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ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells
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Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit 5 6
ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells
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Bruna da Silva Félix Nornberg, Carolina Reyes Batista, Daniela Volcan Almeida, Gilma Santos Trindade, Luis Fernando Marins ⇑
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Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Brazil
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Article history: Received 2 July 2014 Accepted 18 September 2014 Available online xxxx Keywords: ABC transporters Methyl parathion Multixenobiotic resistance (MXR) Verapamil
a b s t r a c t The multi-xenobiotics resistance (MXR) mechanisms are the first line of defense against toxic substances in aquatic organisms and present great importance in the adaptation related to contaminated environments. Methyl parathion (MP) is a widely used organophosphate pesticide, which has been associated to various toxic effects in organisms. In the present work, we studied the main genes related to efflux transporters in zebrafish liver (ZFL) cells exposed to MP with and without an inhibitor of ABC transporters (verapamil). The results concerning transporters activity showed that the MXR mechanism is activated to detoxify from methyl parathion. The toxic effects of MP on ZFL cells were increased in the presence of the efflux transporter inhibitor, once cell viability was significantly decreased in co-exposure experiments. The combined exposure to MP and the inhibitor caused an increase in gene expression of P-gp1 (Abcb1) and MRP4 (Abcc4), suggesting that these transporters isoforms are associated with MP efflux. In general, the expression of genes related to the antioxidant defense system (ADS) was significantly increased in ZFL cells co-exposed to MP and verapamil. These data provide useful insights for better understanding of MP detoxification mechanism in fish hepatocytes. Ó 2014 Published by Elsevier Ltd.
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1. Introduction
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Aquatic organisms are constantly exposed to many pollutants dissolved in the water and great part of these organisms can tolerate exposure to these compounds. Kurelec (1992) has shown that this tolerance capacity is related to the multi-xenobiotics mechanism (MXR), which is considered the first line of defense against toxic compounds (Epel, 1998). MXR is widely distributed in biological systems, from unicellular organisms to vertebrates (Annilo et al., 2006). It is mediated by the transmembrane transporters proteins from ATP-binding cassette (ABC) family, which are responsible for the excretion of many xeno/endobiotics from the cell by ATP hydrolysis (Bard, 2000). ABC transporters constitute a highly conserved superfamily of proteins among vertebrates and exert many functions in the organisms. In zebrafish, the ABC transporter genes are organized in 8 sub-families, which are designated from ABCA to ABCH (Dean and Annilo, 2005). Some ABC transporters from ABCB, ABCC and ABCG subfamilies have been reported
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⇑ Corresponding author at: Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Av. Itália km 8, 96203-900 Rio Grande, RS, Brazil. E-mail address: [email protected] (L.F. Marins).
with functions related to MXR (Buss and Callaghan, 2008; Zucchi et al., 2010; Faria et al., 2011). The ABCB1 (P-gp, MDR1), members of the ABCC (MRP) subfamily and breast cancer resistance protein (BCRP, ABCG2) are toxicologically the most relevant proteins in efflux of xenobiotics in mammals (Doyle and Ross, 2003). P-gp may act eliminating hydroxylated xenobiotics metabolites after its modification by phase I enzymes (cytochromes P450), or leading non-metabolized xenobiotics out of the cell (Bard, 2000; Xu et al., 2005). MRPs have been reported to promote resistance against heavy metals in some mammals and fish species (Zucchi et al., 2010; Long et al., 2011). Besides, MRP and P-gp present significant overlap in substrate specificities, particularly evincing affinity for compounds conjugated with glutathione, glucuronate or sulfate (Cole et al., 1994; Buss and Callaghan, 2008). ABCG2 is a widely studied transporter in humans due to its capacity to grant resistance to chemotherapics used against breast cancer. Concerning fish species, Abcg2 expression was detected in liver of rainbow trout Oncorhynchus mykiss (Zaja et al., 2008a). In zebrafish, four paralogs of Abcg2 gene were already identified (Annilo et al., 2006). ABCB1 and ABCG2 transporters are located in the apical membrane of cells, facilitating the movement of substances and promoting the excretion of xenobiotics. ABCB1 substrates are amphiphilic
http://dx.doi.org/10.1016/j.tiv.2014.09.010 0887-2333/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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or moderately hydrophobic, with a low molecular weight, positively charged or neutral, including both endogenous and exogenous compounds. Thus, the number of potential substrates of Pg-p can be estimated at several hundred (Smital et al., 2003, 2004; Buss and Callaghan, 2008). According to Ito et al. (2005), Abcg2 is expressed in different organs involved in absorption (small intestine), distribution (placenta and blood brain barrier) and elimination (liver), indicating an important role of ABCG2 in excretion of xenobiotics. In mammals, the localization of ABCC4 (MRP4) and ABCC1 (MRP1) in polarized cells is variable. These transporters can be localized on the apical or basolateral membranes depending on the cells type (Luckenbach et al., 2014). Moreover, there are reports about MRP4 expression in some human organs such as prostate, testis, ovary, intestine, pancreas and lung (Sturm and Segner, 2005). ABCC1 is found in lung, testis, kidney, placenta, heart and skeletal muscle (Deeley et al., 2006; Bakos and Homolya, 2007). In fish, the notable Abcc1 and Abcc4 gene expression was found in kidney and high Abcc4 gene expression in gonads (Loncar et al., 2010). Therefore, the implication of these transporters in xenobiotics detoxification in aquatic organisms requires more studies. The synergistic detoxification activity of ABC transporters and enzymes from the antioxidant defense system (ADS) has been suggested (Bard, 2000; Xu et al., 2005). Increased expression of these transporters, as well as phase II enzymes, has been reported in aquatic organisms exposed to different contaminants (Bresolin et al., 2005; Leslie et al., 2005). One important component that modulates these two processes is the NRF2 transcription factor (nuclear factor erythroid 2 – relative to factor 2). This molecule is a key regulator of a cytoprotective network against exposure to electrophilic and oxidative chemicals, by controlling the transcriptional activity of genes coding for ADS enzymes, such as glutathione-S-transferase (GST), glutamate cysteine ligase (GCL), and superoxide dismutase (SOD), as well as for efflux transporters proteins (Osburn and Kensler, 2008; Niture et al., 2010). Several classes of contaminants may induce the MXR mechanism, such as herbicides, fungicides and insecticides (Buss and Callaghan, 2008). Among the most commonly used pesticides are the organophosphates, which contain phosphate groups derived from phosphoric acid. Methyl parathion (MP) is an organophosphate pesticide, which acts by inhibiting acetylcholinesterase (Ecobichon et al., 1990; Küster, 2005). It is highly toxic after conversion to paraoxon by oxygenation in the endoplasmic reticulum of hepatocytes. MP has been reported as a substrate for efflux transporters, acting as an inducer or inhibitor (Sreeramulu et al., 2007). Many types of fish cell lines have been established and applied to test the toxicity of pesticides, including hepatoma cells PLHC-1 (Knauer et al., 2007), hepatic cells RTL-W1 from rainbow trout (Babin and Tarazona, 2005) and zebrafish liver (ZFL) cells (Huang and Huang, 2012). However, there are few studies relating the toxicity of pesticides with ABC efflux transporters in fish cell lines. In this context, the goal of this study was to identify ABC efflux transporters potentially involved in detoxification of methyl parathion in zebrafish liver cells.
