Scientific Reports volume 12, Article number: 20102 (2022 ) Cite this article
Naef AA Qasem, Ramy H. Mohammed & Dahiru U. Lawal 4-Tert-Octylphenol Diethoxylate
Michael P. Murphy, Hülya Bayir, … Barry Halliwell
4-tOP is a component of non-ionic surfactants alkyl phenol polyethoxylates while TCS is an antibacterial present in personal care products. The two compounds can co-exist in environmental matrices and their high levels have been recorded thereby affording humans and wildlife sufficient exposure for toxicity. The compounds are environmental toxicants following their demonstrated endocrine disruption1,2. Data are accumulating linking environmental factors via endocrine disrupting molecules to the increase in metabolic syndrome3. Cells such as β-cells which express oestrogen receptors are affected by endocrine disruptors such as 4-tOP and TCS4. Reports of Ajao et al.5 and Weatherly et al.6 suggested that TCS targets mitochondria in the β-cells but that of 4-tOP with respect to mitochondrial toxicity has not been precisely defined. Following the two compounds ability to coexist in environmental matrix, the resulting toxicity in vitro or in vivo deserves elucidation. INS-1, the rat insulinoma cell line has been widely applied as an in vitro model for β-cell metabolism. We have used the model to present biochemical evidence of toxicity in INS-1 cells by the two household endocrine disruptors individually and in mixture.
Cell viability was assessed by a principle of enzymatic reduction of 3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) to formazan. The treated cells respectively were loaded with 20 µL 0.5 mg/mL MTT and incubated at 37 °C for 3 h. The MTT was removed and 200 µL DMSO/well added to solubilize the formazan crystals. The absorbance of the purple-coloured formazan was measured at 540 nm on microplate reader. IC50, (the median inhibitory concentration) was calculated with GraphPad Prism software. Data were expressed as a percentage of control.
Glucose uptake was determined in INS-1 cells by monitoring the levels of glucose remaining in the culture medium after a selected treatment period. The cell density was 30 000 per well; one row was without cells to serve as maximum glucose content. Spent medium was removed and the cells were treated with 100 µL of the test compounds at concentrations indicated in section “Treatment of INS-1 cells”. The cells were incubated for a further 48 h and 5µL spent culture medium was thereafter transferred to a new plate of 96 wells and 200 µL glucose oxidase reagent added. Incubation was at 37 °C for 20 min and absorbance was read at 520 nm. Glucose uptake was obtained as the difference in glucose concentration between wells without cells and those with cells and respective treatments.
The spent medium in section “Treatment of INS-1 cells” was removed and 50 µL CellROX staining solution was added to each well according to the manufacturer’s instructions and incubated at 37 °C for 30 min. Images from nine sites per well were acquired using filter sets appropriate for DAPI (4′, 6-diamidino-2-phenylindole) and TRITC (tetramethylrhodamine-isothiocyanate) which were the fluorophors. Image acquisition was done on ImageXpress Micro XLS and analyzed using the Multi-Wavelength Scoring software (Molecular Devices). In another experiment 50 µL ThiolTracker Violet staining solution was put in each well and incubated at 37 °C for 20 min. Images from nine sites per well were acquired using filter sets appropriate for DAPI and CY5 (amine reactive fluorescent dye). Image acquisition and data analysis were as done for the CellROX orange reagent. Excitation/Emission wavelength was at 545/565 nm.
The treated cells were sub-cultured at 4:3. Annexin V-FITC staining assay was performed as recommended by the manufacturer. Melphalan (Mel) and Etoposide (Etop) served as the positive controls. Incubation lasted 48 h. The spent medium was removed and 50 µL PI staining solution was in each well and incubated for 15 min at 37 °C followed by image acquisition and data analysis as in previous experiments. Images from nine sites per well were acquired using filter sets appropriate for DAPI, FITC and Texas red dyes.
Additional 100 µL test compounds were solubilized in the medium and incubated for 48 h. The spent medium was replaced with 100 µL of fix solution (4% formaldehyde in phosphate-buffered saline (PBS) and was fixed overnight; the cells were permeabilized with ice cold methanol, then washed with PBS and blocked using 1% BSA followed by incubation with the primary antibody at 1:200 dilution in blocking buffer for 2 h. The excess antibody was removed by washing with blocking buffer. Incubation was thereafter carried out with secondary antibody (FITC labelled) for 30 min and later washed with blocking buffer to remove excess antibody. Images from nine sites per well were acquired using filter sets appropriate for DAPI and FITC. Image analysis was done as in previous experiments.
Staining solution consisted of 10 mL of PBS to which was added 1 µL MitoTracker Green (MTG) stock (1 mM in DMSO) and 2 µL Hoechst solution (10 mg/mL in DMSO). Spent medium was removed and 50 µL staining solution added to each well (96 wells) followed by incubation at 37 °C for 20 min.
