA). All buffer chemicals and solvents used as mobile phases for HPLC measurements were of HPLC grade and were purchased from Sigma Aldrich (St Louis,MO, USA) or Merck. Double-distilled water was obtained from a Milli-Q Water System (Millipore, Billerica, MA, USA). All buffers used for HPLC analysis were filtered through 0.22 μm nylon filters (Agilent). 2.2. Cell cultures The study was conducted on hepatocytes HepG2, which were cultured (37 °C in 5% CO2) in EMEM medium supplemented with 10% fetal bovine serum (without fatty acid), penicillin (100 U/mL), streptomycin (100 mg/mL) and propylparaben in final concentrations 0,4 ng/ml, 2,3 ng/ml and 4,6 ng/ml (Ye et al., 2008). Control cells were incubated with solvent used to make an appropriate solution of propylparaben (DMSO) to find out influence of solvent on examined processes. Propylparaben concentrations were selected on the basis of results of its concentration in human serum (Ye et al., 2008). After 48 h the cells were collected and a pellet was obtained by centrifugation (250 g for 5 min). Protein concentration was measured by using MicroBCA Protein Assay Kit (Thermo Scientific, Pierce Biotechnology, USA) and ELISA. 2.3. Fluorescent microscopy: imaging of apoptosis and necrosis processes Hepatocytes, in the number of 5 × 105 were incubated with propylparaben solutions on microscope slides according to the aforementioned procedure. After the end of cultivation, the cells were rinsed with PBS. Next, the cells were suspended in a binding buffer and stained with 1 ng/mL Annexin V-FITC and 5 ng/mL propidium iodide for 30 min in the dark. Cells that are viable are Annexin V-FITC and PI negative; cells that are in early apoptosis are Annexin V-FITC positive and PI negative; and cells that are in late apoptosis or already dead are both Annexin V-FITC and PI positive (Vermes et al., 1995). A dual-pass FITC/rhodamine filter set was applied (Olimpus FluoView SV100).
Isolation of adenine nucleotides from cells was performed according Baranowska-Bosiacka et al. (2009). The HPLC separations were performed on an Agilent Technologies 1260 liquid chromatograph, consisting of model G1379B degasser, a model G1312B bin pump, a model G1316A column oven and a model G1315CDAD VL +. Samples were injected using a model G1329B. An Agilent ChemStation software (Agilent Technologies, Cheadle, UK), were used for instrument control and data acquisition and analysis. The separation was completed on a Thermo Scientific Hypersil BDS C18 column 100 × 4.6 mm 3 μm (cat no. 28103–104630). The temperature of column oven was set at 20 °C. A dual mobile phase gradient was used to achieve appropriate separation of all analytes of interest. Mobile phase A contained 150 mM KH2PO4/K2HPO4, 150 mM KCl pH 6.0. Mobile phase B had the same final concentrations as mobile phase A, except for the addition of 15% acetonitrile (v/v). The elution was performed with a linear gradient. The flow rate was 1.0 mL/min. The sample injection volume was 20 mL. The DAD detector monitored peaks by adsorption at 254 nm. 2.6. Statistical analysis The obtained results were analyzed statistically using the software package Statistica 10 (Statsoft, Poland). Arithmetical mean and the standard deviation (SD) were found for each of the studied parameters. The distribution of results for individual variables was obtained with the Shapiro–Wilk W test. As most of the distributions deviated from the normal Gauss distribution, non-parametric tests were used for further analyses. For related samples, significance was first checked with Friedmann's analysis of variance, and significant results were subjected to the Wilcoxon matched-pair test. The level of significance was p ≤ 0.05. 3. Results 3.1. Apoptosis and necrosis Immunohistochemical analysis using Annexin V and propidium iodide was performed for the qualitative determination of apoptosis or necrosis of hepatocytes treated with PP. Only a small number of cells underwent necrosis while the majority of cells were subject to apoptosis. Comparative analysis of confocal microscope images showed that the cell death was caused by the presence of PP in the culture medium, while the intensity of the process increased together with the increase in PP level (Fig. 1A). 3.2. Superoxide anion production Application of propylparaben in a hepatocyte culture resulted in an increased production of superoxide anions by the treated cells. The images show an increase in the intensity of red fluorescence that reflects the process of O− 2 production, which rose together with PP
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Fig. 1. Imaging of A) apoptosis by Annexin V-FITC (n = 3) and B) ROS production by MitoSOX Red (n = 3) both with fluorescence microscopy of human hepatocytes (HepG2) after a 48 h incubation with increasing concentrations of propylparaben.
concentration. The most intense production of O− 2 was observed at a concentration of PP at 4.6 ng/ml (Fig. 1B). 3.3. Analysis of adenine nucleotide levels Analysis of results obtained by high performance liquid chromatography (HPLC) suggests that an increase in PP is accompanied by a decrease in cellular ATP. In cells treated with the lowest PP level, i.e. 0.4 ng/ml, the average ATP level was 28.28 nM/mg protein. This level was 11% higher than in control cells, where the average of seven replicates was 25.56 nM/mg protein. However, statistical analysis did not confirm a statistical significance compared to the control, as p N 0.05. In samples where PP level was 2.3 ng/ml, the decrease in ATP was statistically significant compared to the control and the average ATP level fell to 21.13 nM/mg protein, 17% lower than for the control. For samples with PP at 4.6 ng/ml, average cellular ATP was only 3.19 nM/mg protein, i.e. about 88% lower than in cells cultured without the addition of PP. The statistical significance of this result was observed at p ≤ 0.02 (Fig. 2A).