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2. Materials and methods
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2.1. Chemicals
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Methyl parathion (MP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), verapamil (VER) and Rhodamina123 (Rho-123) were purchased from Sigma (Brazil). TRIzol reagent, DNAse I Amplification Grade, Quant-iTTM RNA assay kit, Platinum SYBR Green qPCR SuperMix-UDG and 20,70-dichlorofluorescin diacetate (H2DCF-DA) were obtained from Invitrogen (Brazil). High
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Capacity cDNA Reverse Transcription was acquired from Applied Biosystems (Brazil). All tissue-culture products were from Gibco (Brazil). Zebrafish hepatocytes (line ZFL; CRL2643) were obtained from American Type Culture Collection (ATCC).
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2.2. Growth and treatment of cells
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Adult zebrafish liver cell line (ATCCCRL-2643) were cultivated in RPMI 1640 medium supplemented with sodium bicarbonate (0.2 g L1), L-glutamine (0.3 g L1), HEPES (25 mM) and b-mercaptoethanol (5 105 M), with 10% fetal bovine serum, 100 IU/ml penicillin and 100 lg mL1 streptomycin. Cells were grown in cell culture flasks at 28 °C. One day prior to experiments, cells were seeded in 24 or 96 well plates, in a density of 106 cells or 5 105 cells, into 1 or 0.2 mL of medium per well, respectively.
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2.3. MTT assay
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The MTT test assesses cell metabolism based on the ability of the mitochondrial succinate–tetrazolium reductase system to convert the yellow compound MTT to a blue formazan dye. The amount of dye produced is proportional to the number of live metabolically active cells. The assay was carried out as previously described (Mosmann, 1983). Briefly, cells were seeded in 96-well microplates and incubated overnight. Subsequently, the cells were treated with MP at concentrations of 0.13, 1.3, 6.5, 13, 26 and 130 lM for 24 h. The MP was dissolved in acetone (control group) and added to a final concentration of 0.01% (v/v). Selection of MP concentrations was based on Brazilian legislation (CONAMA, 2012), being the concentration of 0.13 lM indicated the maximum values permitted for freshwater. Intending to verify the involvement of the efflux transporters in the detoxification from MP, the same experimental design was carried out adding 30 lM of VER (dissolved in 96% ethanol), which was used as inhibitor of efflux transporters. Control group was exposed to acetone and VER and the final concentration of the solvents never exceeded 0.01%. MTT (5 mg mL1) dissolved in phosphate-buffered saline (PBS) was added (20 ll) to the culture wells, and plates were incubated for 4 h at 28 °C. Supernatant was removed and 200 ll of DMSO was added to each well and read at 490 nm in EL800 microplate reader (BIO-TEK Instrument, Winooski, VT). Each experiment was performed in triplicate.
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2.4. Determination of intracellular rhodamine 123
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For the transport activity measurements, the accumulation of Rho-123 was observed in ZFL cells exposed to MP with or without inhibitor of ABC transporters (VER). Hepatocytes were cultured at 28 °C for 24 h in 24-well culture plates, as previously explained. Subsequently, cells were treated with acetone (control group) or MP at concentrations of 0.13 and 1.3 lM for 24 h. The same experimental design was carried out adding VER (30 lM). After exposure, cells were washed in PBS and incubated in the dark at 28 °C for 1 h in 1 mL of fresh medium containing Rho-123 (300 ng mL1) with or without VER (Ludescher et al., 1998). Next, cells were Q2 washed twice in PBS, resuspended in 1 mL of fresh medium and incubated for 30 min for Rho-123 efflux. After this, cells were washed again and then fluorescence accumulation was verified using a microplate reader (Victor2, Perkin Elmer Wallac, Boston, MA) at 485 and 590 nm of excitation and emission, respectively. These experiments were replicated three times. Therefore, efflux transporters activity was expressed as the inverse of Rho-123 fluorescence accumulation per number of cells. The results were expressed as percentage of fluorescence in relation to control group.
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Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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2.5. Gene expression analysis
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For gene expression, hepatocytes of zebrafish were exposed to 0.13 and 1.3 lM of MP and acetone (control group), with or without VER (30 lM) for 24 h in 24-well culture plate. These experiments were replicated four times and each sample was composed by a pool of four wells. Total RNA was isolated, for later qRT-PCR analysis, by Trizol Reagent (Invitrogen), following the manufacturer’s instructions. Genomic DNA was degraded with Deoxyribonuclease I (DNAse I) and total RNA was quantified by a QubitTM fluorometer using the Quant-iT™ RNA assay kit (Invitrogen). First-strand cDNA was synthesized from approximately 1 lg of total RNA using High Capacity cDNA Reverse Transcription kit (Invitrogen), according to the manufacturer’s instructions. The expression of ADS and efflux transporters genes was analyzed by real-time RT-PCR (7500 Real Time System, Applied Biosystems, Brazil), using SYBR GREEN PCR Master Mix™ (Invitrogen), according to manufacturer’s protocol. Each sample (n = 4) was analyzed in triplicate. Specific primers for each gene (Table 1) were designed using the Primer-BLAST tool from GenBank (http:// www.ncbi.nlm.nih.gov). Serial dilutions were performed in order to determine the qPCR reaction efficiency. Gene expression was carried out for 50 °C/2 min, 95 °C/2 min, followed by 40 cycles at 95 °C/15 s and 60 °C/30 s. Target gene expression was normalized by the expression of the elongation factor-1 alpha (ef1-a) gene.
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2.6. Data analysis
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The software RESTÓ was applied in the relative quantification of gene expression analyses (Pfaffl et al., 2002), by realizing a paired comparison between control group and treatments. The adopted alpha was 0.05 and results were expressed as median ± standard error One-way ANOVA was carried out, followed by an a posteriori means comparison (Newman–Keuls test, p < 0.05), for efflux transporters ac.tivity and cell viability assay. ANOVA assumptions (normality and homogeneity of variance) were verified prior to ANOVA analysis.
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3. Results
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3.1. Cell viability
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MTT assay for cells viability are shown in Fig. 1. Cells treated with 13, 26 and 130 lM of MP had significant (p < 0.05) viability decrease (83.1 ± 4.8, 73 ± 5.4 and 69 ± 3.2%, respectively) compared with control group (100 ± 4.9%). However, when verapamil was added, a significant (p < 0.05) decrease was observed at the concentrations 6.5, 13, 26 and 130 lM (67.2 ± 9.0, 51 ± 5.4, 47 ± 7.1 and 40 ± 1.3%, respectively) when compared with control group (100 ± 14%). No significant (p > 0.05) differences in viability was verified in hepatocytes treated with 0.13 and 1.3 lM of MP, with (78 ± 2.5 and 77 ± 5.3%, respectively) or without verapamil
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Fig. 1. ZFL cells viability exposed to controls and different concentrations of methyl parathion (0.13, 1.3, 6.5, 13, 26 and 130 lM) with 30 lM verapamil (filled circles and continuous line) and without verapamil (pierced circles and dotted line) for 24 h exposure. Values are expressed as percentages of viable cells in relation to control groups. Different symbols (⁄ and #) indicate significant differences (p < 0.05) between treatments and their respective control groups. Data are expressed as mean ± S.E (n = 3, from independent experiments).