TMRE is a cell permeable cationic dye that accumulates in the mitochondrial matrix according to mitochondrial membrane potential (ΔψM). Staining solution was prepared by adding 5 mL PBS, 50 µL TMRE working solution and 2 µL Hoechst 33342 solution to 5 mL of RPMI. The spent medium was removed and 50 µL staining solution was put in each well and incubated for 30 min at 37 °C. Image acquisition and data analysis were done as indicated.
Preliminary investigation showed that the test compounds had suitable solubility properties for cell-based assays. Both compounds were completely soluble in DMSO up to 50 mM.
Figure 1 and Table 1 depict the cell viability in the presence of single or combined concentrations of the test compounds after 48 h. IC50 value was lowest in triclosan treated group. Each compound induced loss of cell viability at concentration above 50 μM.
Concentration dependent cytotoxicity of 4-tert-octylphenol, triclosan and equimolar ratio mixture of both compounds in INS-1 cells treated for 48 h.
Differential cytotoxicity of the three treatments in glucose versus galactose medium was reported in Fig. 2, which yielded the IC50 values listed in Table 2. Both TCS and DNP exhibited higher toxicity in the galactose medium than in the glucose medium while 4-tOP and the combination exposure (Mix) presented a reverse case though of little difference. All the IC50 values showed very high positive correlation (R2 > 0.9) (Table 2).
Figure 3 shows the trend of glucose consumption by INS-1 cells. Glucose consumption appeared to be concentration dependent; increased uptake was induced by low concentration of the test substances and vice versa, relative to the Blank. The mixtures (Mix) presented a reverse scenario in which those cells exposed to the high concentration of Mix displayed high glucose uptake and low Mix evoked reduced glucose uptake. These conditions were however not statistically significant when compared with the Blank (Fig. 3). Only cells exposed to Etop showed significant increase in glucose uptake relative to the Blank.
Effects of the test substances on cellular glucose uptake. p value* = 0.021, n = 5. TCS High/Low, Triclosan High/Low concentration; 4-tOP High/Low, 4-tert-Octylphenol high/low concentration; Mix High/Low, TCS + 4-tOP high/low concentration.
Estimates of the activity of the reactive oxygen species. p values * = 0.04, ** = 0.0064, *** = 0.0038, n = 3.
The cellular levels of reduced glutathione (GSH) were estimated using ThioltrackerViolet dye in evaluating oxidative stress (Fig. 5). As observed with ROS, GSH metabolism in the cells treated with the test compounds or their mixtures (Mix) appeared (p > 0.05) to be concentration dependent when compared with the Blank (Fig. 5). There was a general reduction of GSH in the exposed cells. Only high TCS concentration (p = 0.0157) as well as the two positive controls, Mel (p = 0.0001) and Etop (p = 0.0001) showed significant reduction of GSH when compared with the Blank. However, all the test substances or their Mix showed significant increase in GSH concentration relative to each of the two controls.
Mechanism of cell death was studied using Annexin V-FITC, PI and Hoechst staining techniques (Fig. 6). The high proportions of live cells remaining after administration of TCS (81.2%) and 4-tOP (81.4%) at their low concentrations when compared with the Blank were not statistically significant (p = 0.22 and 0.24 respectively) (Fig. 6). Significantly reduced live cells were recorded following administrations of high concentration of TCS (43.7%, p = 0.0001) and of 4-tOP (73.8%, p = 0.0009), low (77.9%, p = 0.022) or high (41.7%, p = 0.0001) concentration of Mix as well as Mel (34.5%, p = < 0.0001) and Etop (42.5%, p = < 0.0001) when compared with the Blank. The reduction of live cells from 81.2% to 43.7% by TCS was strongly significant (p = < 0.0001), such substantial reduction was not recorded for cells exposed to 4-tOP. For the Mix, the reduction of the live cells following increase in concentration was substantially significant (p = < 0.0001). Cell reduction by the low Mix but not by the high Mix, was significant when compared with Mel or Etop (p = < 0.0001). Although Mel reduced more live cells than Etop at equal concentrations, the difference was not significant (p = 0.26) (Fig. 6).
Proportion of live cells after treatments. p values * = 0.023, *** = 0.0009, * = 0.0001, n = 4.
Significantly higher proportion of cells exposed to TCS (16.6%) and Mix (18.6%) at their high concentrations as well as Mel (15.7%) and Etop (17.4%) died by apoptosis than the Blank (Fig. 7). 4-tOP failed to significantly induce apoptosis in the cells when compared with the Blank.