In contrast to ATP, other adenine nucleotides in the analyzed hepatocytes showed levels inversely dependent on the dose of PP. At the lowest PP concentration (0.4 ng/ml) ADP rose to 9.13 nM/mg, 38% higher than the control. The increase was statistically significant (p ≤ 0.02). At the next PP level, 2.3 ng/ml, average cellular ADP was 8.97 nM/mg; 55% higher than the control – also statistically significant (p ≤ 0.02). At the highest PP concentration tested (4.6 ng/ml), the concentration of ADP was 6.50 nM/mg protein, 13% higher than the concentration measured for the control. This increase, however, was not statistically significant (Fig. 2B). AMP was another adenine nucleotide analyzed in this study. At a PP level of 0.4 ng/ml the average AMP level was 3.95 nM/mg, a 17% increase relative to control and statistically insignificant (p N 0.05). At a PP level of 2.3 ng/ml, AMP reached 4.07 nM/mg protein, 20% higher than the control, again not statistically significant. In the case of cells cultured in the presence of PP at 4.6 ng/ml, the concentration of AMP was 14.03 nM/mg, a 314% increase compared to control cells. This value achieved statistical significance (p ≤ 0.05) (Fig. 2C).
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Fig. 2. Effect of increasing concentrations of propylparaben on the levels of ATP (A), ADP (B) and AMP(C) in human hepatocytes (HepG2) after 48 h of incubation. *p-Value ≤ 0.05; **p-value ≤ 0.02, significant difference vs control (n = 7).
Fig. 2A, B and C show the correlation between the concentrations of adenosine tri-, di- and monophosphates in the tested HepG2 cell line and the concentration of PP added to the cultures. 4. Discussion Nowadays, contact with preservatives from the paraben group is almost inevitable. Their ubiquitous occurrence is reflected in their detectable presence in human urine and blood (Calafat et al., 2010; Frederiksen et al., 2011; Kang et al., 2013). Therefore, it is necessary to know the exact molecular activity of PBs and determine the effects of chronic exposure to these compounds. In connection with previous reports on the negative activity of PBs on liver cells, we decided to evaluate the effect of propylparaben on the synthesis of adenine nucleotides and superoxide anion, and apoptosis and necrosis occurring in HepG2 line cells treated with this compound. Immunohistochemical evaluation of superoxide anion synthesis showed an increase dependent on PP concentration. The increased
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oxidative stress, a consequence of the enhanced production of O− 2 , may cause a number of irregularities at the molecular level, and thus may have negative consequences for the whole body (Afonso et al., 2007; Emerit et al., 2004; Granot and Kohen, 2004). The superoxide anion is able to penetrate cell membranes and is durable enough to circulate in the cellular environment and activate a cascade of free radical reactions, thus interacting with organelles distant from the site of its origin (Bartosz, 2003). On the other hand oxidative species, particularly HO(*), were the most important reactive oxygen species mediating photocatalytic degradation of PPB, and PPB degradation was found to be significantly affected by pH because it was controlled by the radical reaction mechanism. The estrogenic activity decreased as PPB was degraded, while the acute toxicity at three trophic levels first increased slowly and then decreased rapidly as the total organic carbon decreased during photocatalytic degradation (Fang et al., 2013). In 2011, Shah and Verma (Shah and Verma, 2011) observed that mouse liver homogenates which received different concentrations of butylparaben, had reduced concentrations of antioxidants and elevated levels of malondialdehyde (MDA), one of the major products of lipid peroxidation. The results indicated an increase of oxidative stress caused by reactive oxygen species after exposure to butylparaben. Similar conclusions were reached by Popa et al. (2011), who studied the concentration of MDA and 2,3-dihydroxybenzoic acid (2,3-DHBA) in the blood serum of rats exposed to methylparaben. Our analysis performed with a propyl ester of p-hydroxybenzoic acid shows that this compound is also capable of promoting increased oxidative stress by the production of O− 2 in liver cells in vitro. In addition, in the exposed animals observed by Shah and Verma (2011), a decrease in the concentration of superoxide dismutase (SOD) (an enzyme that catalyzes dismutation of superoxide anion (Johnson and Giulivi, 2005) could be linked with an excessive production of O− 2 , observed in our experiment. This is evidence of impaired antioxidant mechanisms in cells exposed to PBs. The excess of free radicals, uncontrollable by the cell defense mechanisms, may contribute to damage to the mitochondria and the uncoupling of the respiratory chain (Paradies et al., 2002). As can be observed in the graphs, PP used at a concentration of 4.6 ng/ml caused a significant decrease in ATP in the HepG2 cell line. In addition, a corresponding increase in ADP and AMP might suggest the initiation of cellular defense mechanisms against energy loss, although not reflected in the compensation of high-energy particle deficiency. The results confirm recent research by Nakagawa and Moldéus, 1998, who observed a decrease in cell viability, reduction in the mitochondrial membrane potential, as well as reduced total concentration of adenine nucleotides and ATP in freshly isolated rat hepatocytes