(96 ± 4.1 and 98 ± 2.5%, respectively) when compared with control groups. Therefore, for efflux transporters activity and gene expression experiments, these sublethal concentrations were adopted.
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3.2. Functional analysis of the efflux transporters
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Zebrafish hepatocytes exposed to MP concentrations of 0.13 and 1.3 lM (Fig. 2) have shown a significant increase (p < 0.05) of ABC transporters activity (177.8 ± 37.4 and 188.6 ± 23.1, respectively) when compared with control group (100 ± 13.5%). However, when the inhibitor was added, a significant decrease was observed at MP concentrations of 0.13 and 1.3 lM (65 ± 17.3 and 37.2 ± 11.1, respectively), when compared with the control group (116.0 ± 17.6%), as shown in Fig. 2.
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3.3. Gene expression
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The genes analyzed in the present study are involved in the defense system against xenobiotics and are grouped according to their functions: ABC transporters (Abcb1, Abcc1, Abcc4 and Abcg2a; Fig. 3) and antioxidant defense system – ADS (Gst1a, Gclc, Nrf2 and Cu-Zn SOD; Fig. 4). No significant differences (p > 0.05) were found in the expression of Abcb1, Abcc1, Abcc4 and Abcg2a genes in zebrafish hepatocytes exposed to MP for 24 h (Fig. 3a). However, coexposure to MP + VER (Fig. 3b) presented significant induction (p < 0.05) of 4.4-fold in the expression of the gene coding for Abcb1 at the highest MP concentration. Also, a significant increase of 3.4and 4.8-fold induction was observed in the gene expression of Abcc4 at MP + VER concentrations of 0.13 and 1.3 lM, respectively. On the other hand, a significant decrease of approximately 80% in Abcc1 gene expression, at both tested MP concentrations, in the presence of the inhibitor (Fig. 3c).
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Table 1 Genes and PCR primers used for quantitative PCR analysis. Genes
Foward primer
Reverse primer
GenBank
Ef1a Nrf2 Gclc Cu,Zn-Sod Gst1a Abcb1/MDR1 Abcc1/MRP1 Abcc4/MRP4 Abcg2a/BCRP
50 -GGGCAAGGGCTCCTTCAA-30 50 -TGTTGGTTCGGAGGCTCTTAA-30 50 -ACGGCATTCCCCAGGTTAG-30 50 -GGAAGAGCCGGTTGAAATATTG-30 50 -CCCCAAAATACAGGCGTTTC-30 50 -CCTCGGTCAAACGTCTCCAA-30 50 -ATGCTGCCCGGCGAACATCC-30 50 -CACCTTGGGAAACTCAAAAACG-30 50 -GGAACAACAGCGGCAGTGT-30
50 -CGCTCGGCCTTCAGTTTG-30 50 -AGGCCATGTCCACACGTACA-30 50 -TTTTCAACAGGTGTGGGTTTGTA-3’ 50 -AGCGGGCTAAGTGCTTTCAG-30 50 -GCCCGGCTGGAGGAACT-30 50 -CTTTATGGGCGGCTCCTCTA-30 50 -AGGTCCATGAACCGCTGCGC-30 50 -GAAGGTTATCAGGCCACGATTT-30 50 -TGCCCTGCTCCTGAAAAAAC-30
NM_131263.1 NM_182889 NM_199277 NM_131294 NM_213394 NM_001114583 XM_001341859 NM_001007038 NM_001042775
Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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Fig. 2. Efflux transporters activity in ZFL cells exposed to MP (0.13 and 1.3 lM) without verapamil and with 30 lM verapamil (MP + VER). Control group values were set as 100%. MP control group is treated with acetone, while MP + VER control group is treated with acetone + VER. Data are expressed as a percentage of control culture fluorescence corrected by cell number ± S.E. (n = 3, from independent experiments). Asterisk indicates significant differences from control group (p < 0.05).
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Regarding ADS genes, a significant induction was found for Nrf2 and Cu-Zn SOD at the higher MP concentration (2.2- and 1.6-fold, respectively; Fig. 4a and d). Co-exposure of the cells to MP + VER has also increased genes Nrf2 and Cu-Zn SOD gene expression at the higher MP concentration, evincing a 4.8- and 2.5-fold induction, respectively (Fig. 4a and d). In this sense, we have also found a significant induction of 3.6-fold in Gclc gene at the higher MP + VER concentration (Fig. 4c). However, it was observed a Gst1a downregulation of about 50% at the higher MP concentration without VER (Fig. 4b).
4. Discussion
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Organophosphate pesticides are environmental pollutants known to inhibit the catalytic activity of acetylcholinesterase (AChE). Methyl parathion (O,O-dimethyl O-4-nitro-phenylphosphoro-thioate) is widely used in agriculture due to its low costs and high efficiency in eliminating plagues (Ma et al., 2003). Once it is a commonly used pesticide, the toxicity effects of MP have been thoroughly studied. Many studies have shown that this pesticide may cause DNA damage in human and rats’ spermatozoa (Arredondo et al., 2008; Piña-Guzmán et al., 2009). Regarding fish species, MP has been shown to induce oxidative stress, resulting in lipid peroxidation, increase of anti-oxidant enzymes, as well as decrease of glutathione levels (Monteiro et al., 2006). However, there are few reports in literature relating the MP toxicity to the MXR mechanism in aquatic organisms. Therefore, the aim of the present work was to identify efflux transporters genes potentially involved in detoxification from MP. We have firstly tested the cell viability when challenged with different MP concentrations (Fig. 1). Our results have shown that MP causes dose-dependent cell death. Huang and Huang (2012) used zebrafish cells exposed to high MP concentration (0–600 lM) and demonstrated a decrease of cell viability of approximately 20% at the concentration of 100 lM, supporting our results. Subsequently, the activity of the efflux transporters was tested by applying concentrations that did not alter cell viability (0.13 and 1.3 lM de MP). It is important to notice that the lower concentration used here is allowed by the Brazilian law for freshwater. For the efflux transporters activity test, it was used fluorescent Rho123, which has been reported as a substrate for both P-gp and some MRPs (Sturm et al.,
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Fig. 3. Gene expression of efflux transporters Abcb1 (a), Abcc4 (b), Abcc1 (c) and Abcg2a (d) in ZFL cells treated with MP or MP + VER (30 lM) for 24 hours. For the MP treatment, control group was treated with acetone. For the MP + VER treatment, control group was exposed to acetone and VER. The values are relative to the control group and expressed as median ± standard error (n = 4, from independent experiments). Significant differences (p < 0.05) between control and treated cells are indicated by asterisks.
Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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Fig. 4. Expression of ADS-related genes Nrf2 (a), Gst1a (b), Gclc (c) and Cu-Zn SOD (d) in ZFL cells treated with MP or MP + VER (30 lM) for 24 hours. For the MP treatment, control group was treated with acetone. For the MP + VER treatment, control group was exposed to acetone and VER. The values are relative to the control group and expressed as median ± standard error (n = 4, from independent experiments). Significant differences (p < 0.05) between control and treated cells are indicated by asterisks.
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2001a,b; Saengkhae et al., 2003; Zaja et al., 2008b). Our study has demonstrated an increase in the efflux transporters activity at both MP concentration tested (Fig. 2), indicating that this pesticide could be directly or indirectly acting as a substrate for ABC transporters. Buss and Callaghan (2008) suggested that MP act as both, a P-gp substrate and inhibitor, and this different response might be directly related to the applied MP concentration. Corroborating our results, Sreeramulu et al. (2007) observed an increase of P-gp ATPase activity in human cells exposed to MP at similar concentrations of those used in our study. In addition, the same authors have showed that concentrations higher than 100 lM induce a decrease of 25% in P-gp ATPase activity. Although there are studies relating MP toxicity to P-gp activity, not many information is available about the relation between expression of ABC transporter genes and MP detoxification. In this sense, we have analyzed the gene expression of four ABC transporters (Abcb1, Abcc1, Abcc4 and Abcg2a), which have been reported to act in the defense against diverse toxic compounds. Despite the observed increase in activity, there were no significant differences in the expression of those genes (Fig. 3). Recent studies applying a proteomic approach in ZFL cells as well as in brain of zebrafish exposed to MP did not find any alteration in the ABC transporters (Huang and Huang, 2011, 2012; Ling et al., 2012). We have considered two hypothetical scenarios which could be resulting in this response. Firstly, this response could be related to the time needed for gene activation, which could be prior to 24 h. Secondly, the chosen concentrations for the pesticide are too low and, consequently, the increase in the transporters activity could be enough to extrude the MP and its metabolites of cells without implying in an increase of transcription.
The competitive inhibition of efflux transporters is considered an evidence of the MXR functional activity in cells (Toomey and Epel, 1993; Zaja et al., 2007). In the present study, we have evaluated the cell viability, activity, and gene expression using verapamil, an inhibitor of calcium channels (Ford and Hait, 1990), as a competitive inhibitor to modulate MP efflux in ZFL cells. Verapamil has been described to affect P-gp activity (Sturm et al., 2001a). However, Zaja et al. (2008b) have shown that, at high concentrations, this drug loses its specificity and inhibits transporters from other families such as MRPs. Thus, we have used a high verapamil concentration (30 lM), which did not affect the cell viability (data not shown), intending to inhibit both P-gp and MRPs. We have presented here that MP + VER co-exposure caused a significant decrease in ZFL cells viability (Fig. 1) at the concentration of 6.5 lM and no effect in the absence of the inhibitor. This result suggests that, when decreasing the efflux activity, there is an intracellular increase of MP. This confirms that MXR acts as a first line of defense against toxic compounds and that the sensitization of this mechanism directly affects cell viability (Epel, 1998; Bard, 2000; Buss and Callaghan, 2008; Faria et al., 2011). The effect of verapamil as an inhibitor was verified by the evaluation of the ABC transporters activity in ZFL cells exposed to MP + VER. Significant decrease of 35% and 63% was observed at the MP concentrations of 0.13 and 1.3 lM, respectively. Dealing with this stressful situation generated by efflux inhibition and increasing of intracellular MP, we have evaluated the response of the ABC transporter genes. The results have demonstrated a significant increase of the Abcb1 gene transcripts at the higher concentration applied (Fig. 3a). Many studies have shown that Pgp is activated by pesticides (Srinivas et al., 2004; Sreeramulu
Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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et al., 2007; Aurade et al., 2010). Besides, it is known that P-gp is the main transporter involved in the efflux of non-modified xenobiotics (Bard, 2000; Smital et al., 2003; Zucchi et al., 2010). Therefore, the increase of Abcb1 gene expression could be related to the increase of intracellular MP after the inhibition in attempt to extrude this xenobiotic still not metabolized. The expression analysis of the Abcg2a gene did no reveal any difference in the presence of VER. This result suggests that this gene might not be related to the mechanism of MP efflux in ZFL cells at the tested concentrations. On the other hand, a significant increase was detected in Abcc4 gene transcripts at both MP + VER concentrations tested. ABCC4 has not been associated with the detoxification from pesticides, but it has been related to metals such as antimony (Leprohon et al., 2009) and cadmium (Long et al., 2011). This transporter belongs to the MRP subfamily, which has affinity for compounds conjugated with glutathione (Smital et al., 2004; Dean and Annilo, 2005; Annilo et al., 2006). Thus, our results suggest that the increase of Abcc4 gene expression could be related to increase of metabolized MP. Intending to confirm this hypothesis, genes involved with the antioxidant defense system were analyzed (Fig. 4). It was observed a twofold increase in the expression of Nrf2 and Cu-Zn SOD genes at the highest MP concentration tested in cells treated with verapamil in relation to cells without the inhibitor. The increased gene expression of a transcriptional factor such as nrf2 implies in the up-regulation of genes involved in phase II and III of detoxification mechanisms (Li et al., 2008; Niture et al., 2010). We also found an increase of Gclc gene expression in ZFL cells treated with VER and 1.3 lM MP, suggesting that glutathione is being used for detoxification, as observed in the liver of trout exposed to MP (Isik and Celik, 2008). Another ADS gene analyzed, Gst1a, showed significant decrease at the MP concentration of 1.3 lM in the treatment without VER. This result may be related to the GST isoform tested. Trute et al. (2007) demonstrated that the mu-like GST isoform could also catalyze the conjugation of MP metabolites with GSH in rat, mice and human. On the contrary, Abel et al. (2004) observed that Gst1a (hGSTA1-1) is the most implied isoform in the conjugation of MP metabolites in human liver. Additionally, it was observed a decrease in Abcc1 gene transcription levels after exposure to MP + VER. Possibly, this diminishing was caused by a preference in the recruitment of more specific transporters to the detoxification from MP, and may be associated with energy saving. Loncar et al. (2010), while analyzing the gene expression in the liver of the rainbow trout, have found extremely low levels of this transporter. Such fact suggests that this isoform might have minor relevance in the mechanisms of cell efflux in fish liver. In conclusion, our study was the first to identify the efflux transporters related to MP detoxification in ZFL cells. The increased expression of the Abcb1 and Abcc4 genes after MP + VER exposure has shown that ABCB1 and ABCC4 transporters are related to extrusion of non-conjugated and conjugated MP, respectively. Considering that the MXR mechanism is a primary defense against toxic agents, the presence of inhibitors of this mechanism (chemosensitization) in the environment might have ecotoxicological relevance. The inhibition of efflux transporters by environmental pollutants may result in an increased susceptibility of the organQ3 isms to certain toxic substances. Conflict of Interest
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The authors declare that there are no conflicts of interest.
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Achard et al. (2004).
Acknowledgements
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This work was supported by Brazilian CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). L.F. Marins is a research fellow from CNPq (Proc. No. 304675/2011-3).
