2-EH and DEHP were purchased from Sigma Aldrich (St. Louis, MO) and diluted with dimethyl sulfoxide (DMSO) 1% for the working solutions (0.1% final DMSO concentration to cells) which were kept in sterile conditions and refrigerated until utilized. Positive controls: DEHP, camptothecin, and chloroquine for autophagy assays were purchased from Sigma Aldrich. DMSO 0.1% and culture media were used as a vehicle negative control.

The human lymphoblast cell line TK6 was obtained from the American Type Culture Collection (Manassas, VA. ATCC CRL-8015). Cell cultures were maintained in RPMI 1640 culture media with 10% fetal bovine serum (ATCC). Cells were maintained at 37˚C with 5% CO₂.

TK6 Cells were seeded (30,000 cells/well) on fluorometry compatible 96 well plates (NUNC, Rochester, NY) then exposed to the experimental compounds at ten different doses (in triplicates) for 24 hours. After the incubation period, the Prestoblue cell viability reagent (Invitrogen, Carlsbad, California) was added to all wells. This reagent is reduced by metabolically active cells to its fluorescent form and the fluorescence produced by the reduced form is analyzed using a Fluorostar Optima fluorescence reader (BMG lab tech, Cary, NC) using the 485/520 nm filters. Results were analyzed using the MARS data analysis software (BMG). Data with a coefficient of determination (R) lower than 98 was not considered. This growth inhibition dose was used in all subsequent experiments.

The annexin V assay is a common assay used in the detection of apoptosis. This assay measures the migration phosphatidylserine (PS) to the exterior surface of cells, a common event in apoptotic cells [11] . Approximately 3.0 × 10⁶ cells were treated for 24 hours with the 24-hour GI₅₀ dose for each test compound and the controls camptothecin 10 µM, and vehicle (DMSO). After the exposure period, cells were stained with annexin V conjugate, and propidium iodide (Biotium, Hayward, CA). These stained cells are then analyzed using the Nucleo Counter NC3000 system annexin V assay (Chemometec, Allerød, Denmark). Fluorescence of cells is detected and categorized on fluorescence emitted. A one-way ANOVA with post hoc Tukey test was also performed for all cell death assays (GraphPad Prism v.6).

Mitochondrial membrane permeabilization (MMP) is a key event in the apoptotic process [12] . A total of 1 × 10⁶ cells were exposed to the test compounds and the controls at their GI₅₀ doses. Cells suspensions were stained with 200 μg/ml of 5, 5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl benzimidazolo carbocyanine iodide (JC-1). Fluorescence was analyzed using a Fluorostar Optima fluorescence reader (BMG lab tech, Cary, NC) using the 485/520 nm filters. Results were analyzed using the fluorometer’s MARS data analysis software (BMG).

Caspase activation has been used as a key endpoint to identify cell death via apoptosis. In brief, apoptosis activation via caspases which can be grouped into two major categories; initiators and effectors. We have selected as endpoints Caspases 3 and 7 as the effectors, and Caspases 8 and 9 as the initiators. Once this signaling is activated, the cell proceeds with activating its enzymatic degradation [13] . Here, cell cultures were exposed to the test compounds as described previously and stained with the Fluorescent Labeled Inhibitors of Caspases (FLICA) purchased from (Immunochemistry Technologies, Bloomington Min.). This compound binds to the active caspase enzymes and emits green fluorescence at 488 - 492 nm peaking at 515 - 535 nm. Higher fluorescence will represent a higher number of cells with active caspase 3 & 7.

DNA Fragmentation is another common endpoint used to measure apoptotic responses on cells [14] . This event mediated by nucleases generates small fragments of DNA, which can be quantified by examination of DNA content and examining cells containing less than 1 DNA equivalent. The NC3000 assay for DNA fragmentation was used and is based on the removal of small DNA fragments and the retention of higher weight fragments stained with the DNA marker 4’,6-diamidino-2-phenylindole (DAPI). Cells were fixed with 70%, ethanol then stained with 1 µg/ml DAPI, and analyzed using the NC3000 instrument which measures DAPI fluorescence intensity measuring and quantifying high molecular weight DNA fragments.

Changes in the cell cycle can provide useful insights into drug effects. Some compounds may cause cells to become arrested in a particular stage [15] . To study 2-EH effects, the NC3000 fixed cell cycle assay was performed. This DAPI based assay uses ethanol-fixed cells then stained with 1 µg/ml DAPI and analyzed to measure by fluorescence DNA content and determination of cell cycle stage.

Autophagy is a key cellular process for maintaining homeostasis, and involves the degradation of subcellular components [16] [17] . During this process, cellular components reach lysosomes for degradation [18] . It has been documented that phthalates can cause autophagic cell death with mitochondrial effects [19] . The Premo autophagy sensor kit was purchased from Thermo Fisher (Waltham, MA). This probe binds to the LC3B protein, which is used to detect autophagic membranes. Approximately, 4.0 × 10⁴ cells were treated using the Premo kit following manufacturer’s specifications and plated in 96 well plates then is analyzed using a Fluorostar Optima fluorescence reader (BMG lab tech, Cary, NC) using the 485/520 nm filter.

Cathepsins are a group of proteases present in lysosomes and present during autophagy [20] . These enzymes degrade intracellular and extracellular materials into their basic forms reusable by the cells [21] . The green cathepsin B assay (Immunochemistry Technologies, Bloomington MN) was used to detect cells with elevated cathepsin B levels. Cells were stained following the manufacturer’s specifications and analyzed using a Fluorostar Optima fluorescence reader (BMG lab tech, Cary, NC) using the 485/520 nm filter.

Growth inhibition 50% (GI₅₀) is commonly used as a measure of the inhibition of 50% of exposed cells to a substance. The growth inhibition dose of 4.4 µM for 2-EH and 76 µM for DEHP on TK6 cells were determined after a 24-hour exposure (Table 1). The measurement of fluorescence corresponding to viable metabolically active cells was measured at 24 hours. The 2-EH compound presented a dose-dependent inhibition of all tested doses (Figure 2).

Results for annexin V assay revealed statistically significant (p < 0.05) induction of apoptosis when compared to the negative vehicle control (Figure 3). The negative control presented an average of 12% apoptotic cells whereas the positive camptothecin control presented a 52.6% average. The experimental compounds present late apoptotic events with 25% for the parent DEHP and 78.6% for the 2-EH samples.

Mitochondrial health was assessed after exposure to the phthalates and the

controls. Statistically significant (p < 0.05) differences were detected on all test compounds when comparing to the negative control (Figure 4). Mitochondrial membrane health was assessed by measuring the green fluorescence (Fluorescence Standard Units or FSU) of JC-1 stained cells. Cells with an intact mitochondrial membrane will emit orange fluorescence, whereas cells with compromised membranes will fluoresce green. Negative samples reflected an average of 3380 FSU while the positive camptothecin control presented an average of 2130 FSU. The phthalate samples reflected values of 1797 FSU for DEHP and 556 FSU for 2-EH. The 2-EH presented the least number of cells with intact mitochondrial membrane potential (ΔΨ_m).

Figure 5 presents our DNA fragmentation results which presented an average of 14.2% for the negative control and 45.6% for the positive control. DEHP presented an average of 25.6% which was not statistically significant when compared to the negative control and 2-EH (75.3%) presented a majority of cells with fragmented DNA indicating cells undergoing the cell death process.