Proportion of cell death by apoptosis following treatments. p values *** = 0.0002, ** = 0.0006, **** = 0.0001, n = 4.
Proportion of cell death by necrosis after treatments. p values * = 0.048, ** = 0.0032, n = 4.
Caspase 3 activation. p values * = 0.012, **** = 0.0001, n = 3.
Changes in mitochondrial membrane potential in the cells exposed to the test substances. p values **** = 0.0001, Mel *** = 0.0009, Etop **** = 0.0001, n = 4.
The loss of MMP by Mel and Etop was very substantial when compared with TCS or Mix (Fig. 11). In the analysis of glucose stimulated intracellular calcium influx (Fig. 12). Diazoxide and Glibenclamide significantly increased intracellular calcium flux when compared with the Blank in direct contrast to the test substances which reduced the flux.
Effect of the test substances on glucose stimulated intracellular calcium influx. Diazoxide and Glibenclamide as positive controls. p values **** = 0.0001, n = 4.
High content analysis or high content screening (HCA/HCS) is an automated imaging-based methodology used to assess cellular-based toxicity7,8. The cytotoxicity test aside testing for cell viability, also determined the affordability of studying the selected compounds in an in vitro biological system comprising INS-1 cells in DMSO as a medium9. Reduction of MTT occurs by mitochondrial dehydrogenase in active cells, the level of activity is therefore a measure of the viability of the cells. The result showed that TCS was more reactive to INS-1 cells than HepG2 human liver cells (IC50 24.5 μM)10. The toxicity of the combination was lower compared to that of TCS but increased toxicity compared with that of 4-tOP, meaning that the mixture effect was probably antagonistic4,11,12,13. It may be that the constituent compounds attacked different molecular targets14.
Oxidation of galactose to pyruvate via glycolysis yields no net ATP forcing cells to rely on mitochondrial oxidative phosphorylation (the Crabtree effect) to produce sufficient ATP for survival15,16. Mitochondrial toxins display greater toxicity when cultured in galactose medium than in glucose medium and hence a ratio (fold value) greater than 1 is indicative of potential mitochondrial toxicity17,18 meaning that TCS as well as DNP is a mitochondrial toxin supporting previous reports that both compounds are mitochondrial uncouplers6,19. The fold value for 4-tOP and TO was less than 1 implying negative response to mitochondrial toxicity. The observed increase in glucose consumption by 4-tOP and Mix was therefore an adaptive or compensatory mechanism rather than a mitochondrial toxicity.
CellROX oxidative stress reagents are fluorogenic probes for detecting reactive oxygen species (ROS) in live cells. ROS which include superoxide anion (O2·−) and H2O2 are generated by aerobic cells during electron transfer in the mitochondria. The role of ROS in β-cell function is complex as ROS also regulate insulin release26. GSH is a cellular antioxidant molecule which participates in catalytic cycles of antioxidant enzymes such as glutathione peroxidase and glutathione reductase. The substantial reduction of GSH concentration by high TCS was probably a response to the oxidative challenge. The increased activity of ROS with a corresponding reduction in GSH concentration recorded in the cells exposed to low 4-tOP, Mel and Etop might be that the cells were under oxidative stress27,28,29. Following extreme sensitivity of beta cells to oxidative attack30, it is customary to supplement INS-1 cell culture medium with antioxidants such as GSH. This was added to the RPMI medium at 3.2 µM. It might thus be expected that such condition could lead to an under-estimation of pro-oxidant effects of the test compounds when assessed in vitro. The consequences of redox imbalance are membrane lipid peroxidation, DNA damage, protein oxidation and interference of signal transduction which contribute significantly to beta-cell dysfunction and death. Oxidative stress leads to apoptosis by activating the intrinsic apoptosis pathway31. Detection of cell surface phosphatidylserine (PS) by Annexin V, a 35 kDa Ca2+—dependent phospholipid binding protein with very high affinity (KD = 7 nM) is the methodology of choice for detecting apoptosis32. Annexin V binding assay detects early phases of apoptosis on cellular membrane level before the loss of cell membrane integrity33. Loss of phospholipid asymmetry is an early phenomenon in apoptosis34. The test discriminates intact live cells (FITC−/PI−), apoptotic cells (FITC+/PI−) and necrotic cells (FITC+/PI+). Of the three classes of the test substances (TCS, 4-tOP, Mix), the proportion of dead cells were comparable with high TCS concentration and high Mix lending credence to the result of the viability test. The results from PS asymmetry seemed to suggest that TCS and Mix induced cell death by apoptosis while 4-tOP was by necrosis. However, 4-tOP has been reported to induce apoptosis in the neuronal progenitor cells in mouse brain35, in rat Sertoli cells36, in the testicular germ cells of prepubertal rats37, and in human embryo stem cells38 showing that the compound operates different toxicity mechanisms in different cells as presumed for TCS39,40.