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Contents lists available at ScienceDirect
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ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells
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Bruna da Silva Félix Nornberg, Carolina Reyes Batista, Daniela Volcan Almeida, Gilma Santos Trindade, Luis Fernando Marins ⇑
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Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Brazil
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Article history: Received 2 July 2014 Accepted 18 September 2014 Available online xxxx Keywords: ABC transporters Methyl parathion Multixenobiotic resistance (MXR) Verapamil
a b s t r a c t The multi-xenobiotics resistance (MXR) mechanisms are the first line of defense against toxic substances in aquatic organisms and present great importance in the adaptation related to contaminated environments. Methyl parathion (MP) is a widely used organophosphate pesticide, which has been associated to various toxic effects in organisms. In the present work, we studied the main genes related to efflux transporters in zebrafish liver (ZFL) cells exposed to MP with and without an inhibitor of ABC transporters (verapamil). The results concerning transporters activity showed that the MXR mechanism is activated to detoxify from methyl parathion. The toxic effects of MP on ZFL cells were increased in the presence of the efflux transporter inhibitor, once cell viability was significantly decreased in co-exposure experiments. The combined exposure to MP and the inhibitor caused an increase in gene expression of P-gp1 (Abcb1) and MRP4 (Abcc4), suggesting that these transporters isoforms are associated with MP efflux. In general, the expression of genes related to the antioxidant defense system (ADS) was significantly increased in ZFL cells co-exposed to MP and verapamil. These data provide useful insights for better understanding of MP detoxification mechanism in fish hepatocytes. Ó 2014 Published by Elsevier Ltd.
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1. Introduction
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Aquatic organisms are constantly exposed to many pollutants dissolved in the water and great part of these organisms can tolerate exposure to these compounds. Kurelec (1992) has shown that this tolerance capacity is related to the multi-xenobiotics mechanism (MXR), which is considered the first line of defense against toxic compounds (Epel, 1998). MXR is widely distributed in biological systems, from unicellular organisms to vertebrates (Annilo et al., 2006). It is mediated by the transmembrane transporters proteins from ATP-binding cassette (ABC) family, which are responsible for the excretion of many xeno/endobiotics from the cell by ATP hydrolysis (Bard, 2000). ABC transporters constitute a highly conserved superfamily of proteins among vertebrates and exert many functions in the organisms. In zebrafish, the ABC transporter genes are organized in 8 sub-families, which are designated from ABCA to ABCH (Dean and Annilo, 2005). Some ABC transporters from ABCB, ABCC and ABCG subfamilies have been reported
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⇑ Corresponding author at: Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Av. Itália km 8, 96203-900 Rio Grande, RS, Brazil. E-mail address: [email protected] (L.F. Marins).
with functions related to MXR (Buss and Callaghan, 2008; Zucchi et al., 2010; Faria et al., 2011). The ABCB1 (P-gp, MDR1), members of the ABCC (MRP) subfamily and breast cancer resistance protein (BCRP, ABCG2) are toxicologically the most relevant proteins in efflux of xenobiotics in mammals (Doyle and Ross, 2003). P-gp may act eliminating hydroxylated xenobiotics metabolites after its modification by phase I enzymes (cytochromes P450), or leading non-metabolized xenobiotics out of the cell (Bard, 2000; Xu et al., 2005). MRPs have been reported to promote resistance against heavy metals in some mammals and fish species (Zucchi et al., 2010; Long et al., 2011). Besides, MRP and P-gp present significant overlap in substrate specificities, particularly evincing affinity for compounds conjugated with glutathione, glucuronate or sulfate (Cole et al., 1994; Buss and Callaghan, 2008). ABCG2 is a widely studied transporter in humans due to its capacity to grant resistance to chemotherapics used against breast cancer. Concerning fish species, Abcg2 expression was detected in liver of rainbow trout Oncorhynchus mykiss (Zaja et al., 2008a). In zebrafish, four paralogs of Abcg2 gene were already identified (Annilo et al., 2006). ABCB1 and ABCG2 transporters are located in the apical membrane of cells, facilitating the movement of substances and promoting the excretion of xenobiotics. ABCB1 substrates are amphiphilic
http://dx.doi.org/10.1016/j.tiv.2014.09.010 0887-2333/Ó 2014 Published by Elsevier Ltd.
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or moderately hydrophobic, with a low molecular weight, positively charged or neutral, including both endogenous and exogenous compounds. Thus, the number of potential substrates of Pg-p can be estimated at several hundred (Smital et al., 2003, 2004; Buss and Callaghan, 2008). According to Ito et al. (2005), Abcg2 is expressed in different organs involved in absorption (small intestine), distribution (placenta and blood brain barrier) and elimination (liver), indicating an important role of ABCG2 in excretion of xenobiotics. In mammals, the localization of ABCC4 (MRP4) and ABCC1 (MRP1) in polarized cells is variable. These transporters can be localized on the apical or basolateral membranes depending on the cells type (Luckenbach et al., 2014). Moreover, there are reports about MRP4 expression in some human organs such as prostate, testis, ovary, intestine, pancreas and lung (Sturm and Segner, 2005). ABCC1 is found in lung, testis, kidney, placenta, heart and skeletal muscle (Deeley et al., 2006; Bakos and Homolya, 2007). In fish, the notable Abcc1 and Abcc4 gene expression was found in kidney and high Abcc4 gene expression in gonads (Loncar et al., 2010). Therefore, the implication of these transporters in xenobiotics detoxification in aquatic organisms requires more studies. The synergistic detoxification activity of ABC transporters and enzymes from the antioxidant defense system (ADS) has been suggested (Bard, 2000; Xu et al., 2005). Increased expression of these transporters, as well as phase II enzymes, has been reported in aquatic organisms exposed to different contaminants (Bresolin et al., 2005; Leslie et al., 2005). One important component that modulates these two processes is the NRF2 transcription factor (nuclear factor erythroid 2 – relative to factor 2). This molecule is a key regulator of a cytoprotective network against exposure to electrophilic and oxidative chemicals, by controlling the transcriptional activity of genes coding for ADS enzymes, such as glutathione-S-transferase (GST), glutamate cysteine ligase (GCL), and superoxide dismutase (SOD), as well as for efflux transporters proteins (Osburn and Kensler, 2008; Niture et al., 2010). Several classes of contaminants may induce the MXR mechanism, such as herbicides, fungicides and insecticides (Buss and Callaghan, 2008). Among the most commonly used pesticides are the organophosphates, which contain phosphate groups derived from phosphoric acid. Methyl parathion (MP) is an organophosphate pesticide, which acts by inhibiting acetylcholinesterase (Ecobichon et al., 1990; Küster, 2005). It is highly toxic after conversion to paraoxon by oxygenation in the endoplasmic reticulum of hepatocytes. MP has been reported as a substrate for efflux transporters, acting as an inducer or inhibitor (Sreeramulu et al., 2007). Many types of fish cell lines have been established and applied to test the toxicity of pesticides, including hepatoma cells PLHC-1 (Knauer et al., 2007), hepatic cells RTL-W1 from rainbow trout (Babin and Tarazona, 2005) and zebrafish liver (ZFL) cells (Huang and Huang, 2012). However, there are few studies relating the toxicity of pesticides with ABC efflux transporters in fish cell lines. In this context, the goal of this study was to identify ABC efflux transporters potentially involved in detoxification of methyl parathion in zebrafish liver cells.