incubated with various PBs. However, compared to our analysis, the concentrations of PBs used by Nakagawa and Moldéus, (1998) were very high (0.5–2.0 mM), which corresponds to 90–360 mg/l (Błędzka et al., 2014), which proves that they may adversely affect the energy status of liver cells even at lower concentrations, i.e. in the ranges detected in the blood of people exposed to PBs (Ye et al., 2008; Frederiksen et al., 2011; Sandanger et al., 2011). However, it should be borne in mind that the actual exposure of this organ to this xenobiotic in vivo is much smaller than is the case in cells cultured in vitro. The reason for this is the greater mass of the organ and the greater number of cells in the liver than in a cell culture. Both phenomena demonstrated that intensification of oxidative stress and a reduction in energy reserves in the HepG2 cell line can lead to lower cell viability. Our study confirms the results obtained in other studies (Nakagawa and Moldéus, 1998; Pérez Martín et al., 2010; Martín et al., 2014), and determines how cell death occurs. Analysis of confocal microscopy images indicates that the hepatocytes treated with various concentrations of PP, were subject mainly to apoptosis, at 4.6 ng/ml it concerned most cells in the culture. Necrosis was observed at each PP level but included only a small number of cultured cells; this, however, may have depended not on the presence of PP in the culture medium but on the specificity of the cell line
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(http://www.hepg2.com/). The tested hepatocytes are susceptible to the formation of cell aggregates in the culture, which may cut off access to the necessary nutrients from cells located within the aggregate, and results in an environment rich in toxic by-products of cellular metabolism, which may in turn trigger necrosis. This does not change the fact that the HepG2 cell culture in the presence of PP leads to a PP concentration-dependent reduction in cell viability, and this process occurs primarily via apoptosis. Probably apoptosis process noted in PP-treated hepatocytes is caused by induction of DNA double-strand breaks and oxidative damage underlies the cytostatic effect what was observed by Pérez Martín et al. (2010) in treated Vero cells. Additional the authors noticed that at the lowest concentration tested, changes in cell-proliferation rates rather than in cell viability were observed. A significant and dose-dependent decline in the percentage of mitotic cells was observed, mainly due to cell-cycle arrest at the G0/G1 phase (Pérez Martín et al., 2010; Martín et al., 2014).These cytotoxic effects on cells are greater when in experiments simultaneous were used propylparaben and butylated hydroxyanisole (Martin et al., 2014). The findings indicated that using mixtures of parabens it potentiate their single pro-oxidant effect what could change their safety assessment. The obtained results allow a conclusion that propylparaben, one of the two most commonly used parabens in manufacturing, can be toxic to liver cells. The toxicity of this compound is associated with the increased production of superoxide anions, which can contribute to a reduced concentration of superoxide dismutase in vivo and impairment of the body's antioxidant mechanisms (Kang et al., 2013; Nakagawa and Moldéus, 1998). A further reduction in the potential of mitochondrial membranes (Nakagawa and Moldéus, 1998) and uncoupling of the respiratory chain resulting in a reduction in ATP concentration, may consequently lead to cell death via apoptosis induced by mitochondrial damage. In our study, these adverse effects of PP on the liver cells were best visible at the highest concentration (4.6 ng/ml). This value was twice the highest level recorded by Ye et al. (2008) for the concentration of free PP in human blood. The other measurements of PB concentrations in the blood (Frederiksen et al., 2011; Sandanger et al., 2011) also probably do not reflect the actual scale of exposure to these compounds, as Sandanger et al. (Sandanger et al., 2011) analyzed samples derived only from women, while Frederiksen et al. (2011) studied only men. Therefore, in order to assess actual exposure to the esters of p-hydroxybenzoic acid, large-scale studies are needed to reliably determine the concentrations of these compounds in the blood of people who have regular contact with PBs. In addition, more research is needed on the molecular activity of PBs and the possible effects of exposure to specific concentrations of these xenobiotics. Conflict of interest statement This study was supported by the statutory budget of the Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Poland. Transparency document The Transparency document associated with this article can be found, in online version. References Abbas, S., Greige-Gerges, H., Karam, N., Piet, M.H., Netter, P., Magdalou, J., 2010. Metabolism of parabens (4-hydroxybenzoic acid esters) by hepatic esterases and UDP-glucuronosyltransferases in man. Drug Metab. Pharmacokinet. 25, 568–577. Afonso, V., Champy, R., Mitrovic, D., Collin, P., Lomri, A., 2007. Reactive oxygen species and superoxide dismutases: role in joint diseases. Joint Bone Spine 74, 324–329. Andersen, F.A., 2008. Final amended report on the safety assessment of methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, isobutylparaben, and benzylparaben as used in cosmetic products. Int. J. Toxicol. 27, 1–82.
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