MTG is a green-fluorescent (excitation/emission 490/520 nm) mitochondria-selective stain believed to localize to mitochondria regardless of mitochondrial membrane potential48. The dye binds covalently with mitochondrial proteins through reaction with free sulphydryl groups of cysteine residues49. The increased staining by this dye therefore suggests increased mitochondrial staining. The increased mitochondrial mass as recorded with the positive controls suggests an increase in mitochondrial protein50,51 which may constitute a cell endogenous response to compensate for the mitochondria-related ‘deficits’52. It appears all the test substances at the concentrations applied, had no substantial effect on the mitochondrial mass.
The significant loss of MMP by TCS, Mix, Mel and Etop-exposed cells suggests the involvement of mitochondria in the apoptosis53,54. Failure of 4-tOP to reduce MMP affirms the substantial difference in the mechanism of toxicity between the two compounds (TCS and 4-tOP) in INS-1 cells. Loss of MMP leads to an opening of the mitochondrial permeability transition pore and subsequent leakage of intermembrane proteins, including cytochrome C that facilitates the induction of apoptosis through apoptosome formation55.
To evaluate the functionality of the β-cells in the presence of the compounds, glucose- stimulated insulin secretion was selected as a molecular signature to assess intact β-cells specific function. To this end, an in vitro system was established in which changes in the cellular calcium levels with or without glucose served as an indicator of cell function with respect to insulin secretion. In the presence of glucose, a significant increase in the cellular calcium levels was evident, thus confirming the rational of the experimental design. The reduction of the intracellular calcium influx by the test substances means a reduction of glucose-stimulated insulin release which was not substantial. Glibenclamide, a sulphonylurea and oral hypoglycaemic drug primarily stimulates insulin secretion by blocking the K-ATP channels and depolarizes the pancreatic β-cells which explains its increased calcium flux56. Diazoxide, a benzo thiadiazine derivative operates in contrast to glibenclamide mode of action. The drug exerts its effects through binding to the SUR subunit of the K-ATP channels, resulting in the opening of the channels and increased potassium entry into the cell (hyperpolarisation of the cell membrane) thus inhibiting insulin secretion57. It is therefore unexplainable at this time the observed increase in calcium staining by diazoxide. Elevation of the intracellular free calcium concentration is a key event in insulin secretion by pancreatic β-cells. Increased glucose metabolism, a major stimulus to β-cells, ultimately results in elevation of the ATP/ADP ratio causing plasma membrane depolarization through the closing of K-ATP channels. This leads to the entry of calcium through the opening of voltage-gated Ca2+ channels, which are located on plasma membranes58. Since mitochondria produce the most of glucose-derived ATP, mitochondrial dysfunction will impact negatively on glucose-stimulated insulin secretion. The summary of the results is presented in Table 3.
The study appears to support previous report that TCS is a weak mitochondrial toxin6 that combined exposure of INS-1 to the two compounds did not result in synergistic but probably antagonistic effects. The results confirmed apoptosis as the mode of cell death via a mitochondria-mediated pathway by TCS and Mix. Contrariwise, 4-tOP induced cell death by necrosis which was not mediated by mitochondrial pathway. Although the two compounds have been reported to be oestrogenic59,60 but they seemed to have targeted different sub cellular molecules in INS-1 cell. The adverse impact of the compounds on β-cell function probably provides an insight into the environmental role in the aetiology of non-communicable diseases like diabetes. While TCS acts mitochondrially, the molecular target of 4-tOP in INS-1 cell still needs to be precisely defined. The recorded increase in Ca2+ influx by diazoxide which contrasted with its primary role of inhibiting insulin secretion need be re-investigated.
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The authors are grateful for the financial support received from the University of Sharjah, the South Africa Medical Research Council and from Nigeria Tertiary Education Trust Fund.
Biochemistry Department, Faculty of Basic Medical Sciences, Ladoke Akintola University of Technology, Ogbomoso, Nigeria
SAMRC-Microbial Water Quality Monitoring Centre, University of Fort Hare, Alice, 5700, South Africa
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L.W.B. and A.I. conceived the project. L.W.B. performed the experiments and wrote the main manuscript. All authors reviewed the manuscript.
Correspondence to Lamidi WB Olaniyan.
The authors declare no competing interests.
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Olaniyan, L.W.B., Okoh, A.I. In vitro biochemical assessment of mixture effects of two endocrine disruptors on INS-1 cells. Sci Rep 12, 20102 (2022). https://doi.org/10.1038/s41598-022-20655-0
DOI: https://doi.org/10.1038/s41598-022-20655-0
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