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2. Materials and methods
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2.1. Chemicals
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Methyl parathion (MP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), verapamil (VER) and Rhodamina123 (Rho-123) were purchased from Sigma (Brazil). TRIzol reagent, DNAse I Amplification Grade, Quant-iTTM RNA assay kit, Platinum SYBR Green qPCR SuperMix-UDG and 20,70-dichlorofluorescin diacetate (H2DCF-DA) were obtained from Invitrogen (Brazil). High
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Capacity cDNA Reverse Transcription was acquired from Applied Biosystems (Brazil). All tissue-culture products were from Gibco (Brazil). Zebrafish hepatocytes (line ZFL; CRL2643) were obtained from American Type Culture Collection (ATCC).
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2.2. Growth and treatment of cells
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Adult zebrafish liver cell line (ATCCCRL-2643) were cultivated in RPMI 1640 medium supplemented with sodium bicarbonate (0.2 g L1), L-glutamine (0.3 g L1), HEPES (25 mM) and b-mercaptoethanol (5 105 M), with 10% fetal bovine serum, 100 IU/ml penicillin and 100 lg mL1 streptomycin. Cells were grown in cell culture flasks at 28 °C. One day prior to experiments, cells were seeded in 24 or 96 well plates, in a density of 106 cells or 5 105 cells, into 1 or 0.2 mL of medium per well, respectively.
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2.3. MTT assay
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The MTT test assesses cell metabolism based on the ability of the mitochondrial succinate–tetrazolium reductase system to convert the yellow compound MTT to a blue formazan dye. The amount of dye produced is proportional to the number of live metabolically active cells. The assay was carried out as previously described (Mosmann, 1983). Briefly, cells were seeded in 96-well microplates and incubated overnight. Subsequently, the cells were treated with MP at concentrations of 0.13, 1.3, 6.5, 13, 26 and 130 lM for 24 h. The MP was dissolved in acetone (control group) and added to a final concentration of 0.01% (v/v). Selection of MP concentrations was based on Brazilian legislation (CONAMA, 2012), being the concentration of 0.13 lM indicated the maximum values permitted for freshwater. Intending to verify the involvement of the efflux transporters in the detoxification from MP, the same experimental design was carried out adding 30 lM of VER (dissolved in 96% ethanol), which was used as inhibitor of efflux transporters. Control group was exposed to acetone and VER and the final concentration of the solvents never exceeded 0.01%. MTT (5 mg mL1) dissolved in phosphate-buffered saline (PBS) was added (20 ll) to the culture wells, and plates were incubated for 4 h at 28 °C. Supernatant was removed and 200 ll of DMSO was added to each well and read at 490 nm in EL800 microplate reader (BIO-TEK Instrument, Winooski, VT). Each experiment was performed in triplicate.
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2.4. Determination of intracellular rhodamine 123
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For the transport activity measurements, the accumulation of Rho-123 was observed in ZFL cells exposed to MP with or without inhibitor of ABC transporters (VER). Hepatocytes were cultured at 28 °C for 24 h in 24-well culture plates, as previously explained. Subsequently, cells were treated with acetone (control group) or MP at concentrations of 0.13 and 1.3 lM for 24 h. The same experimental design was carried out adding VER (30 lM). After exposure, cells were washed in PBS and incubated in the dark at 28 °C for 1 h in 1 mL of fresh medium containing Rho-123 (300 ng mL1) with or without VER (Ludescher et al., 1998). Next, cells were Q2 washed twice in PBS, resuspended in 1 mL of fresh medium and incubated for 30 min for Rho-123 efflux. After this, cells were washed again and then fluorescence accumulation was verified using a microplate reader (Victor2, Perkin Elmer Wallac, Boston, MA) at 485 and 590 nm of excitation and emission, respectively. These experiments were replicated three times. Therefore, efflux transporters activity was expressed as the inverse of Rho-123 fluorescence accumulation per number of cells. The results were expressed as percentage of fluorescence in relation to control group.
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2.5. Gene expression analysis
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For gene expression, hepatocytes of zebrafish were exposed to 0.13 and 1.3 lM of MP and acetone (control group), with or without VER (30 lM) for 24 h in 24-well culture plate. These experiments were replicated four times and each sample was composed by a pool of four wells. Total RNA was isolated, for later qRT-PCR analysis, by Trizol Reagent (Invitrogen), following the manufacturer’s instructions. Genomic DNA was degraded with Deoxyribonuclease I (DNAse I) and total RNA was quantified by a QubitTM fluorometer using the Quant-iT™ RNA assay kit (Invitrogen). First-strand cDNA was synthesized from approximately 1 lg of total RNA using High Capacity cDNA Reverse Transcription kit (Invitrogen), according to the manufacturer’s instructions. The expression of ADS and efflux transporters genes was analyzed by real-time RT-PCR (7500 Real Time System, Applied Biosystems, Brazil), using SYBR GREEN PCR Master Mix™ (Invitrogen), according to manufacturer’s protocol. Each sample (n = 4) was analyzed in triplicate. Specific primers for each gene (Table 1) were designed using the Primer-BLAST tool from GenBank (http:// www.ncbi.nlm.nih.gov). Serial dilutions were performed in order to determine the qPCR reaction efficiency. Gene expression was carried out for 50 °C/2 min, 95 °C/2 min, followed by 40 cycles at 95 °C/15 s and 60 °C/30 s. Target gene expression was normalized by the expression of the elongation factor-1 alpha (ef1-a) gene.
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2.6. Data analysis
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The software RESTÓ was applied in the relative quantification of gene expression analyses (Pfaffl et al., 2002), by realizing a paired comparison between control group and treatments. The adopted alpha was 0.05 and results were expressed as median ± standard error One-way ANOVA was carried out, followed by an a posteriori means comparison (Newman–Keuls test, p < 0.05), for efflux transporters ac.tivity and cell viability assay. ANOVA assumptions (normality and homogeneity of variance) were verified prior to ANOVA analysis.
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3. Results
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3.1. Cell viability
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MTT assay for cells viability are shown in Fig. 1. Cells treated with 13, 26 and 130 lM of MP had significant (p < 0.05) viability decrease (83.1 ± 4.8, 73 ± 5.4 and 69 ± 3.2%, respectively) compared with control group (100 ± 4.9%). However, when verapamil was added, a significant (p < 0.05) decrease was observed at the concentrations 6.5, 13, 26 and 130 lM (67.2 ± 9.0, 51 ± 5.4, 47 ± 7.1 and 40 ± 1.3%, respectively) when compared with control group (100 ± 14%). No significant (p > 0.05) differences in viability was verified in hepatocytes treated with 0.13 and 1.3 lM of MP, with (78 ± 2.5 and 77 ± 5.3%, respectively) or without verapamil
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Fig. 1. ZFL cells viability exposed to controls and different concentrations of methyl parathion (0.13, 1.3, 6.5, 13, 26 and 130 lM) with 30 lM verapamil (filled circles and continuous line) and without verapamil (pierced circles and dotted line) for 24 h exposure. Values are expressed as percentages of viable cells in relation to control groups. Different symbols (⁄ and #) indicate significant differences (p < 0.05) between treatments and their respective control groups. Data are expressed as mean ± S.E (n = 3, from independent experiments).
