Abl1 inhibitory contaminants leach from plastic tubes

Abstract

Plastic materials are widely used in research laboratories. Disposable plasticware facilitates life science research in the storage, transportation and manipulation of biological samples. However, recent findings have shown that some disposable plasticwares release bioactive contaminants. The bioactive leachates from plastic tubes, used as Abl1 catalytic incubator in this report, were noticed to interfere with the activity of Abl1. Extraction of these bioactive leachates was performed, and their inhibitory activity against Abl1 and cytotoxicity were tested. Results indicated that the tube extracts had no significant cytotoxicity but could inhibit the activity of Abl1. Therefore, these bioactive leachates from plastic tubes might be a specific inhibitor of tyrosine kinase.

• Abl1
• bioactive leachate
• inhibitory activity
• plasticware

Introduction

Plastic materials are widely used in contemporary life science research laboratories. Disposable plasticware is routinely used for the storage and manipulation of biological samples. The unique physicochemical nature of these plastic materials provides the necessary and desirable performance characteristics. However, recent scientific reports have noticed that agents released from disposable plastic labwares affect laboratory experiments leading to erroneous results.

Most laboratory disposables are manufactured from polypropylene, polyethylene, polystyrene or polyallomer. The pipette tips and microfuge tubes are commonly manufactured from softer plastics, such as polypropylene and polyethylene. A variety of additives are essential to make plastics processable and to assure their end-use properties. These additives include supplements, such as slip agents (including oleamide, erucamide, stearamide), biocides [including di(2-hydroxyethyl)-methyldodecylammonium (DiHEMDA)], plasticizers (phthalates) and some heavy metals. Several past studies have demonstrated that these additives and other chemicals can leach from plastic labwares and interfere with biomedical experiments.

Belaiche et al.1 reported that nonylphenol ethoxylate (NPE) plastic additives leach from disposable laboratory plasticware and inhibit the mitochondrial respiratory chain (MRC) complex I. NPE compounds, non-ionic surfactants from the Tergitol series are used to increase surface activity, and provide excellent all-purpose detergency and wetting, as well as solubilization and emulsification. In this report1, NPE-10 and NPE-9 were identified in the extract of blue tips. The IC₅₀ values of NPE-10 and NPE-9 against MRC complex I were determined at 4 and 3.7 µmol/L. The inhibition of oxygen consumption in whole mitochondria by NPE-9 and NPE-10 indicated that these compounds penetrate the inner mitochondrial membrane. The test of leachate from blue tips on growing fibroblasts in culture confirmed that these substances penetrate whole cell membranes.

McDonald et al.2 reported that disposable laboratory plasticware (pipette tips, microfuge tubes) leached the processing agents DiHEMDA and oleamide into buffers, resulting in pronounced inhibition of human monoamine oxidase-B (hMAO-B). DiHEMDA is a quaternary ammonium biocide. Many such biocides bind substantially to proteins and DNA and have recently been linked with fertility problems in mice3. Oleamide, a fatty acid primary amide, is an endogenous signaling molecule that binds to numerous receptor and channel proteins4.

An active leachate from polypropylene has been extracted and identified as erucamide by Watson et al.5. They indicated that tip extracts prepared in DMSO, as well as a commercially obtained sample of erucamide, were active in a cell-based functional bioassay of a known G-protein-coupled fatty acid receptor. Erucamide, a long-chain mono-unsaturated fatty acid amide, is also an endogenous molecule. It was also reported to be the major bovine angiogenic lipid as assessed by chorioallantoic membrane assay6.

Absorbance spectroscopy is routinely used to monitor the concentrations of nucleic acids and proteins within solutions and assess changes in their structures. These biological samples are usually manipulated and stored in small microfuge tubes. Lewis et al.7 found that leached compounds from these plastic tubes absorbed UV light strongly at 220 nm and 260 nm, which are the wavelengths normally used to detect and quantitate proteins and DNA. During incubation (e.g. enzymatic assay at 37 °C or DNA denaturation at 95 °C), additives used in the manufacturing process can leach out of the plastic and contaminate the biological samples. In this report, we describe that a specific batch of microcentrifuge tubes (BT) were inhibitory towards an Abl1 assay.