(96 ± 4.1 and 98 ± 2.5%, respectively) when compared with control groups. Therefore, for efflux transporters activity and gene expression experiments, these sublethal concentrations were adopted.
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3.2. Functional analysis of the efflux transporters
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Zebrafish hepatocytes exposed to MP concentrations of 0.13 and 1.3 lM (Fig. 2) have shown a significant increase (p < 0.05) of ABC transporters activity (177.8 ± 37.4 and 188.6 ± 23.1, respectively) when compared with control group (100 ± 13.5%). However, when the inhibitor was added, a significant decrease was observed at MP concentrations of 0.13 and 1.3 lM (65 ± 17.3 and 37.2 ± 11.1, respectively), when compared with the control group (116.0 ± 17.6%), as shown in Fig. 2.
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3.3. Gene expression
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The genes analyzed in the present study are involved in the defense system against xenobiotics and are grouped according to their functions: ABC transporters (Abcb1, Abcc1, Abcc4 and Abcg2a; Fig. 3) and antioxidant defense system – ADS (Gst1a, Gclc, Nrf2 and Cu-Zn SOD; Fig. 4). No significant differences (p > 0.05) were found in the expression of Abcb1, Abcc1, Abcc4 and Abcg2a genes in zebrafish hepatocytes exposed to MP for 24 h (Fig. 3a). However, coexposure to MP + VER (Fig. 3b) presented significant induction (p < 0.05) of 4.4-fold in the expression of the gene coding for Abcb1 at the highest MP concentration. Also, a significant increase of 3.4and 4.8-fold induction was observed in the gene expression of Abcc4 at MP + VER concentrations of 0.13 and 1.3 lM, respectively. On the other hand, a significant decrease of approximately 80% in Abcc1 gene expression, at both tested MP concentrations, in the presence of the inhibitor (Fig. 3c).
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Table 1 Genes and PCR primers used for quantitative PCR analysis. Genes
Foward primer
Reverse primer
GenBank
Ef1a Nrf2 Gclc Cu,Zn-Sod Gst1a Abcb1/MDR1 Abcc1/MRP1 Abcc4/MRP4 Abcg2a/BCRP
50 -GGGCAAGGGCTCCTTCAA-30 50 -TGTTGGTTCGGAGGCTCTTAA-30 50 -ACGGCATTCCCCAGGTTAG-30 50 -GGAAGAGCCGGTTGAAATATTG-30 50 -CCCCAAAATACAGGCGTTTC-30 50 -CCTCGGTCAAACGTCTCCAA-30 50 -ATGCTGCCCGGCGAACATCC-30 50 -CACCTTGGGAAACTCAAAAACG-30 50 -GGAACAACAGCGGCAGTGT-30
50 -CGCTCGGCCTTCAGTTTG-30 50 -AGGCCATGTCCACACGTACA-30 50 -TTTTCAACAGGTGTGGGTTTGTA-3’ 50 -AGCGGGCTAAGTGCTTTCAG-30 50 -GCCCGGCTGGAGGAACT-30 50 -CTTTATGGGCGGCTCCTCTA-30 50 -AGGTCCATGAACCGCTGCGC-30 50 -GAAGGTTATCAGGCCACGATTT-30 50 -TGCCCTGCTCCTGAAAAAAC-30
NM_131263.1 NM_182889 NM_199277 NM_131294 NM_213394 NM_001114583 XM_001341859 NM_001007038 NM_001042775
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Fig. 2. Efflux transporters activity in ZFL cells exposed to MP (0.13 and 1.3 lM) without verapamil and with 30 lM verapamil (MP + VER). Control group values were set as 100%. MP control group is treated with acetone, while MP + VER control group is treated with acetone + VER. Data are expressed as a percentage of control culture fluorescence corrected by cell number ± S.E. (n = 3, from independent experiments). Asterisk indicates significant differences from control group (p < 0.05).
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Regarding ADS genes, a significant induction was found for Nrf2 and Cu-Zn SOD at the higher MP concentration (2.2- and 1.6-fold, respectively; Fig. 4a and d). Co-exposure of the cells to MP + VER has also increased genes Nrf2 and Cu-Zn SOD gene expression at the higher MP concentration, evincing a 4.8- and 2.5-fold induction, respectively (Fig. 4a and d). In this sense, we have also found a significant induction of 3.6-fold in Gclc gene at the higher MP + VER concentration (Fig. 4c). However, it was observed a Gst1a downregulation of about 50% at the higher MP concentration without VER (Fig. 4b).
4. Discussion
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Organophosphate pesticides are environmental pollutants known to inhibit the catalytic activity of acetylcholinesterase (AChE). Methyl parathion (O,O-dimethyl O-4-nitro-phenylphosphoro-thioate) is widely used in agriculture due to its low costs and high efficiency in eliminating plagues (Ma et al., 2003). Once it is a commonly used pesticide, the toxicity effects of MP have been thoroughly studied. Many studies have shown that this pesticide may cause DNA damage in human and rats’ spermatozoa (Arredondo et al., 2008; Piña-Guzmán et al., 2009). Regarding fish species, MP has been shown to induce oxidative stress, resulting in lipid peroxidation, increase of anti-oxidant enzymes, as well as decrease of glutathione levels (Monteiro et al., 2006). However, there are few reports in literature relating the MP toxicity to the MXR mechanism in aquatic organisms. Therefore, the aim of the present work was to identify efflux transporters genes potentially involved in detoxification from MP. We have firstly tested the cell viability when challenged with different MP concentrations (Fig. 1). Our results have shown that MP causes dose-dependent cell death. Huang and Huang (2012) used zebrafish cells exposed to high MP concentration (0–600 lM) and demonstrated a decrease of cell viability of approximately 20% at the concentration of 100 lM, supporting our results. Subsequently, the activity of the efflux transporters was tested by applying concentrations that did not alter cell viability (0.13 and 1.3 lM de MP). It is important to notice that the lower concentration used here is allowed by the Brazilian law for freshwater. For the efflux transporters activity test, it was used fluorescent Rho123, which has been reported as a substrate for both P-gp and some MRPs (Sturm et al.,
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Fig. 3. Gene expression of efflux transporters Abcb1 (a), Abcc4 (b), Abcc1 (c) and Abcg2a (d) in ZFL cells treated with MP or MP + VER (30 lM) for 24 hours. For the MP treatment, control group was treated with acetone. For the MP + VER treatment, control group was exposed to acetone and VER. The values are relative to the control group and expressed as median ± standard error (n = 4, from independent experiments). Significant differences (p < 0.05) between control and treated cells are indicated by asterisks.
Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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Fig. 4. Expression of ADS-related genes Nrf2 (a), Gst1a (b), Gclc (c) and Cu-Zn SOD (d) in ZFL cells treated with MP or MP + VER (30 lM) for 24 hours. For the MP treatment, control group was treated with acetone. For the MP + VER treatment, control group was exposed to acetone and VER. The values are relative to the control group and expressed as median ± standard error (n = 4, from independent experiments). Significant differences (p < 0.05) between control and treated cells are indicated by asterisks.
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2001a,b; Saengkhae et al., 2003; Zaja et al., 2008b). Our study has demonstrated an increase in the efflux transporters activity at both MP concentration tested (Fig. 2), indicating that this pesticide could be directly or indirectly acting as a substrate for ABC transporters. Buss and Callaghan (2008) suggested that MP act as both, a P-gp substrate and inhibitor, and this different response might be directly related to the applied MP concentration. Corroborating our results, Sreeramulu et al. (2007) observed an increase of P-gp ATPase activity in human cells exposed to MP at similar concentrations of those used in our study. In addition, the same authors have showed that concentrations higher than 100 lM induce a decrease of 25% in P-gp ATPase activity. Although there are studies relating MP toxicity to P-gp activity, not many information is available about the relation between expression of ABC transporter genes and MP detoxification. In this sense, we have analyzed the gene expression of four ABC transporters (Abcb1, Abcc1, Abcc4 and Abcg2a), which have been reported to act in the defense against diverse toxic compounds. Despite the observed increase in activity, there were no significant differences in the expression of those genes (Fig. 3). Recent studies applying a proteomic approach in ZFL cells as well as in brain of zebrafish exposed to MP did not find any alteration in the ABC transporters (Huang and Huang, 2011, 2012; Ling et al., 2012). We have considered two hypothetical scenarios which could be resulting in this response. Firstly, this response could be related to the time needed for gene activation, which could be prior to 24 h. Secondly, the chosen concentrations for the pesticide are too low and, consequently, the increase in the transporters activity could be enough to extrude the MP and its metabolites of cells without implying in an increase of transcription.
The competitive inhibition of efflux transporters is considered an evidence of the MXR functional activity in cells (Toomey and Epel, 1993; Zaja et al., 2007). In the present study, we have evaluated the cell viability, activity, and gene expression using verapamil, an inhibitor of calcium channels (Ford and Hait, 1990), as a competitive inhibitor to modulate MP efflux in ZFL cells. Verapamil has been described to affect P-gp activity (Sturm et al., 2001a). However, Zaja et al. (2008b) have shown that, at high concentrations, this drug loses its specificity and inhibits transporters from other families such as MRPs. Thus, we have used a high verapamil concentration (30 lM), which did not affect the cell viability (data not shown), intending to inhibit both P-gp and MRPs. We have presented here that MP + VER co-exposure caused a significant decrease in ZFL cells viability (Fig. 1) at the concentration of 6.5 lM and no effect in the absence of the inhibitor. This result suggests that, when decreasing the efflux activity, there is an intracellular increase of MP. This confirms that MXR acts as a first line of defense against toxic compounds and that the sensitization of this mechanism directly affects cell viability (Epel, 1998; Bard, 2000; Buss and Callaghan, 2008; Faria et al., 2011). The effect of verapamil as an inhibitor was verified by the evaluation of the ABC transporters activity in ZFL cells exposed to MP + VER. Significant decrease of 35% and 63% was observed at the MP concentrations of 0.13 and 1.3 lM, respectively. Dealing with this stressful situation generated by efflux inhibition and increasing of intracellular MP, we have evaluated the response of the ABC transporter genes. The results have demonstrated a significant increase of the Abcb1 gene transcripts at the higher concentration applied (Fig. 3a). Many studies have shown that Pgp is activated by pesticides (Srinivas et al., 2004; Sreeramulu
Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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et al., 2007; Aurade et al., 2010). Besides, it is known that P-gp is the main transporter involved in the efflux of non-modified xenobiotics (Bard, 2000; Smital et al., 2003; Zucchi et al., 2010). Therefore, the increase of Abcb1 gene expression could be related to the increase of intracellular MP after the inhibition in attempt to extrude this xenobiotic still not metabolized. The expression analysis of the Abcg2a gene did no reveal any difference in the presence of VER. This result suggests that this gene might not be related to the mechanism of MP efflux in ZFL cells at the tested concentrations. On the other hand, a significant increase was detected in Abcc4 gene transcripts at both MP + VER concentrations tested. ABCC4 has not been associated with the detoxification from pesticides, but it has been related to metals such as antimony (Leprohon et al., 2009) and cadmium (Long et al., 2011). This transporter belongs to the MRP subfamily, which has affinity for compounds conjugated with glutathione (Smital et al., 2004; Dean and Annilo, 2005; Annilo et al., 2006). Thus, our results suggest that the increase of Abcc4 gene expression could be related to increase of metabolized MP. Intending to confirm this hypothesis, genes involved with the antioxidant defense system were analyzed (Fig. 4). It was observed a twofold increase in the expression of Nrf2 and Cu-Zn SOD genes at the highest MP concentration tested in cells treated with verapamil in relation to cells without the inhibitor. The increased gene expression of a transcriptional factor such as nrf2 implies in the up-regulation of genes involved in phase II and III of detoxification mechanisms (Li et al., 2008; Niture et al., 2010). We also found an increase of Gclc gene expression in ZFL cells treated with VER and 1.3 lM MP, suggesting that glutathione is being used for detoxification, as observed in the liver of trout exposed to MP (Isik and Celik, 2008). Another ADS gene analyzed, Gst1a, showed significant decrease at the MP concentration of 1.3 lM in the treatment without VER. This result may be related to the GST isoform tested. Trute et al. (2007) demonstrated that the mu-like GST isoform could also catalyze the conjugation of MP metabolites with GSH in rat, mice and human. On the contrary, Abel et al. (2004) observed that Gst1a (hGSTA1-1) is the most implied isoform in the conjugation of MP metabolites in human liver. Additionally, it was observed a decrease in Abcc1 gene transcription levels after exposure to MP + VER. Possibly, this diminishing was caused by a preference in the recruitment of more specific transporters to the detoxification from MP, and may be associated with energy saving. Loncar et al. (2010), while analyzing the gene expression in the liver of the rainbow trout, have found extremely low levels of this transporter. Such fact suggests that this isoform might have minor relevance in the mechanisms of cell efflux in fish liver. In conclusion, our study was the first to identify the efflux transporters related to MP detoxification in ZFL cells. The increased expression of the Abcb1 and Abcc4 genes after MP + VER exposure has shown that ABCB1 and ABCC4 transporters are related to extrusion of non-conjugated and conjugated MP, respectively. Considering that the MXR mechanism is a primary defense against toxic agents, the presence of inhibitors of this mechanism (chemosensitization) in the environment might have ecotoxicological relevance. The inhibition of efflux transporters by environmental pollutants may result in an increased susceptibility of the organQ3 isms to certain toxic substances. Conflict of Interest
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The authors declare that there are no conflicts of interest.
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Achard et al. (2004).
Acknowledgements
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This work was supported by Brazilian CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). L.F. Marins is a research fellow from CNPq (Proc. No. 304675/2011-3).
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Please cite this article in press as: da Silva Félix Nornberg, B., et al. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.09.010
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