Experimental

Extraction of tubes and Abl1 inhibition assay

Twenty microcentrifuge tubes (Eppendorf) were cut into pieces and then extracted with water at 37 °C for 6 h, and the extraction solution was dried with nitrogen flow and then redissolved in 2 mL reaction buffer. The extraction solution was added into Abl1 reaction tubes in different volumes: 0, 25.2, 63 and 126 µL. The total volume of reaction mixture was 150 µL. The inhibition assays were conducted in 15 mM Tris-HCl buffer (pH 7.5) containing dithiothreitol (DTT, 1 mM) and MgCl₂ (5 mM) in the presence of ATP (35 µM) and the peptide substrate Abltide (KKGEAIYAAPFA-NH₂, 35 µM), Abl1 (0.1 µg protein/mL) and inhibitors at 30 °C in a total volume of 150 µL for 15 min. The reaction mixtures were analyzed by LC/MS8 and LC-UV9. The extraction and assay were repeated at least three times.

The LC-ESI-MS analysis was monitored by positive ESI ionization mode. HPLC was performed using a P680 HPLC pump from Dionex (Sunnyvale, CA) and it was coupled to a LCQ ion trap mass spectrometer (Thermo, Waltham, MA). A voltage of 4.5 kV applied to the ESI needle resulted in a distinct signal. The temperature of the heated capillary was set at 250 °C. The number of ions stored in the ion trap was regulated by Auto gain control (AGC). Nitrogen was supplied by Air Liquide (Liège, Belgium), used as sheath and auxiliary gas at a flow rate of 70 arbitrary units (arb) and 5 arb, respectively. Helium (Brussels, Belgium) was used as damping gas and as collision gas. The voltages across the capillary and octapole lenses were tuned by an automated procedure to maximize the signal for the ion of interest. The capillary voltage was set at 25 V and the tube lens offset voltage at −25 V. Octapole 1 offset voltage, octapole 2 offset voltage and the interoctapole lens voltage were set at −3, −6 and −12 V, respectively.

Cytotoxicity test of tube extraction

Cells used were immortalized mouse embryonic fibroblasts (MEF). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin solution (10 000 units of penicillin and 10 mg of streptomycin in 1 mL 0.9% NaCl) in a humidified atmosphere of 5% CO₂, 95% air at 37 °C. Cytotoxicity induced by the tube extraction was assessed by MTT assay10 and Neutral Red assay (NR)11. Analyses were performed in triplicate.

Results and discussion

The initial indication of a problem in the compound-handling process was the observation of no reaction in the Abl1 assay. This was unexpected because the assay had been performed weekly for approximately half a year without any issue. To identify the source of the problem, we examined our entire compound-handling process, including the disposable tubes used to prepare the Abl1 assay. It was noticed that the problem occurred just after a new batch of microcentrifuge tubes (Eppendorf, Safe-Lock Tubes 0.5 mL; lot: X131259G) were used. So, two groups of Abl1 assays were performed using new and old batches of microcentrifuge tubes. The results suggested that the unexpected result could be attributed to the new batch of microcentrifuge tubes (BT). The reasons might be the adsorption of Abl1 on the surface of the tubes or some bioactive leachates from the tubes. Because the enzymatic reaction was completely blocked when the BT were used, the possibility of the adsorption problem could be ruled out. The extraction and attempts for identification of the leaching compound were then performed. To ensure the trustworthy outcome of the Abl1 assay, higher quality labwares [Eppendorf, LoRetention PCR-clean tubes (GT) and tips] were used in the subsequent assays.

To confirm the inhibitory activity of compounds leaching from tubes, inhibition assays were performed using different volumes of tube extraction solutions. Optimized LC-UV and LC/MS methods were used to quantify Abl1 phosphorylated peptides with the presence of different concentrations of BT extract (BTE) in the reaction mixture. In order to get sufficient signal, the incubation time was doubled to 30 min when UV was used as the detector. Figures 1 (LC/MS chromatogram) and 2 (LC-UV chromatogram) show that there is a negative correlation between the activity of Abl1 and the volume of extract added. This observation indicates that the extract of BT has an inhibitory activity on Abl1. So, an effort was done to try identifying the leaching compounds and find potential inhibitors of Abl1.

Figure 1. Comparison of LC/MS chromatograms of Abl1 reaction solutions with different volumes of extract solution of BT: (a) 0 µL; (b) 126 µL; (c) 63 µL; (d) 25.2 µL. LC separation was performed on a Symmetry® C-18 column (150 × 2.1 mm i.d., particle size 5 µm) (Waters, Milford, MA) at room temperature (23 °C) with a flow rate of 0.2 mL/min. Mobile phase A (mpA) was 0.1% trifluoroacetic acid (TFA) in 25% acetonitrile; mobile phase B (mpB) was water. Gradient program: 0–2 min, 69% of mpA (isocratic); 2–6 min, 69 to 80% of mpA (linear gradient); 6–10 min, 69% of mpA (isocratic). A: peak area of p-Abltide; P: p-Abltide; S: Abltide.

Figure 2. Comparison of LC-UV chromatograms of Abl1 reaction solutions with different volumes of extract solution of eppendorf plastic tubes: 4 (0 µL); 3 (25.2 µL); 2 (63 µL); 1 (126 µL). Analysis was carried out at 210 nm with an Alltima C18 5 µm, 250 mm × 4.6 mm column (Alltech Associates, Lokeren, Belgium). The temperature of the column was kept at 30 °C, while the temperature of the sampler chamber was set to 4 °C. The injection volume was 10 µL. Mobile phase was prepared by combining water, acetonitrile and TFA in a 78:22:0.05 (v/v/v) ratio. The mobile phases were degassed by sparging with helium for 2 min. The flow rate was set to 0.5 mL/min. p-Abltide is shown in the dashed rectangle.

To identify the active contaminant, we extracted samples of the BT and GT for UV and MS analysis. When LC-UV was performed, two wavelengths (λ=210 nm and λ=254 nm) were used. Figure 3(a) and (b) indicate that more peaks appear on the chromatogram of both BTE and GT extract (GTE) compared to the blank. Both figures also show the difference between BTE and GTE. It indicates some compounds did leach from the tubes, especially the BT gave more leaching not only in terms of the number of compounds but also in terms of the amount. So a LC-ESI-MS analysis was performed to identify those peaks. According to the information from LC-ESI-MS, the peak around 27 min had a m/z of 1108.7. Although this signal with m/z of 1108.7 was present in both BTE and GTE samples, the intensity in BTE was 12.6-fold higher than GTE. The UV chromatograms (Figure 3a and b) also indicated that the concentration of the potential inhibitor with m/z of 1108.7 was much higher in BTE than in GTE. A q-TOF mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA) was also used to try and identify BTE by direct infusion. The data obtained by q-TOF mass spectrometry suggested that two other potential compounds with m/z of 261.0533 and 301.1412 were also present in the BTE. These two signals were also present in GTE based on the LC-ESI-MS data and the EIC chromatogram indicated that the mass response for m/z of 261.0533 was about 1.9-fold higher in GTE than in BTE, and the mass response for m/z of 301.1412 was almost same in GTE and in BTE (1.1-fold higher). The result suggested the compound with m/z of 261.0533 could not be responsible for the inhibitory effect of BTE. Therefore, the peak with m/z of 1108.7 will be further studied in the future work. Cytotoxicity test results based on NR and MTT assay indicated that there was no apparent cytotoxicity when the cells were treated with GTE or BTE. Four different concentrations (0.3, 1.0, 3.3 and 10 µL of GTE or BTE were added into the cell test wells with a final volume of 100 µL) of tube extract were used to test cytotoxicity. Every test was performed in triplicate. MTT assay results show the percentage viability was from 94.3% ± 2.4% to 99.5% ± 3.0% for GTE and from 91.0% ± 5.9% to 99.0% ± 5.5% for BTE. The results indicated that both GTE and BTE did not induce cytotoxicity in fibroblasts after incubation for 24 h. NR results confirmed this conclusion.

Figure 3. LC-UV chromatograms of microcentrifuge tubes extract with λ = 210 nm (a) and λ = 254 nm (b). Trace 1 represents freshly prepared blank of reaction buffer, trace 2 is GTE and trace 3 BTE. DTT1 and DTT2 represent the reduced and oxidized forms of DTT, respectively. Due to air oxidation, the reduced form of DTT was converted to the oxidized form. The time from preparation to injection was different for the blank solution and the tube extraction solutions, which caused one extra peak (DTT1) in the chromatogram of trace 1. LC separation conditions were identical to those mentioned in Figure 2, except the flow rate was 0.2 mL/min, which is comparable to the LC/MS method.

To conclude, the leachates from a specific batch of plastic microcentrifuge tubes were capable of interfering with the activity of Abl1. But these leachates did not show cytotoxicity. Therefore, the leachates from plasticware might interfere with the activity of specific enzymes. It is thus highly advised to exclude the interference of plasticwares first when a bio-reaction test is performed.

Declaration of interest

The purchase of the quadrupole orthogonal acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA) was made possible by the support of the Hercules Foundation of the Flemish Government (grant 20100225–7).

The authors have declared no conflict of interest.